\r\n\tFrequently, bullous diseases are misdiagnosed, and the precise diagnosis requires a skilled dermatologist. Nowadays, histopathology is the standard method which requires supplemented methods including direct immunofluorescence (DIF) confirming the origin and location of autoantibody. Indirect immunofluorescence (IIF) is filled up with ELISA allowing the precise type of autoantibody playing the mainstay role in the pathogenesis of a disease. Follow up of serum titer of autoantibody is important in the management of disease, especially in pemphigus group. Established time of free clinical appearance and negative detection of autoantibody using ELISA or IIF allow confirming a loss of production of autoantibody using DIF. Negative results in these examinations allow discontinuing the immunosuppressive therapy in patients with pemphigus and bullous pemphigoid groups. In dermatitis herpetiformis, the pathogenesis differs, and the immunosuppressive therapy and/or gluten-free diet should be used for the whole lifetime. The book will provide a comprehensive overview of the current diagnostic skills and methods, management of immunosuppressive therapy and topical therapy, which may allow decreasing the systemic administration of immunosuppressive medications.
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:null,priceUsd:null,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"f91d25ba59038b77804ec2af35ef8447",bookSignature:"Dr. Danka Svecova",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8703.jpg",keywords:" Pemphigus Vulgaris, Paraneoplastic Pemphigus, Pemphigus Foliaceus, Pemphigoid Gestationis, Bullous Pemphigoid, Iga Pemphigus, Mucous Membrane Pemphigoid, Linear Iga Dermatosis, Epidemiology, Etiology and Pathophysiology, Histopathology, Immunopathology",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"November 29th 2018",dateEndSecondStepPublish:"December 20th 2018",dateEndThirdStepPublish:"February 18th 2019",dateEndFourthStepPublish:"May 9th 2019",dateEndFifthStepPublish:"July 8th 2019",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"209275",title:"Dr.",name:"Danka",middleName:null,surname:"Svecova",slug:"danka-svecova",fullName:"Danka Svecova",profilePictureURL:"https://mts.intechopen.com/storage/users/209275/images/system/209275.jpg",biography:"Dr.Danka Svecova, Medicine Doctor (PhD., dermatology), is a professor of Dermato-venerology, Head of Bullous Disorders Unit at Dept. of Dermatovenerology, University Hospital and Faculty of Medicine, Comenius University in Bratislava, Slovakia. \r\nShe is a board member of the Division Committee for Dermatovenerology Dissertation for Ph.D. She is a member of a committee for Probation of Specialization for Dermatovenerology. \r\nShe participated in research on Skin Allergology and Immunology under the supervision of Professor Akira Ohkawara, at Hokkaido University in Sapporo, Japan. She held several grant projects on fungal infection, therapy of psoriasis, borreliosis, immunomodulatory and anti-inflammatory efficacy of normal polyphenols, immunogenetic determination to psoriasis Vulgaris and pemphigus Vulgaris, GWAS (Genome-Wide Association Study) on pemphigus vulgaris in cooperation Harvard Medical School in USA and Anhui Medical University in China under supervision of Professor Liangdan Sun.",institutionString:"Comenius University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Comenius University",institutionURL:null,country:{name:"Slovakia"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"175",title:"Dermatology",slug:"dermatology"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"225753",firstName:"Marina",lastName:"Dusevic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/225753/images/7224_n.png",email:"marina.d@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. 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1. Introduction
1.1. Morphology
The thyroid is an endocrine gland formed by two lobes (Figure 1A L) located on each side of the trachea and the larynx; they are joined by an isthmus (ultimo-branchial origin) (Figure 1A clear arrow) located between the trachea’s second and third cartilage rings (Figure 1A black arrow).
1.1.1. Histology
A fibrous connective tissue capsule covers each lobe from where the septa go inside, partially dividing the glandular parenchyma containing a very developed network of capillaries surrounding the follicles and irrigating the glandular parenchyma. The gland also contains adipocytes, nerve fibres, mastocytes and occasionally lymphocytes and macrophages [2].
The thyroid parenchyma mainly consists of follicles which are the thyroid’s functional unit (Figure 1B). It has an oval or spherical structure whose wall consists of a layer of cubic epithelial cells (thyrocytes) (Figure 1B black arrow) surrounding a viscous solution of proteins called colloid (Figure 1B Co) [2,3] containing 80% thyroglobulin (Tg) or thyroid hormone [4,5]. Follicle size varies according to an individual’s age, its localisation in the gland and animal species; for example, diameter varies from 50 to 150 μm in rats and mice where peripheral follicles are larger than the central ones (Figure 1A L), whilst diameter varies from 150 to 500 μm in humans and pigs, the largest ones occurring towards the inside of the gland, even though their location could vary [6,2]. As well as follicle cells or thyrocytes, it has been found that 1% to 2% of neural crest cells in different mammals’ thyroids are parafollicular or clear cells, appearing clearer in different types of histological staining. These are located at the base of follicles but do not come into contact with the colloid and secrete calcitonin [7].
Figure 1.
A. Histological cross-section of mouse trachea, showing cartilage (black arrow) and mouse thyroid lobes (L) and isthmus (clear arrow) adhering to connective tissue between cartilage and thyroid. The thyroid parenchyma consists of follicles which look like spherical or oval structures. B. The appearance of pig follicles in the lobe’s central region. Each follicle consists of simple cubic epithelium (black arrow) limits the follicle centre full of colloid (Co) which follicular cells or thyrocytes secrete. The capillaries surrounding the follicles can be seen (clear arrow). C. Rat thyrocyte ultra-structure or cytology. The rugose endoplasmic reticulum (RER) can be seen around the nucleus (N) and Golgi complex in supranuclear position; these organelles and lysosomes (L) occupy the cells’ base region. Different vesicles can be seen at apical level, from exocytosis (ex) being denser than electrons and endocytosis (en) and having the same density as electrons and colloid (Co), and the apical membrane forming microvellosities (M) in contact with colloid in the centre of the follicles. The binding complex can be observed in the lateral membrane at apical level: tight junctions (TJ) followed by belt desomosome (BD) and spot desmosome (SD). The thyrocytes’ basement membrane or basal lamina (white arrow) is in close contact with the endothelium’s (E) fenestrated capillaries’ basement membrane (black arrow); the endothelium cell nucleus (EN) can be seen (A and B H-E, MO. Scale bar A 1mm, B 40 µm. C TEM. 7300X).
1.1.2. Ultra-structure
Endocrine gland cells are usually not polarised with their central nucleus and organelles; however, thyrocytes are exceptionally polarised endocrine cells and the nucleus and organelles are located in the cell’s basal region like exocrine epithelium cells. This is due to the larynx’s embryonic development by polarised cells involuting inside it. Such morphological and exocrine functional characteristics are conserved within follicles for thyroid hormone synthesis, storing and secretion (Figure 1C). The thyrocytes’ basal membrane is directly related to the follicles’ basement membrane or basal lamina (Figure 1C white arrow), in turn, being in direct contact with the fenestrated capillaries’ endothelial (Figure 1C E) basement membrane (Figure 1C black arrow). The thyrocytes’ apical membrane is in direct contact with colloid forming microvellosities (Figure 1C M) whose length and amount vary according to a gland’s functional state [8]. Exocytotic vesicles (Figure 1C ex) can be seen in the apical region, some of them being more electron dense than less electron dense endocytic vesicles (Figure 1C en) and some coated at the base of microvellosities [5].
The thyrocytes’ lateral membranes have binding complexes in the apical region formed by tight junction, belt desmosome and spot desmosome (Figure 1C TJ, BD, SD) isolating and separating colloid from the intercellular spaces and basolateral apical membranes [9].
The thyrocytes’ nucleus is surrounded by abundant RER in the cells’ basal region (Figure 1C RER) and the GC is in the supra-nuclear region in normal physiological conditions (Figure 1C N). The lysosomes are located in the thyrocytes’ basal media region (Figure 1C L). The mitochondria are distributed throughout the whole cell.
The thyrocytes form depends on their functional state; they are cubic in normal conditions (euthyroid morphology) and have the aforementioned ultra-structure (Figure 1C). Without thyrotrophic or thyrotropin-stimulating hormone (TSH), endocytic vesicles disappear at the beginning and exocytosis increases and microvellosities become reduced. The lysosomes increase in size and have very heterogeneous content following several days without TSH; the follicular cavity increases after a few days and cells become thin and atrophied because the RER and GC become reduced. Such cells disappear following 20 or 30 days’ suppression of TSH (hypothyroid morphology). A rapid increase in exocytic vesicles (micropendocytosis) occurs when the gland is stimulated by TSH and the apical membrane forms cytoplasmic expansions or pseudopods forming large macroendocytic vesicles called colloid droplets; such vesicles merge with lysosomes which migrate to the cells’ apical region [10]. When TSH stimulation is sustained for more than 5 days, microvellosities’ length and amount increase, follicular cavities become reduced and the thyrocytes become cylindrical and hypertrophied because the RER and GC increase, occupying almost the whole of a cell (hyperthyroid morphology) [2,8,10,11].
2.2. Function
T3 (3,5,3’-triiodine-thyronine) and T4 thyroid hormone (3,5,3’,5’- tetraiodine-thyronine) synthesis reflects thyrocytes’ follicular morphology and ultra-structure which can be divided into 3 stages (Figure 2 circles): stage 1, Tg synthesis and colloid secretion at intracellular level; stage 2, iodide accumulation and its organification regarding Tg in colloid at thyrocyte extracellular level; and stage 3, endocytosis and intracellular hormone secretion [5,12].
Figure 2.
A. A diagram of a thyroid follicle surrounded by capillaries (Cap); Co: colloid. B. A diagram of a thyrocyte showing the physiology of thyroid hormone synthesis. Iodide is captured in the basement membrane by the sodium/iodide symporter or Na+/I- symporter (NIS) and rapidly transported to colloid, mainly via pendrin (P); it is used there for thyroid hormone synthesis. Tg is synthsised in the RER and N-osidic glycosylation culminates in the GC; Tg is secreted to colloid by exocytic vesicles (green circle 1). Once in colloid, thyroperoxidase (blue) with thyroid oxidase 1 or 2 (red) fixes iodide to Tg (red circle 2) forming iodine-thyronine on Tg (Tg-I). When thyroid hormones are required, Tg-I is endocyted by microvesicles or macrovesicles (blue circle 3) when TSH stimulates the thyroid. Tg-I becomes degraded by lysosomal enzymes (L) releasing T3 and T4 into the blood stream. N: nucleus; RER: rugose endoplasmic reticle; GC: Golgi complex; I-: ion iodide; NIS: Na+/I- symporter; EeE: early endosome; Pre: prelysosome or late endosome; L: lysosome; BMs: basements membranes or basals laminas; T3 and T4: thyroid hormones. Diagram modified from Spinel (2003) [12].
The first stage occurs at intracellular level, Tg is synthesised and glycosylated (N-osidic glycosylation) in RER and then glycosylation in culminates in the GC. Tg is transported in exocytic vesicles which emerge from the GC (Figure 2 green circle 1) and is released to the colloid [5]. Iodide accumulation takes place in the basal membrane via the sodium/iodide symporter (NIS) or Na+/I- symporter (Figure 2 NIS) [13]. It then passes through colloid via pendrin (I-/Cl- apical exchanger) [14] (Figure 2 P), regulated by the apical region’s ClC-5 channel [15].
The second stage happens at extracellular level. Thyroperoxidase (TPO) is found in the base of microvellosities anchored to the apical membrane which oxides iodide (Figure 2 blue) and thyroid oxidases 1 or 2 (Duox1 and 2) forming H2O2 (Figure 2 red). TPO fixes one or two iodines on specific Tg thyrosins in colloid (iodide organification), thereby forming mono- and di-iodinethyrosins (Figure 2 circle 2). TPO couples the iodinethyrosins, producing iodine-thyronine or T3 and T4 hormones on Tg19S or thyroid prohormone (Figure 2 Tg-I); in both processes TPO reduces H2O2 [16,17].
The third stage is intracellular. Tg19S is endocyted [10] (Figure 2 blue circle 3) and degraded in prelysosomes [18] and in lysosomes (Figure2 Pre, L), releasing the hormones which become diffused through the basal membrane to the blood stream [19] where they are transported by three families of blood proteins to an organism’s cells [20,21].
Due to the thyroid’s morphological characteristics and its function, “it is an exquisitely regulated gland”, [22]. Its function is essentially controlled by the hypothalamus-hypophysis and also by the nervous system and other thyroid systems [22]. Thyroid gland function and growth is controlled by TSH secreted by adenohypophysis thyreotropic cells. TSH secretion is stimulated by thyrotropin releasing hormones (TRH) secreted by the hypothalamus. TSH and TRH concentration in circulation are regulated by T3 and T4 concentration; thyroid and TSH concentrations are regulated by iodide concentration in the blood stream obtained during daily intake [2,3,22,23,24,25].
TSH mainly activates the AMPc route which stimulates transcription factors (CREB, TTF-1 and -2, PAX8) and culminates by activating the transcription and expression of molecules implicated in T3 and T4 hormone synthesis (i.e. NIS in basal membrane, Tg in RER and its exocytosis, TPO and Douxs in the apical membrane and H2O2 formation). TSH’s effect can be shown by increased T3 and T4 in the blood stream [26,27].
Normal iodide in circulation ranges from 10E-9 to 10E-7 M. Concentrations of this ion greater than 10E-5 inhibit T3 and T4 organification and synthesis during the first 48 h (called the Wolff-Chaikoff effect) [1], regardless of TSH concentration. Iodide organification inhibition directly depends on iodide intrathyroid accumulation [28]. This thyroid auto-regulatory effect happens when inorganic iodine concentration in blood exceeds a set threshold (overload) and the gland blocks iodine’s organic binding for 48 h [1]. The gland adapts once such 48 h have elapsed and organified iodide escapes, producing new hormones [29]; NIS expression becomes reduced at this time, as does iodide capture [30]. It has been suggested that there is a reduction in the function of the molecules implicated in organification and hormone formation: TPO, Duox 1 and 2, pendrin and Tg [30,31]. Thyrocytes in culture in the presence of 10-E3 M iodide reduce NIS expression and inhibit TPO and Tg synthesis [32]. Such reduction of NIS does not happen in hypothyroid mice, nor is Duox 1 and 2, TPO, pendrin and Tg gene expression modified [33]. An excess of iodide leads to iodide organification inhibition depending on TPO and not on NIS. TSH effects become reduced in the presence of strong concentrations of iodide, resulting in them adopting antagonic roles [34].
2. Thyroid culture
2.1. Introduction
Thyroid tissue fragments were kept on glass in saline solution, or in vitro (as this involved a glass vessel). Established the neuron cell theory, it has since been established that the unit of life is a cell (i.e. cell theory 1910) and cell culture or in vitro study began [35]. Cell cultures were then developed, thereby leading to studying cell functions in controlled conditions and different descriptions of culture mediums, supports and conditions have been developed from 1910. Fibroblasts in culture leave a matrix on culture surface on which endothelium cells from blood capillaries can be cultured; this has been called an extracellular matrix (ECM) [36]. Extracellular supports close to the ECM surrounding cells in vivo (such as collagen, laminin, fibronectin or matrigel) are currently being used [37].
New culture techniques were developed in 1975 in view of the close structure-function relationship, recognising the importance of organs’ functional units, such as isolating and culturing isles of Langerhans from the pancreas [38] or “acini” regions from the lactant mammary gland or epithelial structures which needed to be conserved in polarised cell culture [39]. Thyroid follicle incubations and cultures could also be mentioned here.
A brief description of the most pertinent techniques for culturing the thyroid, isolated thyrocytes and/or thyroid follicles is given below.
2.2. Organotypical culture or organ culture
Organ culture or organotypical culture consists of culturing an organ’s fragments or explants. Regarding the thyroid, this began with 2 to 3 h incubations (the term usually used to refer to cultures lasting less than 24h) of sheep thyroid fragments in the presence of radioactive iodide thereby demonstrating in vitro the ion’s incorpoproportion into diiodinethyronine (DIT) and T4 [40]. When the transmission electron microscope was developed in the 1970s, this led to an ultra-structural description of thyrocytes in vitro; the first descriptions of thyrocytes’ morphological changes in different culture conditions were made. It was shown that organ culture thyrocytes had reduced RER and GC in the absence of TSH [41,42]. Thyrocyte follicular architecture and ultra-structure were rapidly lost in some of the models which were described. Approaching the 1990s attempts were made to use very small fragments (less than 1mm3) in organ cultures (called mini organ cultures) which lasted 2 to 3 days without necrosis, exhibited iodide, sulphate and phosphate transport, synthesised a 19S Tg (normally glycosylated and iodised) [43] and were maintained for up to 7 days without cell death when coated with collagen [44]
2.3. Isolation and monolayer culture or cell culture
Monolayer culture (better known as cell culture) mainly deals with a single cell type. This implies tissue dissociation by enzymatic digestion or mechanical action and the isolation of cellular types by different sepaproportion methods. Isolated cells are placed on different types of supports where they adhere and proliferate in a single layer until reaching confluence (called primary culture). Secondary culture consists of sowing cells removed from the primary culture in fresh recipients and so on. The term passage is used to indicate the number of successive secondary culture sowings, thus the 1st secondary culture is the 1st cell passage. Thyrocytes were first cultured in 1911 [45]. Using this dissociation technique and continuous shaking during culture has shown that sheep thyrocytes concentrate radioactive iodide and incorporate it in iodine-thyronine: MIT, DIT and T4 [46]. Isolated and small cells, aggregates of 10 to 15 thyrocytes, are obtained after dissociation with trypsin [47,48]. One of the greatest drawbacks is the loss of cultured thyrocytes’ cellular polarity when one wishes to study thyroid physiology since such polarity is fundamental in conserving thyrocyte membrane domains, and thus the expression of domain-specific molecules guaranteeing hormone synthesis [49].
Thyrocyte cultures were developed in dual chambers during the 1990s on cubic monolayers as in vivo with binding complexes in the lateral membranes’ apical region, separating in thyrocytes’ the apical membrane domains from the basolateral membrane domains, TSH favouring such cellular polarisation [50]. This model has demonstrated that ion flow is determined by thyrocytes’ polarity, thereby corroborating the fact that ion channels are different in both thyrocytes membrane domains when thyrocytes’ cubic form is conserved. A new channel has been described for the thyrocytes’ apical membrane [51]; this new channel is CLC5 which is located in the apical region in vivo and it has been proposed that thyrocytes have a position in the apical membrane for controlling pendrin, the I-/Cl- transporter [15].
The foregoing has shown the importance of conserving cell polarity and cubic form in thyrocyte culture for studying the gland’s physiology and biochemistry.
Cell lines are continually growing and indefinitely proliferating cell cultures because they have lost control over their own cell division, contrary to primary and secondary cell cultures which die after a finite number of passes or subcultures, as is genetically determined in normal cells. Thethyrocyte cell line was described [52,53]; Fischer rat thyroid cell line or FRTL is most used around the world as it has a more similar ultra-structure to thyrocytes and synthesised Tg. These have been very useful in studying gene expression, cytoskeleton modification with different factors, iodide flow and that of other ions.
Such studies have led to advances being made in knowledge regarding some precise processes but there are limitations for extrapolating this to the gland in vivo because they are cells which lost certain control over their tissue of origin.
2.4. Pseudofollicles
Some of the main problems involved in thyroid physiology in vitro studies are the loss of follicle architecture, thyrocyte polarity and T3 and T4 hormone synthesis [54]. As mentioned above, the follicle lumen disappears in the monolayer with its colloid and the thyrocyte membrane domain polarity necessary for carrying out hormone synthesis [55,56]. However, tridimensional structures can be induced by covering monolayers with ECM elements, they become re-organised into two- to four-cell structures around the intercellular cavity in the presence of TSH, called pseudofollicles by some authors and “follicles” by others [54,56,57,58,59,60,61,62]. These pseudofollicles are unstable, short-duration structures and do not reproduced in vitro the function of iodide incorporation in Tg or synthesis of T3 or T4, and shown that follicle structures’ thyrocyte polarity is necessary for studying thyroid physiology and the molecular processes implicated in such function [63].
3. Follicles
A new culture technique was developed from 1965 to 1980 where the functional unit of different epithelial organs, isles of Langerhans in the pancreas [64,65] or the acinar region of the mammary gland [39] are isolated and cultivated, conserving in vivo morphology.
It is clear that conserving multicellular structures in vitro forming exocrine glands’ functional units depends on preserving apical-basal polarity for imitating in vivo functions; this is why attempts at characterising the factors generating polarity and which molecules allow maintaining 3D structures in vitro in mammary gland acini [66], the endocrine pancreas [67] or kidneys [68] are continued.
The 1980s saw the beginning of 24-h thyroid follicle cultures [23,69,70,71,72,73,74] on agarose, to avoid cell adhesion and monolayer formation [75]. Such incubations showed that iodide organification and H2O2 production took place in colloid [23,72] and that rat thyrocitos of open and closed follicles incubated for 12 h on agarose conserved their apical-basal polarity and in vivo ultra-structure, in basal region nucleus surrounded by RER, supranuclear GC, in apical region vesicles’ and microvellosities. As well as responding to TSH-forming pseudopods [16,23,75], synthetic TSH peptides bound in the basolateral membrane [76]. Closed follicles were maintained for up to 3 days [69] and responded to thyrocytes’ TSH, increasing RER [23]. TSH has stimulated pig and human open follicle thyrocyte function in culture during the first two days [49,74]. Pig open follicles cultured for 48 h with forskolin have been used for determining the function of H2O2 formation which is important in hormone synthesis [77]. The presence of TSH- or forskolin-induced cyclic adenosine monophosphate (AMPc) route stimulants has been seen to be indispensable in these cultures and has reiterated the importance of conserving follicle structure in culture for promoting hormone formation, as happens with thyroid gland apical membrane in vivo. It is so important that specific genes have been described which govern follicle formation and maintain follicle architecture, the gene transcribing the thyroid-specific enhancer-binding protein (T/ebp or Nkx2.1) regulating the transcription of genes implicated in hormone synthesis: NIS, TPO, TSH receptor and Tg and re-organised transfecteds thyrocites in follicles [78]. Human goitre follicle structure has been covered with collagen to preserve it longer and cavities in human [79,80] and mouse thyrocyte [78] thin cell bilayers have persisted 2 days more. Collagen’s importance in preserving epithelial cells’ apical-basal polarity has been described for obtaining MDKC (Madin-Darby canine kidney epithelial cell line) cell 3D cultures, but hepatic growth factor (HGF) was added to cultures [81].
The methodology developed by our group for reproducibly obtaining closed follicles, conserving their architecture and function in culture analogously to that of the gland in vivo is described below.
4. Method used
Most of the first work on rat follicle culture was open and became disorganised during the first days of culture [16,23,72]. We based our approach on these rat thyroid dissociation techniques. We describe the importance of isolating closed follicles, their culture and long-term response to TSH and iodide (9 and 12 days) and to increasing doses of iodide. Details are given of the isolation methodology, the morphological study of these isolated follicles and in culture at morphological level by inverted (IM) optical (OM), electron (TEM) and (CM) confocal microscope and their functional study: iodide accumulation and organification, Tg, T3, T4 synthesis and NIS localisation.
4.1. Isolation and closed follicle culture
Wistar rat (200g) and ICR mouse (30g) thyroid was used; the animals were obtained from the Universidad Nacional de Colombia’s Bioterium. Pig Cialta and strain 769 thyroid was provided by two slaughterhouses in Bogotá. Some having sub-clinical hyperthyroidism due to β- energetic injection, having T3 (1.34 ng/dL) and T4 (107.0 ng/dL) within the normal range and excessively low TSH (<0.005 mUI/mL), were called hypothyroidic, as morphologically and functionally described in mice [33], whilst the others were called euthyroidic. The animals were handled according to Colombian considerations for animals being used in research and care of animals for domestic consumption.
The methodology mainly involved rat thyroid and was corroborated in mouse thyroid. Obtaining human thyroid fragments is difficult; pig was thus used due to its similarity with human metabolism [82,83,84,85,86], even though it is hoped to begin cultures with human thyroid in the near future. The differences between rodents and pig had to be considered. General metabolism regulated by the thyroid gland in rodents is 10 times greater than that in pigs and humans. Follicle diameter ranges from 50 to 150 µm in rodents, whilst this is 150 to 500 µm in pigs and humans. Rodent lobes range from 3 to 5 mm3 at their widest whilst this is 3 to 5 cm3 in pigs and humans, meaning that many rodents must be sacrificed for each experiment; 30% to 40% of a pig’s lobe is used. Rodents’ capsule is thin and the parenchyma does not have large amounts of connective tissue; this capsule is thick in pigs and connective tissue septa are very abundant and extensive. Shaving razors are used for stereoscopic micro-dissection. The capsule of rat and mice lobes is eliminated; each is cut in two along its major axis whilst pig lobes are opened in two with a scalpel and cut into 7 to 10 mm3 fragments. Connective tissue is then eliminated as far as possible using stereoscopic microdissection with razors without affecting the parenchyma. Around 3 mm3 fragments are obtained (similar to rodent fragments) without connective tissue visible by stereoscope.
The thyroid fragments are put together and washed 3 times with COON medium [55], enzymatically dissociated with collagenase II which digests collagen (250 U/mL rodents; 400 U/mL pig) and 2 μg/mL DNase 1 (nb, the original article [82] read “2 mg/ml DNase I” when it should have been 2 µg/ml). Dead cells form aggregates which are avoided with DNase which only dead cells’ DNA digest and become fragmented; live follicle cells or fragments become attached to these aggregates if DNase is not added [23].
Thyroid fragments become dissociated in enzyme solution in COON medium at 37ºC and being shaken at 140 oscillations per minute; without delay, they are mechanically dissociated in this solution, aspirating and expelling enzyme solution containing thyroid fragments with 20 mL pipettes (3 to 5mm distal diameter; extreme for liquid entry and exit from pipettes), 10 times. The technique with rodent fragments continues with 10mL pipettes (1.5 mm distal diameter), 10 times. Such pipette dissociation is done at 10 min intervals during enzyme dissociation (i.e. the supernatant containing isolated follicles is collected every 10 min after pipette dissociation and fresh enzyme solution added for the following 10 min). This is done three times x 10 min with rodent thyroid and 6 x 10 min with pig thyroid; this is a modification of already described dissociation [16,23,72] and is most important for avoiding follicle opening. Most follicles are isolated during a second dissociation for rats and mice and in a third and fourth for pigs.
The follicles isolated during each 10 min interval are washed 3 times with COON + 2% foetal calf serum (FCS) spun at 50g for rodent follicles and 30g for pig follicles. This must be done in a free rotor centrifuge using low centrifugal force, otherwise centrifugal pressure opens up many follicles. All the follicles are placed together and filtered through 100 μm pore diameter mesh for rodents; those for pigs are left to decant at 1g for 10 min. Dissociation pre-incubation or recuperation time is continued for 4 h for rats and mice and 12 h for pigs in COON medium + 0.5% FCS in a 95% air - 5% CO2 atmosphere and 100% humidity on 1% agarose type I (less grouping than with agarose type II) to avoid cells adhering to the support [44,75]. Culture medium (the same as pre-incubation) is changed for aspiration with follicles; it is spun at 50g for rodents and 30g for pigs. The supernatant is skimmed off and fresh medium added to begin culture in the same ambient conditions and on agarose type I; the same is done for changing medium when making the culture.
Undissociated fragments (around 0.8 mm3) remaining after enzyme dissociation are washed 3 times with COON + 2 % FCS and cultured in the same conditions as for follicles but with 2% FCS. This has been called mini organ culture, according to Bauer and Herzog [43].
Different supports have been tried for maintaining follicular architecture, such as glass, plastic, collagen or collagen coated (1mg/mL), and 1%, 5% and 10% FCS concentration. Closed follicles are conserved in culture; however, thyrocyte monolayers grow proportionally to serum concentration in the medium. Monolayer growth should be avoided because this increases iodide accumulation values and interferes with analysis of iodide organification function; fresh enzyme dissociation must be carried out to recover the follicles [82,86]. When follicular cell fragments are cultured in collagen they do not reform follicles as has been described for human [79,80] and mouse thyrocytes [78]; some MDKC epithelial cells are organised in follicle-like structures requiring HGF [81]. The foregoing meant that the use of glass, plastic and collagen for follicle culture was rejected and agarose type I used instead.
Follicles’ functional and morphological state was controlled before beginning the cultures by 5% Trypan blue exclusion exam [87], cell viability was determined by IM which also allowed visualising open or closed follicle architecture. Thyrocyte viability is usually around 100% immediately after isolation and before beginning pre-incubation. Cells which do not exclude Trypan blue are usually endothelium cells bound to follicle periphery (Figure 3B solid black arrows) or follicular fragment aggregate thyrocytes (Figure 3B circle).
Iodide accumulation and organification is determined (5 μCi/mL Na125I, 4 h) for ensuring a high percentage of closed follicles; the importance of this control before beginning culture is described and discussed 125I in 6.1. Importance of obtaining closed follicles.
Follicles are culture for 0, 1, 3, 6, 9 and 12 days with TSH and without TSH (1 and 0.1 mU/mL rat; 1 mU/mL pig) in the same conditions as for pre-incubation. Culture medium is changed during these days aspirating it with the follicles and spinning at 50g for rat and 30 g pig, discarding the supernatant which usually contains cell and follicle fragments. The follicles are examined by IM during each stage. 5 μCi/mL Na125I is added each culture day 4 h before collecting the follicles for morphological and functional studies.
The follicles are cultured for 1 day with and without TSH (0.1 mU/mL rats; 1 mU/mL pigs) for studying the effect of different iodide concentrations. The medium is changed and the follicles cultured for 0.5, 3, 8, 12, 24 and 48 h with 10E-10, 10E-7, 10E-5, 10E-3 M Na127I and Na125I 5 µCi/mL (kindly donated by Manuel E Patarroyo) with and without TSH. The follicles are collected after such treatment for morphological and functional analysis.
4.2. Morphological studies
Morphological study involves impregnating follicles in Epon resin; follicles are spun at 300g for dehydratation before being impregnated in the resin [23,82,83]. Semi-fine, autoradiographed slices are observed by OM and ultra-fine slices by TEM.
Protein synthesis and NIS expression reduce excess iodide [30,31,32] and (bearing in mind that NIS has not been described in closed follicle cultures) the presence of NIS is determined in culture in the presence of different iodide and TSH concentrations, with anti-NIS/GS antibodies (1:500, kindly donated by Thierry Pourcher) and Alexa 488 anti-rabbit secondary antibodies (1:1,000). The nuclei are visualised using DAPI/PBS (1:9,000). Follicles are compressed on commercial laminas as their diameter is greater than that of cells; a 1 mm high chamber was constructed to enable observing by CM without follicular compression.
4.3. Functional studies
Iodide organification (which is essential in thyroid hormone synthesis) becomes lost during the first days in the thyroid culture models described to date. Functional analysis of follicles in culture for determining whether follicular architecture is conserved has advantages over other cultures regarding hormone synthesis.
Follicles’ accumulated and organified 125I-radiactivity is determined by γ well counter. Rat follicles are washed 3 times for 5 min at 50g and at 30g for pig follicles with COON + Na127I (cold) 100 times the concentration of radioactive iodide used in culture and the radioactivity arising from accumulated (A) is determined. 10% trichloroacetic acid + Na127I 100 times the radioactive iodide concentration used in culture is then added and the radioactivity present in protein precipitate corresponding to protein binding iodide (PBI) or iodide organification (O) is determined.
The precipitate is used for determining the amount of DNA by diphenylamine method [88], Tg19S by HPLC and iodine-thyronine (MIT, DIT, T3, rT3 and T4) in Tg by inverse-phase HPLC [89]. The results are expressed in % iodide dose in μg DNA. Follicle structure does not allow the number of cells to be counted and statistical analysis requires having a parameter letting the results be homogenised; based on 1 pg DNA/mL equals 2E5 cells, the number of cells present in cultures can be determined and the results statistically correlated [82].
4.4. Statistical analysis
Data given in the text are expressed as mean standard deviation for N values. Significant differences are established between some times for A and O variables by Student’s t-test.
5. Results
Closed follicular architecture is indispensable in agarose culture since open follicles become dissociated and cells die; it is thus essential to begin with maximum closed follicles possible to avoid this. The following item gives the criteria determined important for beginning culture. Then, the long-term culture results in which closed and isolated follicle morphology and function were compared. Last, the morphological and functional results in the presence of different doses of iodide and TSH.
5.1. The importance of obtaining closed follicles for culturing them
Many isolated cells and follicular fragments were obtained when thyrocytes became detached after 30 contiguous min, instead of 10 minute enzyme dissociation time (Figure 3A); these thyrocytes were eliminated 24 h later when culture medium was changed. If some fragments persisted after 1 or 2 days’ culture they became dissociated and thyrocytes died because they could not adhere to the agarose covered culture support [75] since normal cells require support for growing in culture. Trypan blue allowed an approximation of follicles’ closed or open state (Figure 3B); those conserving colloid showed up due to birefringence in IM and there was a clear boundary between colloid and cells (Figures 3B star and 3D) whilst those which became resealed lost their colloid birefringence and the boundary between cavity and cells was not clear (Figures 3B clear arrow and 3C). Openings in follicles which did not become resealed appeared (Figure 3B triangles beam) as did colloid loss. Cells which did not exclude the stain were mainly endothelium cells which were found on follicle periphery (Figure 3B black arrows). Follicle fragments could be seen because they lost follicular structure and organisation continued from thyrocytes’ epithelial layer (Figure 3B circle).
Figure 3.
A. A follicle fragment from euthyroidic pig thyroid dissociated for 30 min without interruption pre-incubated for 12 h. Note the contour of thyrocytes which became detached from the follicular fragment and cell waste in culture support. B. Trypan blue for recently isolated rat thyroid follicles by strong dissociation (Strong dissociation, Table 1). Follicles which did not open (star) conserved colloid birefrigence and a clear and continuous boundary between colloid and cells, whilst those which became resealed (clear arrow) lost colloid birefrigence and the boundary between cavity and cells was not clear. Openings could be seen in those which did not reseal (triangles beam). Cells which did not exclude stain were mainly endothelium cells found in follicle periphery (solid arrows). Follicle fragments presented non-viable thyrocyte aggregates (circle). C. Pig euthyroid resealed follicles 12 h pre-incubation. Note that the thyrocytes’ apical boundaries could be distinguished due to a lack of colloid. D. Trypan blue of closed follicle with colloid birefrigence of pig hypothyroids pre-incubated for 12 h; colloid was birefrigent and the boundary between colloid and thyrocytes was clear and continuous (IM. Scale bar: A 20 µm, B 50 µm, C 15 µm, D 70 µm).
Follicle morphology and function were analysed after 4 hours’ labelling with radioactive iodide, varying according to the dissociation procedure used with and without pre-incubation. If pipette dissociation after each 10 min enzyme digestion was done in such a way that hydrodynamic forces were produced with turbulence, this was called strong\n\t\t\t\t\tdissociation, but when this was done slowly without turbulence in the liquid and avoiding air-bubble formation it was called mild\n\t\t\t\t\tdissociation.
Iodide which did not bind to molecules was eliminated in fixation liquid until being impregnated in resin; labelling in autoradiographs (following pre-incubation, added for 2 h in the presence of Na125I 5 μCi/mL) was that which bound to proteins and was equivalent to organified iodide. The number of closed follicles could thus be counted and distinguished from open ones by autoradiographs. Open follicles were numerous in strong dissociation; they could be seen because no organified iodide was concentrated within follicle interior (Figure 4A star) even though histological cross-sections seemed to suggest that the cubic epithelial layer continued (Figure 4A circle). There were much fewer open follicles if dissociation was mild (Figure 4B). Follicles which resealed could be distinguished because, even though the labelling was homogeneous, it was less intense in colloid than in those which remained closed (Figure 4B arrow).
Pre-incubation time was another key aspect for obtaining a maximum of closed follicles at the beginning of culture (4 h for rat follicles and 12 h for pig follicles when this was divided into 4 initial hours in which medium was changed by aspirating the medium with follicles and spinning at 30g x 5 min followed by pre-incubation for 8 h). The pre-incubation period was essential since this time allowed cells to recover from the aggression of the enzyme used for dissociation.
Pre-incubation time following dissociation promoted an increase in closed follicles with two dissociations (strong or mild, 33% and 46%, respectively, Table 1). Open follicles which did become resealed or follicular fragments (Figure 4A) became disorganised and were discarded in the supernatant when changing the medium and spinning to wash them.
Morphological modifications included modification of the amount of iodide accumulated and organified by follicles (Table 1); if dissociation was mild, iodide accumulation (A) and organification (O) values and O/A percentage increased following pre-incubation time, whereas if dissociation was strong then these values were lower.
Figure 4.
Autoradiograph of dissociated follicles (following pre-incubation, they were added for 2 h in the presence of 5 μCi/mL Na125I). Iodide ions which did not bind to proteins were eliminated while washing the histotechnic preparation until being impregnated in resin. Closed follicles were labelled in the follicle centre even though they became resealed (arrow). A. Follicles isolated by strong dissociation (aliquot of follicles from Table 1, strong dissociation with pre-incubation). Follicles seeming to be closed by histological cross-section but which did not accumulate organified iodide in colloid were open follicles (circle). B. Most follicles isolated by mild dissociation (aliquot of follicles from Table 1, mild dissociation with pre-incubation) were closed because of intense labelling within follicular cavities (OM. Toluidine bleu. Scale bar: 25 µm).
Closed follicles counted on autoradiographies corresponded to O/A proportion percentage; thus mild dissociation gave 83% closed follicles and 85% O/A proportion whilst strong dissociation gave 45% closed and 58% O/A proportion (Table 1).
Follicles isolated by mild dissociation, following 2 h with radioactive iodide, accumulated iodide 4.8 times and 7.8 times more with 0.1 and 1 mU/mL TSH, respectively, regarding without TSH. TSH did not modify morphology regarding follicular architecture or O/A proportion (Table 2).
This follicle isolation method can be applied to other thyroid tissues from other species, such as rabbits or humans. The percentage of open follicles was greater in pig or human thyroid follicles and more tissue was obtained per experiment; 12 h was thus allowed for pre-incubation (Figures 4 C and 4D).
Digestion conditions
% follicles closes
A
O
% O/A
Without pre-incubation
Mild dissociation
57 ± 1,4
1.14 ± 0,01
0.89 ± 0,01
78
Strong dissociation
45 ± 5,7
1.84 ± 0,37
1.08 ± 0,14
58
With pre-incubation 4 hours
Mild dissociation
83 ± 1,4
1.93 ± 0,24
1.64 ± 0,17
85
Strong dissociation
60 ± 7,1
1.32 ± 0,17
0.91 ± 0,01
68
Table 1.
Influence of dissociation conditions and pre-incubation time on the percentage of rat thyroid isolated and closed follicles and their function. It can be seen that the percentage of organified iodide on accumulated iodide (O/A) was equivalent to the percentage of closed follicles concentrating radioactive ion determined by follicle count using accumulated grains in follicular colloid in autoradiography of semi-fine cross-sections (Figure 4). Average values for two culture dishes ± SD. Following pre-incubation, 125I- accumulation (A) and incorporation in proteins (O) were determined following 2 h in the presence of Na125I 5 μCi/mL and expressed in µg/dose/µg DNA.
TSH
A
O
% O/A
None
0.73 ± 0,18
0.58 ± 0,11
79
0,1mU/mL
3.48 ± 0,17
2.78 ± 0,04
80
1mU/mL
5.71 ± 0,72
4.98 ± 0,67
87
Table 2.
Influence of TSH on iodide accumulation and organification in isolated rat follicles expressed in µg/dose/µg DNA. Follicles were cultured for 2 h in the presence of Na125I 5 μCi/mL immediately following pre-incubation. TSH was stimulated by Na125I but O/A proportion was around 80% with or without TSH. The value without TSH represented the percentage of closed follicles obtained following correct mechanical dissociation and pre-incubation of follicles. Average values for culture dishes ± SD (these values were representative of 3 experiments).
A good approximation of the percentage of closed follicles following pre-incubation must thus be born in mind (this corresponded to day 0 in our cultures). It can thus be generalised that O/A proportion values should be greater than 80% before beginning culture (day 0, Table 2) and that there should be a potentially high number of closed follicles, even though follicle diameter may vary in each species or come from different thyroid functional states [85,86,90]. Open follicles’ ability to reveal themselves during the course of pre-incubation probably depended on the degree of initial opening. Even though groupings of rat thyroid cells became organised in the presence of TSH and on agarose, they became reorganised into 6- to 10-cell follicles which could be cultured for 3 days [69,73]. In our results, rat or pig follicles which became resealed did not require TSH for conserving their follicular architecture.
5.2. Long-term closed follicle culture
5.2.1. Functional study
On day zero (4 h pre-incubation in the presence of radioactive iodide) follicles accumulated 3.5 ± 2.1% of the dose in the medium without TSH (9 experiments in rats). Organified iodide percentage varied according to closed follicle percentage at the start of each experiment. O/Aprioportion was 57% in experiment 1 and 91% in experiment 2 (Table 3); more than two thirds of organified iodide bound to stable Tg19S (Table 4), the rest of the molecules having lower molecular weight. Capture increased in the presence of TSH regarding without TSH 250 ± 210% (9 experiments in rats), but O/A percentage remained constant. The effect of TSH was evident on A and O, and higher than values without TSH (Tables 2, 3 and 4). Iodide accumulation on Tg19S slightly increased with TSH, but not significantly so (Table 4).
Exp. 1
Exp. 2
TSH
0.0
0.1
1.0
0.0
0.1
1.0
Culture, day
0
57
46
69
91
93
94
1
69
75
71
78
94
93
3
64
85
85
84
86
87
6
0
58
83
60
51
84
9
2
51
71
59
78
91
12
0
54
82
50
73
90
Table 3.
Evolution of 125I- organification/accumulation in rat follicle culture. Comparing two experiments where rat closed follicle percentage varied during the course of two experiments (Exp. 1 and Exp. 2). 80% of follicles were closed at the start of culture counted in autoradiographs of culture aliquots in experiment 2 (cf 65% in experiment 1). Na125I 5 μCi/mL was added 4 h before collecting follicles during each day of the experiment, expressed in %/dose/μg DNA. Each value was the average of two samples or culture dishes. Experiment 2 was representative of 7 independent experiments. TSH: mU/mL.
Following one day’s culture without TSH, accumulation was low at the start of culture; it did not become modified, but if it was high it became reduced, whilst incorpoproportion of iodide in Tg became reduced (Table 4). T3 and T4 proportion in regarding Tg was not modified. Iodide accumulation increased in the presence of TSH, O/A percentage remained high and in starting values (Table 3). Iodide incorpoproportion in Tg19S was better (Table 4) and Tg19S content in follicles did not vary.
Accumulation fell abruptly on the third day without TSH when starting with a low percentage of closed follicles, just like O/A percentage, whilst it became reduced with a high percentage of closed follicles, but O/A was maintained. O/A values were high in the presence of TSH and remained higher than 80% (Table 3).
Accumulation and organification values and their O/A percentage differed on the sixth day regarding closed follicle percentage; A and O could not be determined in experiment 1 which began with 57% (Table 3. Exp. 1) whilst experiment 2 began with 94% and became reduced to 60% (Table 3. Exp. 2). There was a reduction in all O/A percentages in rat or pig cultures by the sixth day which also differed with the percentage of closed follicles (Table 5), animal species and functional state at the start of culture. The percentage of iodide on Tg19S also became reduced (Table 4).
A
125I- in
Tg19S*
TSH
0.0
0.1
1.0
0.0
0.1
1.0
Culture. Day
Without NaI
0
5.8
5.5
7.2
59
63
67
1
1.4
2.7
3.1
43
66
65
3
1.3
4.5
8.7
46
50
52
6
0.7
1.2
4.0
25
13
42
9
0.2
0.6
4.0
16
17
55
12
0.2
0.7
8.0
15
25
52
With NaI
0
5.8
7.8
9.3
60
61
66
1
1.1
1.8
3.1
31
58
64
3
0.7
4.2
7.6
49
49
52
6
0.1
1.4
3.4
10
22
39
9
0.2
1.6
2.6
14
32
38
12
0.1
1.9
2.9
11
33
58
Table 4.
Evolution of iodide accumulation (A) and its incorporation in stable Tg19S in rat follicle culture. Effect of adding a small dose of cold iodide (10E-10 M Na127I). A was expressed in %/dose/μg DNA. Radioactivity determined in stable Tg19S was expressed in % 125I- accumulation/dose/μg DNA. The results without NaI were those expressed in experiment 2, Table 3. Na125I 5 μCi/mL 4 h was added before collecting follicles during each day of the experiment. Each value was the average of two samples or culture dishes.
O/A retained the same values on day 9 and 12 as those for day 6 in both experiments without TSH, whilst with TSH this increased in experiment 1 regressed to day 1 values and then maintained similar values to those of the two first days. The percentage of iodide on Tg19S with and without TSH was conserved (Table 4).
The presence of a small dose of iodide (10E-10 M NaI) did not modify iodide accumulation values at the start of culture (Table 4), but responded better to TSH. Tg19S had more T3 and T4 and TSH increased the percentage of T3. O/A percentage increased with 0.1mU/mL TSH on days 6, 9 and 12 whilst it fell with 1 mU/mL (not significantly). Iodide had no effect on stable Tg19S content in follicles; more than two thirds of accumulated iodide was incorporated in Tg19S (Table 4) even in the absence of TSH where iodide accumulation was very low on day 12.
Preserving the function of pig follicle thyrocytes during culture also depended on the percentage of closed follicles since the beginning. However, this differed from those for rats in the absence of TSH, because this began with 43% O/A equivalent to the percentage of closed follicles presenting iodide organification on days 6 and 9 (Table 5, euthyroidic follicles) contrary to rat follicles which was zero (Table 3. Exp. 1). As 7 times more material was obtained from pig thyroids than rat follicles, a greater number of recovered follicles were conserved in changes of medium by centrifuging, or different species having variations in response to the same medium conditions.
Euthyroid
Hipothyroid
Culture, day
TSH
% O/A
N
% O/A
N
0
0
43.4. ±.5.9
4
82.4. ±.7.2
4
1mU/mL
61.5. ±.4.6
4
80.5. ±.3.7
4
1
0
56.94. ±.4.7
3
77.0. ±.3.8
4
1mU/mL
71.4. ±.7.1
2
76.44. ±.3.9
4
3
0
37.4. ±.6.6
2
82.24. ±.4.1
3
1mU/mL
43.2. ±.5.9
2
76.44. ±.5.3
2
6
0
9.16. ±.2.2
2
23.3. ±.9.4
2
1mU/mL
15.0. ±.3.7
2
52.32. ±.4.2
2
9
0
52.66. ±.2.2
2
16.62. ±.1.5
3
1mU/mL
64.8. ±.4.3
2
53.6. ±.5.0
3
Table 5.
Evolution of 125I- organification/accumulation (O/A proportion) percentage for pig follicles. Hypothyroid follicles had high O/A percentage at the start regarding euthyroids (representative results from 4 experiments). This experiment began with less than 50% O/A in euthyroids for comparing with Table 3, experiment 1; however, when 3 experiments began with euthyroid follicles having 80% or more O/A they behaved like those for rat follicles in experiment 2, Table 3. 5 μCi/mL Na125I were added 4 h before collecting follicles on each day of the experiment expressed in %/dose/μg DNA. Average values for the number (N) of culture dishes ± SD.
The difference between euthyroidic rat or pig follicles regarding hypothyroid ones was that hypothyroids responded to TSH on the sixth and ninth culture days, perhaps due to adapting to normal culture conditions, like hypothyroid glands’ response in vivo when the effect induced by hypothyroidism stimulated with TSH was eliminated [24].
We compared isolated follicles’ iodide accumulation and organification with their respective pig mini organ cultures lasting up to 9 days. Euthyroidic tissue became disorganised after the sixth day and functionality could not be determined after this day. Some follicles were conserved in the hypothyroids on the outside of the cultured fragment and presented this function on days 6 and 9 (Table 6). Mini organ culture functional values were higher than those of follicles isolated on the first day, but these were not significant; they were higher from the first day onwards in isolated follicles, maintaining higher values up to day 9 (euthyroidic and hypothyroid) (Table 6).
TSH
A
O
% A/O
N
TSH
A
O
% A/O
N
Euthyroid, follicles
Euthyroid, mini organ culture
0
0.049
0.025
51.6 ± 0.052
4
0
CND
CND
3
1mU/mL
0.074
0.048
65.3 ± 0.043
4
1mU/mL
CND
CND
3
Hypothyroid, follicles
Hypothyroid, mini organ culture
0
0.122
0.0633
36.75 ± 0.67
4
0
0,115
0.025
22.7 ± 1.5
3
1mU/mL
0.122
0.0655
53.6± 0.050
4
1mU/mL
0.723
0.278
38.5 ± 0.067
2
Table 6.
Determining iodide accumulation (A) and organification (O) for isolated follicles and mini organ cultures from the same pig thyroids after 9 days’ culture. 5 μCi/mL Na125I was added 4 h before collecting follicles and mini organ cultures on each day of the experiment expressed in %/dose/μg DNA. Average μg of follicle DNA was 4.5 and 14.7 for mini organ cultures. Values represent the average number (N) of culture dishes ± SD. CND: could not be determined.
It has been described that pig thyroid mini organ cultures enables studying “thyroid tissue structural and functional integrity in vitro [54]”; however, we have considered that studying thyroids in vitro is better done with isolated follicles than using mini organ cultures. Since closed follicles maintain their architecture throughout culture time, thyrocytes are viable and their basement membrane is in direct contact with the medium and not with capillaries whose endothelial cells die rapidly in culture during the first 24 h (Figure CB).
DNA content per culture dish did not show a significant change during 12 days’ rat follicle culture (1.55 ± 0.52 μg/dish, N = 16) or 9 days’ pig follicle culture (5.35 ± 0.36 μg/dish, N = 25).
Even though closed and isolated follicles in culture had differences regarding stable Tg19S, the amount of T3 and T4 and iodide accumulation between different treatments with and without TSH and with or without 10E-10 M NaI, the follicles did have more iodide organification, iodised Tg19S and T3 and T4 at 12 days’ culture, even without TSH [83,86], than in all other culture models published to date except of the group [83,84,85,86]. Such variations were homologous to glands in vivo in the same study conditions.
Our culture system has different characteristics distinguishing it from other models described up to now. Monolayers lose their function on the first day [48], become reorganised in pseudofollicles on the third day and only 2% to 4 % become incorporated or organified in iodine accumulated in poorly iodised Tg (Tg16S) [91], even though higher than 90% O/A with TSH has been reported for a matrigel-covered monolayer culture forming a double cell layer having cavities [61]. Different models mentioning culturing “follicles” [92,93,94,95] have not shown these functions in their results; others culturing pig “follicles” for 2 days, based on Björkman and Ekholm [16] as we, require 5% FCS, 1 mU/mL TSH and non-physiological molecules such as forskolin or 8-(4-chlorophenylthio)-cAMP for maintaining thyrocyte functions [77]. Using closed follicles enables functional parameters to be conserved and measured: iodide accumulation, iodide organification and, particularly, Tg19S equivalent to that in vivo and T3 and T4 formation throughout culture with or without TSH. We have also shown that maintaining closed follicular architecture is an indispensable condition for conserving such thyroid functions in culture in vitro. If follicular architecture is to be conserved, it is not enough to maintain functions at the same values as those at the start of culture as TSH is required and culture becomes improved by adding iodide, as thyroid function in vivo is governed by TSH and iodide.
6.2.2. Morphological study
Pig hypothyroid follicles cultured 1 and 3 days without TSH have a very thin epithelial layer (Figure 5A) like original tissue’s follicle epithelium. The epithelium became cubic after day 6 and was preserved up to day 9 (Figure 5C). It was seen that follicles conserving colloid had a birefrigent aspect when observed by IM, thereby showing that this was conserved during culture. Epithelium thickness increased in the presence of TSH, becoming cubic on the first day of culture (Figure 5B) and being maintained so until day 9.
Figure 5.
Morphological aspect of follicles in culture. A. Hypothyroid follicle 1 day’s culture without TSH; birefringent colloid and thin follicle epithelium can be seen. B. Hypothyroid follicle 1 day’s culture with 1mU/mL TSH; cubic epithelium and thyrocytes’ apical poles typical of a resealed follicle can be seen. C. Hypothyroid follicles in 9 days’ culture without TSH; the epithelium is cubic and colloid birefringence can be seen in all follicles. D. Euthyroid follicle 1 day culture without TSH. A resealed follicle (circle) having irregular contour between thyrocytes and cavity can be seen in the centre. Closed follicles preserve colloid birefringence and regular boundary between thyrocytes and colloid since the start of culture. E Pig euthyroid follicles in the presence of 1 mU/mL TSH; follicular cavities are difficult to distinguish. F. Autoradiography of rat follicles cultured 12 days in the presence of TSH (1 mU/mL) corresponding to experiment 2, Table 3. Follicle cavities are evident due to the organified iodide found only in very narrow follicular cavities (Scale bar: A and D 50 µm, B 25 µm, C 130 µm, E 100 µm, IM. F 150 µm, OM. Toluidina bleu).
Rat or pig euthyroid follicles without TSH kept the same follicular architecture throughout the whole culture time (Figure 5D). Colloidal cavities became reduced from the third day in the presence of TSH and were difficult to distinguish on day 9 and 12 by IM (Figure 5E); however, they could be seen by autoradiography where only iodide bound to molecules could be identified and they were only located in follicles’ very narrow colloidal cavities (Figure 5F). Colloidal cavities’ boundaries could also be seen by labelling thyrocytes’ apical membrane protein SLC5A8 (short-chain fatty acids transporter) [84].
Rat thyrocyte and pig euthyroid follicle ultra-structure in one day culture without TSH (Figure 6A) and with TSH (Figure 6B) was comparable to that for cells in vivo (Figure 1C). They conserved their polarity and organelles, but exocytic vesicles were difficult to distinguish from those from endocytosis. RER was more abundant in the presence of TSH and microvellosities were more evident than without TSH.
Thyrocytes had vacuolated and reduced RER and GC following 3 days’ culture without TSH; the GC could be seen in supra-nuclear position (Figure 6C G) as could numerous autophagic vacuoles (Figure 6C arrow) and secondary lysosomes. This became modified in the presence of TSH, presenting abundant RER and GC (Figure 6D G) and containing more autophagic vacuoles and secondary lysosomes in thyrocytes’ apical region (Figure 6D arrows) than without TSH. RER and GC became more reduced by the sixth day without TSH and thyrocytes became thin. Whilst autophagic vacuoles and secondary lysosomes became reduced with TSH, RER and GC also did so by day 3.
Figure 6.
Ultra-structure of thyrocytes from follicles cultured without TSH A 1 day, C 3 days, D 12 days and with 1 mU/mL TSH B 1 day, D 3 days and F 12 days. Binding complexes were preserved in thyrocytes’ lateral membrane apical region during all the times with and without TSH. A. Thyrocytes conserved their polarisation; microvellosities in contact with electron-dense colloid. Binding complexes were located in lateral membrane’s apical region between cells. The rugose endoplasmic reticulum (RER) was found to be slightly vesiculated. B. Thyrocyte ultra-structure was comparable to without TSH, even though RER was more abundant. C. and D. Ultra-structure was conserved in thyrocytes; the supranuclear Golgi complex (G) and more abundant RER in thyrocytes in the presence of TSH can be seen. Autophagic vacuoles (C. arrow) and secondary lysosomes (D, arrows) with or without TSH can be seen. E. Thyrocyte polarity was conserved. Colloid was electron-dense and separated from follicle exterior. Thyrocytes had exiguous RER and G. F. Organelles were well conserved. RER was well developed and occupied thyrocytes’ apical region (TEM. A and C 7,300 X, B 7,500 X, D 7,438 X, E 9,810 X, F 8,260 X).
Thyrocytes were thin by day 9 and 12 without TSH and had exiguous RER and GC and reduced microvellosities. The nuclei contained very little heterochromatin (Figure 6E). Follicular centres were very narrow in the presence of TSH, thyrocytes had abundant GC and RER reaching the cells’ apical regions (Figure 6F) and the other organelles had the same distribution as on the first days of culture.
Adding 10E-10 M NaI did not modify thyrocytes’ follicular architecture or ultra-structure; however, RER and GC were preserved up to day 12 without TSH.
Pig thyrocyte and hypothyroid follicle ultra-structure had exiguous RER and GC when culture began (Figure 7A), like original gland hypothyroids in vivo, and culture (even without TSH) developed these organelles from the third day of culture [86]. These hypothyroid follicles’ thyrocytes became cubic in the presence of TSH (Figure 5B) from the first day of culture and it was seen that the RER and GC developed and became more evident on the sixth and ninth day of culture (Figure 7B), similar to rat or pig euthyroid follicle response with TSH, but follicular cavity did not become reduced.
Thyrocytes had normal mitochondria in rat follicle and pig euthyroidic and hypothyroid cultures for all culture times.
Figure 7.
Pig hypothyroid follicle culture. A. RER and GC were exiguous and cells were thin 1 day in the absence of TSH. B. 9 days in the presence of 1 mU/mL TSH. Cross-section of thyrocyte follicle basal pole. Abundant RER can be seen around the nucleus (TEM. A 7,800 X, B 9,000 X).
Similar culture models to these have been described. Pig follicle culture has highlighted the importance of thyrocyte polarity, but OM morphological study was limited on day 1 [74,94]. Another, dealing with “normal” human follicle culture of thyroidectomy for goitre requiring TSH [93] had a TEM image showing thyrocytes having the ultra-structure for cells in the process of cell death with lysed mitochondria, without RER or GC. Using this human follicle culture model enabled analyzing the effect of TSH [95] or cytokines [95,96] without presenting culture morphology.
Our results showed that if culture was begun with closed follicles then extracellular matrix support elements were not required [56,57,79,80] nor was TSH for maintaining follicular cavity, as has been described in most pseudofollicle or follicle cultures reconstructed from monolayers, or similar structures called “follicles” [57,50,60,61,62,79,96], thereby demonstrating that if closed follicles are used from the start of culture they conserve their morphology, having the correct polarity as that of their thyrocytes in culture and have a binding complex (tight junction, belt desmosome and spot desmosome) in lateral membranes’ apical part as well as in vivo [9]. Such closed follicles in culture responded to TSH, like other in vitro models [42] or like gland follicles in normal in vivo to TSH stimulus [3,25], epithelium thickness, RER and GC becoming increased, and follicular cavities becoming notably reduced [11,83,90]. Follicle response to the absence of TSH both in vivo [24] and in vitro [42] was also comparable as these organelles became reduced. This effect became reverted in vivo when TSH was added, reactivating thyroid functions [11,33,97].
Pig closed and isolated hypothyroid follicles behaved like the gland in vivo when the hypothyroid effect was deleted [24], RER and GC increased, follicular epithelium became thin to cubic and culture time became faster in the presence of TSH, but follicular cavity did not become reduced during the 9 days of culture.
Our results thus showed that long-term thyroid follicle function and morphology can be maintained in vitro, being equivalent to the gland in vivo.
The next section describes the effect of increasing doses of iodide on closed follicles in culture.
6.3. Closed follicle cultures reproduce the Wolff-Chaikoff effect described in vivo
6.3.1. Functional study
Follicles were cultured for 1 day before starting to analyse the effect of different iodide concentrations.
Follicles accumulated iodide linearly for 6 h in the presence of 10E-10 and 10E-7 M NaI; accumulation was at its maximum after the first 30 min with 10E-5 M and 10E-3 M NaI (Table 7); they became accumulated 100 times more than with 10E-10 M NaI in the presence of 10E-7 M NaI. Whilst high O/A percentages in follicles were maintained in the presence of 10E-10 and 10E-7 M NaI, organification began after 2 h with 10E-5 M, accounting for only 5% of accumulated iodide, these values being maintained for 6 h, whilst O/A proportion was zero for all times with 10E-3 M NaI (Table 7), even though accumulation could have been 100 times greater to that presented by follicles in the presence of 10E-7 M NaI [98].
% O/A
M, NaI
10E-10
10E-7
10E-5
10E-3
Culture, hours
0.5
82
64
0
0
1
84
42
0
0
2
88
90
5
0
3
92
88
3
0
4
95
87
6
0
5
88
89
6
0
6
84
93
4
0
Table 7.
Response of organification percentage regarding iodide accumulation (O/A proportion) by rat follicles cultured in the presence of increasing doses of iodide (NaI, M) and Na125I 5 μCi/mL. The follicles were cultured on agarose with 0.5% FCS for 1 day, medium was changed and culture began with NaI and TSH experimental points. Each value was the average of two samples or culture dishes expressed in %/dose/μg DNA.
Iodide accumulation and organification in rat follicles could be assimilated to biochemical reactions after 30 min with and without TSH and thus define a constant (Km) and maximum speed (Vmax). Accumulation apparent Km without TSH was 5x10E-6 M and 10E-7 M de NaI with TSH; iodide organification apparent Km was 5x10E-7 M and was not modified by the presence of TSH (Figure 8). Iodide accumulation by isolated follicles corresponded well with saturable iodide transport characteristics [15,99] and stimulated TSH thereby reducing Km but not Vmax (Figure 8). It should be stated that organification had to be inhibited and the time between the end of the culture and the moment when accumulation was determined had to be reduced for measuring exact iodide transport parameters. The washing times meant that part of 125I- concentrated in follicles would be released before radioactivity was determined and this would induce under-stimulation of accumulation and perhaps overstimulation of O/A percentage.
Figure 8.
Initial 125I-iodide accumulation (A) and organification (B) for follicles in culture measured following half an hour in the presence of increasing doses of 127-I Na and 5 μCi/mL 125-I Na expressed in -log M with and without TSH. The follicles were cultured on agarose with 0.5% FCS for 1 day, the medium was changed and culture began with NaI and TSH experimental points. The results were the average of two samples and were expressed as percentage of maximum value.
The presence of TSH for 30 min suppressed the inhibiting effect on iodide organification at 10E-5 M, but did not suppress such iodide effect on organification in the presence of 10E-3 M TSH (Table 8).
M, NaI
10E-10
10E-7
Culture
TSH
A
O
% O/A
A
O
% O/A
0.5
-
2,58
1,29
50 ± 5*
360,9
156,34
43 ± 6*
+
8,06
4,52
56 ± 2*
676,69
406,02
60 ± 14*
3
-
10,88
8,16
75 ± 10*
902,26
525,24
58 ± 7
+
14,84
11,81
79 ± 1*
1308,27
1079,24
82 ± 1
8
-
35,97
29,84
83 ± 11
2255,64
1602,81
71 ± 4*
+
32,26
28,06
87 ± 23
1736,84
1371,98
79 ± 10*
12
-
ND
1849,62
1191,13
64 ± 9
+
ND
1353,38
853,23
63 ± 13
24
-
ND
1714,29
1421,05
83 ± 10
+
ND
1759,4
1254,17
71 ± 8
48
-
ND
1917,29
1361,98
71 ± 10*
+
ND
3969,92
3194,23
80 ± 6*
M, NaI
10E-5
10E-3
Culture
TSH
A
O
% O/A
A
O
% O/A
0.5
-
14749,09
0
0 *
233469,39
0
0
+
12480,01
2,63
21 ± 16*
244244,9
0
0
3
-
15272,73
1553,64
10 ± 6
520816,33
0
0
+
17541,82
2987,27
17 ± 2
466938,78
0
0
8
-
16495,45
2397,15
14 ± 5
574693,88
0
0
+
20770,91
4450,91
21 ± 2
790204,08
0
0
12
-
ND
* 718367,35
0
0
+
ND
* 1957551,02
0
0
24
-
ND
* 3071020,41
0
0
+
ND
* 682448,98
0
0
48
-
ND
664489,8
0
0
+
ND
484897,96
0
0
Table 8.
The effect of I- dose on the accumulation (A) and organificación (O) of the rat follicles. Follicles cultured on agarose with 0.5% FCS for 1 day; the medium was changed and culture began with different experimental points. The follicles were keep in culture along the time (culture in hours) with 5 µCi/mL Na125I, with (+) or without 0.1 mU/mL TSH (-) and doses of Na127I (NaI). Each value was the average of two samples expressed in µg/dose/µg DNA. This result was representative of 5 independent experiments. O/A: percentage of the proportion O/A ± SD. *p< 0.05. ND: not determinated.
Follicle culture in the presence of the same iodide dose with 0.1 mU/mL and without TSH led up to 8 h with 10E-10 M and 10E-5 M NaI and up to 48 h with 10E-7 M and 10E-3 M NaI. An iodide accumulation function directly proportional to NaI concentration was reproduced as in table 7.
Accumulation in follicles became saturated after 8 h in the presence of 10E-7 M NaI with and without TSH, becoming slightly reduced at 12 and 24 h and increasing at 48 h; iodide accumulation with TSH was lower at 8, 12 and 24 h than without TSH (significant only at 12 h, p< 0.05). Percentage of the proportion O/A was maintained high values with and without TSH, difference with and without TSH at 30 min and 48 h being significant (Table 8).
Iodide accumulation satuproportion was reproduced in follicles in the presence of 10E-5 M NaI, whether with or without TSH. TSH significantly stimulated organification inhibited by 10E-5 M NaI (p< 0.05) at 30 min, even though O/A percentage was slightly higher than at other times (but was not significant) (Table 8).
Iodide accumulation in follicles in the presence of 10E-3 M NaI with and without TSH reached a plateau at 3 h (Table 8) and was greater in follicles without TSH than with TSH at 12, 24 and 48 h; however, this difference was only significant at 12 and 48 h (p< 0.05). Iodide accumulation became reduced with and without TSH at 48 h to 3 h values, even though without TSH this was greater but not significantly so. Iodide organification was zero with such strong dose of iodide with and without TSH at all times examined (Table 8).
Pig euthyroidic follicles had the same response as rat follicles when culturing for 48 h in the presence of increasing doses of iodide with or without TSH. Organification was zero at all times in the presence of 10-E3 M NaI with and without TSH (Table 9).
M, NaI
10E-10
10E-7
10E-3
Culture
TSH
% O/A
N
% O/A
N
% O/A
N
0.5
-
30.4 ± 0.56
2
30.4 ± 0.63
2
0
3
+
34.7 ± 9.21
2
72.7 ± 2.84
2
0
3
3
-
67.0 ± 1.54
2
63.2 ± 9.14
2
0
3
+
79.5 ± 6.79
2
73.6
1
0
3
12
-
71.8 ± 2.35
2
52.4 ± 2.63
1
0
3
+
78.9 ± 2.63
2
77.6 ± 3.15
2
0
3
24
-
59.7 ± 2.09
2
87.6 ± 1.97
2
0
4
+
68.8 ± 7.13
2
85.7 ± 0.99
2
0
3
Table 9.
The effect of I- dose on the proportion accumulation/organificación pourcentage (% A/O) in euthyroidic pig follicles cultured. Follicles cultured on agarose with 0.5% FCS for 1 day; the medium was changed and culture began with different experimental points. The follicles were keep in culture along the time (culture in hours) with 5 µCi/mL Na125I, with (+) or without 1 mU/mL TSH (-) and doses of Na127I (NaI). Each value was the average of accumulated or organified (O/A) iodide expressed in %/dose/μg DNA per number (N) of culture dishes ± SD.
Pig hypothyroid follicles had a different response to the presence of increasing doses of iodide than rat and pig euthyroidic follicles (Table 10). They accumulated 40 to 100 times more in the three doses than euthyroidic follicles, presenting organification in the presence of 10E-3 M NaI with or without TSH. Iodide accumulation in these follicles in the presence of perchlorate (30 μM) was only inhibited at 12 h in the presence of 10E-3 M NaI and at 3 h in the presence of 10E-7 M NaI (euthyroidic follicles in the presence of 30 μM perchlorate inhibited iodide capture at all concentrations) and O/A percentages in follicles in the presence of 10E-7 M NaI were greater in the absence of TSH than in their presence (Table 10), such value becoming reduced with culture time.
NaI
10E-10 M
10E-7 M
10E-3 M
Culture
TSH
% O/A
N
% O/A
N
% O/A
N
0.5
-
77.9 ± 0.61
4
74.9 ± 0.63
4
6.3 ± 0.63
3
+
57.3 ± 0.78
4
71.6 ± 2.84
4
15.1 ± 0.63
3
3
-
75.8 ± 0.49
4
68.7 ± 9.14
4
5.4 ± 0.63
3
+
60.6 ± 0.68
4
57.2 ± 9.14
4
3.0 ± 0.63
3
12
-
ND
87.3 ± 2.63
4
10.4 ± 0.63
3
+
ND
72.8 ± 3.15
4
3.8 ± 0.63
3
24
-
ND
44.3 ± 1.97
4
11.6 ± 0.63
4
+
ND
28.7 ± 0.99
4
9.7 ± 0.63
3
Table 10.
The effect of I- dose on the proportion accumulation/organificación pourcentage (% A/O) in pig hypothyroid follicles cultured. Follicles cultured on agarose with 0.5% FCS for 1 day; the medium was changed and culture began with different experimental points. The follicles were keep in culture along the time (culture in hours) with 5 µCi/mL Na125I, with (+) or without 1 mU/mL TSH (-) and doses of Na127I (NaI). Each value was the average of iodide accumulated or organified (O/A) iodide expressed in %/dose/μg DNA per number (N) of culture dishes ± SD. ND: not determinated.
Iodide accumulation values in the presence of 10E-3 M NaI in hypothyroid follicles were 2 times greater in follicles in the presence of TSH than without TSH, had organification in similar proportions to euthyroidic follicles in the presence of 10E-5 M NaI. Percentage of the proporction O/A was higher at 30 min without TSH than with TSH; it was lower at the other times. Without TSH increased with culture time whilst values became reduced regarding time with TSH (Table 10). Organification in this case was not nil, but the organified iodine did not exceed 11% for accumulated iodide.
Mouse follicles cultured 3 days in the presence of 10E-7 M NaI was more intense in the presence of reactive species at the boundary between apical membrane microvellosities and colloid and the oxide reduction system became completely closed down with an excess of iodide (10E-4 M NaI) [100]. This proved that an excess of iodide inhibited the enzymes responsible for organification in closed follicles as we have thought should be in the gland in vivo.
Euthyroidic follicles accumulated iodide regarding medium constant proportion concentration. Accumulated iodide was organified in the presence of 10E-10 M and 10E-7 M NaI; organification was extremely reduced in the presence of 10E-5 M and zero with 10E-3 M NaI as described in vivo [1]. Accumulation was greater in follicles without TSH than with TSH in the presence of 10E-3 M NaI at 12, 24 and 48 h; this reduction was similar to that of the gland in vivo [101,102].
Hypothyroid follicles in the presence of 10-3 M NaI accumulated more iodide without TSH than with TSH at the beginning (30 min), even though proportion of O/A percentage was greater at 30 min than at other times; this effect (different to euthyroidic ones) was similar to that described in vivo in hypothyroid glands [28]. They also responded by reproducing the effect observed in animals suffering experimental goitre as, when excess iodide was added to food, they did not respond to TSH [24]. 50% of human endemic goitre has responded to an iodine-rich diet and some of these to TSH [25] as hypothyroid follicles in the presence of TSH and 10E-3 M NaI have responded by becoming less organified than in the absence of TSH. It should be stated that there is no consensus regarding what should be the correct dose for defining low, medium or high iodide concentration in human alimentation [25].
Closed and isolated follicles thus responded to 10E-5 M and 10E-3 M NaI inhibiting iodide organification in a similar way to that described for the gland in vivo in 1948 [1] (i.e. reproducing the Wolff-Chaikoff effect).
6.3.2. Morphological study
Hypothyroid follicles conserved follicular architecture during 48 h of culture in the presence of different doses of iodide with and without TSH (Figures 9A, 9B and 9C). Rat follicles and pig euthyroidic follicles at all iodide doses in the presence or absence of TSH conserved follicular architecture for 48 h (Figure 9D).
Figure 9.
Appearance of pig follicles cultured on agarose with 0.5% FCS for 1 day; the medium was changed and culture lasted 48 h. A. Hypothyroid s follicles in the presence of 10E-10M NaI. B. Hypothyroid follicles in the presence of 10E-7M NaI. C. Hypothyroid follicles in the presence of 10E-3M NaI. D. Euthyroidic follicles in the presence of 10E-3M NaI. The follicular architecture of the gland in vivo was conserved in all cultures; hypothyroids (A, B and C) had thin epithelium and euthyroidic (D) ones cubic epithelium (IM. Scale bar: A, B, C 800 µm, D 200 µm).
The Trypan blue exclusion exam of thyrocytes from follicles cultured with 10E-3 M NaI did not have an alteration to their membranes and excluded the stain, whether being pig euthyroidic follicles cultured 48 h (Figure 10A) or rat ones cultured for 6 days (Figure 10B). The cells of cell aggregates which did not have follicular structure became stained (Figure 1C), the same as isolated cells (Figure 10B) or those found in follicles.
The ultra-structure of thyrocytes in all treatments and times preserved cell polarisation and organelle distribution (Figure 11), like the gland in vivo (Figure 1C); endocytic vesicles can be seen in thyrocytes’ apical region (Figure 11) like follicles after 3 days of culture (Figures 6 C and 6D).
Figure 10.
Trypan blue exclusion exam of culture aliquots for follicles cultured on agarose with 0.5% FCS 1 day; the medium was changed and culturing involved 10E-3 M NaI. A. 2 day rat follicle culture. B. 6 day pig euthyroid follicle culture. The thyrocytes from the follicles excluded the stain whilst isolated cells did not exclude it, whether separated from the follicle (A) or on the follicle (B) (IM, Scale bar A 60 µm, B 15 µm).
Figure 11.
The ultra-structure of rat follicle rat thyrocytes cultured on agarose with 0.5% FCS for 1 day; the medium was changed and 48-h culture involved different experimental points: A. 10E-7 M NaI. B. 10E-3 M NaI + 0.1 mU/mL TSH. Thyrocytes kept their polarisation; microvellosities were in contact with electron-dense colloid. The binding complexes were located in the lateral membrane’s apical region between cells. The RER was slightly vesiculated. Thyrocytes did not have cytological differences regarding iodide dose; follicular centres only became narrowed in the presence of TSH (B) (TEM. A 8,720 X, B 10,720 X)
Follicles in the presence of TSH narrowed their follicle centres at all iodide doses used (Figures 12B and 12D), but did not undergo any ultra-structural modification in strong iodide concentrations: 10E-5M or 10E-3 M NaI. Organelle distribution was comparable to normal gland in vivo (Figure 1C) or follicles in long-term culture (Figures 6C and 6D).
Lysosome fusion occurred in follicles in the presence of 10E-10 M NaI and 0.1 mU/mL TSH for 8 h (Figure 12B) while this did not happen in thyrocytes’ apical region (Figure 12B insert) in the absence of TSH (Figures 12A and 12C). This was also observed en thyrocytes from follicles cultured for 8 h with 10E-3 M NaI and TSH, but colloid droplets were only located in the apical region in this dose (Figure 12D). Thyrocytes also had endocytic vesicles in this dose without TSH but also located in cells’ apical region (Figure 12C). Thyrocytes ultra-structure in the presence of strong concentrations of iodide did not have morphological modifications, or distribution of organelles regarding normal or non-stimulated cells in vivo (Figure 1C) or in vitro (Figures 6C and 6D).
Figure 12.
The ultra-structure of rat follicle thyrocytes cultured on agarose with 0.5% FCS for 1 day; the medium was changed and 8 h of culture involved different experimental points: A. 10-E10 M NaI. B. 10-E10 M NaI + 0.1 mU/mL TSH. C. 10-E3 M NaI. D. 10-E3 M NaI + 0.1 mU/mL TSH. A. The ultra-structure of thyrocytes in the absence of TSH was identical to that of thyrocytes from follicles cultured for 1 day. B. Lysosomes close to the nucleus and few colloid droplets located in the apical pole (insert) were observed in the presence of TSH. C. They were well conserved in the presence of TSH, even in this strong dose of NaI. Endocytic vesicles were present in such strong dose of iodide. D. Colloid droplets were also observed in the presence of TSH, having the same density as colloid and were located in the thyrocytes’ apical pole. Follicle centre was narrow and had abundant microvellosities (TEM. A 8,720 X, B 12,510 X, box 10,720 X, C 7,430 X, box 70,950 X, D 13,450 X).
The thyrocytes from follicles cultured from 30 min up to 6 days in the presence of 10E-3 M NaI did not have cytotoxic signs and all follicular cells were viable, like normal animals’ thyroid glands’ in vivo when fed with excess iodide for 3 weeks [103].
Open human follicle culture in the presence of 10E-3 M NaI with and without TSH for 24 h had ultra-structural alterations related to cytotoxicity [92] involving free radical attack and lipid peroxidation. Excess iodide in pig follicles led to thyrocyte apoptosis because iodine production in lactone became reduced, but did not present morphology. Our results showed that conserving closed follicles did not lead to signs of cell death with 10E-5 M or 10E-3 M NaI or disorganisation or alteration of thyrocyte ultra-structure. The difference with [92] and [94] was that they were open follicle cultures and thyrocytes died simply because they were cultured on a support which inhibited cellular adhesion, like our cell aggregates.
Closed follicles were present (Figure 12), as described for the gland. There was apical membrane turnover between microvellosities which is important for maintaining Tg synthesis and its secretion to colloid [8]. Coated endocytic vesicles (Figure 12C insert) were also present in the base of microvellosities, like micro-endocytosis in vivo [5], and those stimulated by TSH formed pseudopods and colloid droplets (Figures 12B insert and 12C letter DC), called in vivo macro-endocytosis [10]. Thyrocyte fusion with prelysosomes or late endosomes from the lysosome route was also observed (Figure 12B arrows) for Tg degradation and thyroid hormones were formed in vivo [19] and in vitro [18].
Closed rat and pig euthyroidic follicles responded to increasing doses of iodide, as in vivo, thereby producing the Wolff-Chaikoff effect [1], and presented no modifications in thyrocytes’ follicular architecture or ultra-structure, being comparable to a gland in vivo.
6.3.2.1. Na+/I- symporter determination
Many thyrocyte culture studies have described reduced RNAm and NIS protein expression when maintained in the presence of strong iodide concentrations (10E-6 to 10E-4 M of iodide). We wanted to determine NIS in rat follicles cultured with strong iodide concentrations with and without TSH 0.1 mU/mL.
Follicles cultured at 12 h in the presence of 10E-7 M NaI and without TSH had NIS in basolateral membranes (Figure 13A) and labelling was more intense in lateral membranes in the presence of TSH (Figure 13B). In the presence of 10E-3 M NaI NIS was mainly located in vesicles between nucleus and basolateral membranes (Figure 13C), and in the presence of TSH; as well as being presented in vesicles they were observed in basolateral membranes (Figure 13D). It could have been that inhibiting vesicular movement in the presence of 10E-3 M NaI in the apical region (Figures 11C and 11D), as well as inhibiting the movement of the vesicles forming in the basement region with NIS symporter for avoiding excessive iodide entry to thyrocytes.
The NIS symporter was located in the basolateral members in the presence of 10E-10 M NaI at 48 h (E and F), labelling being more intense in the presence of TSH. The NIS symporter was found in the cytoplasmatic vesicles with TSH (F). NIS symporter expression in the presence of 10E-3 M NaI for 48 h (G and H) was so low that confocal microscope parameters (laser intensity, detector gain, scanning time) had to be adjusted again to increase labelling intensity. NIS protein was mainly observed in cytoplasmatic vesicles in these follicles, being more intense than labelling without TSH (G). Basement membranes had exiguous NIS labelling (CM. Scale bar: 10 µm).
Figure 13.
Indirect immunofluorescence of NIS symporter (green) expression and localisation and DAPI-labelled nuclei (blue) in follicles isolated from rats cultured for 1 day on agarose with 0.5% FCS for 1 day; the medium was changed and were cultured for 12 h (A, B, C and D) and for 48 h (E, F, G and H), with different experimental points: NaI with and without 0.1 mU/mL TSH. A. 10E-7 M NaI, B. 10E-7 M NaI + TSH. C. 10E-3 M NaI. D. 10E-3 M NaI + TSH. E. 10E-7 M NaI. F. 10E-7 M NaI + TSH. G. 10E-3 M NaI. H. 10E-3 M NaI + TSH. NIS symporter was located in the basolateral membranes in the presence of 10E-10 M NaI at 12 h (A and B). TSH intensified labelling (B). NIS symporter was located in cytoplasmatic vesicles near thyrocytes’ basolateral membranes in the presence of 10E-3 M NaI at 12 h (C and D). There was more intense labelling in some thyrocytes’ basolateral membranes in the presence of TSH (D). [104]
The NIS symporter in follicles cultured for 48 h in the presence of 10E-7 M NaI were located in vesicles and basolateral membranes (Figure 13E); TSH intensified such labelling (Figure 13F). NIS expression was very reduced regarding the other treatments in the presence of 10E-3 M NaI with and without TSH and microscope parameters had to be readjusted for observing fluorescence. NIS was located in cytoplasmatic vesicles in this strong dose of NaI and without TSH (Figure 13G). TSH was located in vesicles in the base region but labelling was less intense (Figure 13H) than with TSH.
NIS has normally been located in thyrocytes’ basolateral members in vivo [13], and a reduction in its normal expression has been associated with escape from the Wolff-Chaikoff effect [30] following 48 h in the presence of strong iodide concentrations [29]. Being found in vesicles has reduced NIS in its normal position for thyrocytes from follicles in the presence of a strong dose of NaI [104] and has thus suppressed I-transport for thyroid hormone production.
These results were similar in the FRTL5 cell line where the same dose did not alter NIS RNAm percentage, but protein became reduced by 50% and 78 % at 24 and 48 h, respectively [32]. NIS RNAm became reduced in dogs with goitre at 48 h with a comparable iodide dose [105]. This was perhaps presented by reduced AM2Pc levels at 48 h, as excess I- inhibited an increase in AMPc stimulated by TSH in hypophysectomised rats [106] and mice [34], which could have explained the low NIS level during Wolff-Chaikoff effect and in follicles at 48 h in the presence of excess iodide (Figures 13G and 13H). Excess I- inhibited IP3 production and increased Ca2+ flow induced by TSH, which could have led to reduced peroxide production during Wolff-Chaikoff effect [107,108]. The organification observed in the presence of 10E-5 M (Table 8) did not completely inhibit TPO and had no effect on NIS in the presence of 10E-3 M NaI (Figures 13E and 13H), thereby demonstrating that organification depends on TPO and not on NIS as in cells transfected with the TPO gene [34]. TSH did not stimulate the organification of iodide captured in 10E-3 M but did so in 10E-5 M NaI (Tables 8 and 10), as it has been described that the effect of TSH on thyroid physiology becomes reduced in the presence of excess I-, meaning that antagonic roles are assumed in vivo [102].
TSH modulated relative NIS expression and its subcellular localisation in the thyrocytes of isolated and closed follicles in vitro. These results were similar to those found in FRTL5cells, where it has been demonstrated that de novo synthesis [32], half-life time, NIS targeting and/or retention regarding cytoplasmatic membrane requires TSH to be located throughout cell membrane, due to loss of polarity [109].
Thyrocyte disposition in follicles has not been necessary for iodide accumulation, since it has been present in foetal thyroids before follicular lumen formation [110] and also in primary cultures from normal thyrocytes [111] or goitre patients [112] and in the FRTL cell line [113]; however, these cultures have required TSH, hormones and other molecules for maintaining them. Nevertheless, isolating the colloidal cavity from the exterior must be ensured for iodide accumulation and incorporation in Tg, T3 and T4 hormone synthesis, as demonstrated with rat or pig isolated and closed follicle cultures.
Rat and pig follicles thus inhibited iodide organification in the presence of strong concentrations of iodide, i.e. performed the Wolff-Chaikoff effect. Neither thyrocytes’ follicular architecture nor ultra-structure was modified and no sign of cell death was presented. The TSH and iodide effects observed in vivo during the Wolff-Chaikoff effect were reproduced.
7. Conclusion
Loss of follicular structure during the first 24 h of culture has been the main drawback of in vitro thyroid studies and, therefore, hormone synthesis. It is not enough to conserve thyrocytes’ apical-basal polarity in culture in this specific tissue for maintaining colloid’s extracellular functions for the enzymes implicated in iodide fixation on Tg and hormone synthesis.
We have shown that follicular architecture must be conserved in culture, especially the follicular cavity isolated from extracellular medium as this is indispensable for maintaining ultra-structure and the polarity of thyrocytes around the follicular cavity; such premise conserving the idea of Tg19S synthesis usually being glycosylated and iodised, as also T3 and T4 hormone synthesis. Follicular morphological conservation is necessary for reproducing the Wolff-Chaikoff effect in vitro, as has been described in vivo. NIS symporter localisation in thyrocytes depends on I and TSH concentration.
This culture may be used for obtaining follicles from pathologies of human tissue whose epithelium may be thin plate-like cells for in vitro studies in controlled and homologous conditions regarding the pathology in vivo. It will also enable studying normal or pathological thyroid’s physiological, cellular and molecular mechanisms (for example CLC-5 channel) in a homologous model of the gland in vivo.
Acknowledgement
This work was supported by grants from the Colombian Science, Technology and Innovation Department (COLCIENCIAS) programmes Ecos-Nord and Jóvenes Investigadores P 2009-0745, by the Universidad Nacional de Colombia’s Research Department and Fundacion Instituto de Inmunología de Colombia (FIDIC). We would like to extend our most sincere thanks to Marcela Camacho, Marie France van den Hove, Thierry Pourcher, Jean-François Denef, Hernando Curtidor and Manuel E Patarroyo for their support and encouragement for carrying out basic research in Colombia.
We would also like to thank the students who carried out and who are carrying out BSc, MSc and PhD thesis experiments: Sandra Perdomo, Gabriela Delgado, Cristina Zapata, Ricardo Cabezas, Alejandro Ondo, Leslie Leal, Oscar Vivas, Claudia Moreno, Eleonora Bernal, Anyela González, Mauren Ortíz, Carolina Ochoa and Luz Marina Porras; this work could not have been completed without their input.
\n',keywords:null,chapterPDFUrl:"https://cdn.intechopen.com/pdfs/37916.pdf",chapterXML:"https://mts.intechopen.com/source/xml/37916.xml",downloadPdfUrl:"/chapter/pdf-download/37916",previewPdfUrl:"/chapter/pdf-preview/37916",totalDownloads:2209,totalViews:432,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,dateSubmitted:"November 12th 2011",dateReviewed:"April 14th 2012",datePrePublished:null,datePublished:"July 18th 2012",dateFinished:null,readingETA:"0",abstract:null,reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/37916",risUrl:"/chapter/ris/37916",book:{slug:"thyroid-hormone"},signatures:"Clara Spinel, Magnolia Herrera and Jhon Rivera",authors:[{id:"140409",title:"Prof.",name:"Clara",middleName:null,surname:"Spinel",fullName:"Clara Spinel",slug:"clara-spinel",email:"cmspinelg@unal.edu.co",position:null,institution:null},{id:"140928",title:"BSc.",name:"Magnolia",middleName:null,surname:"Herrera",fullName:"Magnolia Herrera",slug:"magnolia-herrera",email:"magno74@yahoo.com",position:null,institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_1_2",title:"1.1. Morphology",level:"2"},{id:"sec_1_3",title:"1.1.1. Histology",level:"3"},{id:"sec_2_3",title:"1.1.2. Ultra-structure",level:"3"},{id:"sec_4_2",title:"2.2. Function",level:"2"},{id:"sec_6",title:"2. Thyroid culture",level:"1"},{id:"sec_6_2",title:"2.1. Introduction",level:"2"},{id:"sec_7_2",title:"2.2. Organotypical culture or organ culture",level:"2"},{id:"sec_8_2",title:"2.3. Isolation and monolayer culture or cell culture",level:"2"},{id:"sec_9_2",title:"2.4. Pseudofollicles",level:"2"},{id:"sec_11",title:"3. Follicles",level:"1"},{id:"sec_12",title:"4. Method used",level:"1"},{id:"sec_12_2",title:"4.1. Isolation and closed follicle culture",level:"2"},{id:"sec_13_2",title:"4.2. Morphological studies",level:"2"},{id:"sec_14_2",title:"4.3. Functional studies",level:"2"},{id:"sec_15_2",title:"4.4. Statistical analysis",level:"2"},{id:"sec_17",title:"5. Results",level:"1"},{id:"sec_17_2",title:"5.1. The importance of obtaining closed follicles for culturing them",level:"2"},{id:"sec_18_2",title:"5.2. Long-term closed follicle culture",level:"2"},{id:"sec_18_3",title:"Table 3.",level:"3"},{id:"sec_19_3",title:"6.2.2. Morphological study",level:"3"},{id:"sec_21_2",title:"6.3. Closed follicle cultures reproduce the Wolff-Chaikoff effect described in vivo",level:"2"},{id:"sec_21_3",title:"Table 7.",level:"3"},{id:"sec_22_3",title:"6.3.2. Morphological study",level:"3"},{id:"sec_25",title:"7. Conclusion",level:"1"},{id:"sec_26",title:"Acknowledgement",level:"1"}],chapterReferences:[{id:"B1",body:'WolffJ.ChaikoffI. L.1948Plasma inorganic iodide as a homeostatic regulator of thyroid function. J Biol Chem. 17455564'},{id:"B2",body:'WollmanS.1980Structure of the thyroid gland. In: Vissher M, editor. The thyroid gland. New York: Raven Press. 119\n\t\t\t'},{id:"B3",body:'WernerC.IngbarH.1971The thyroid. 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Endocrinol. 611420'},{id:"B77",body:'MorandS.ChaaraouiM.KaniewskiJ.DemeD.OhayonR.MSNoel-Hudsonet.al2003Effect of iodide on nicotinamide adenine dinucleotide phosphate oxidase activity and duox2 protein expression in isolated porcine thyroid follicles. Endocrinology 144412418'},{id:"B78",body:'KusakabeT.KawaguchiA.HoshiN.KawaguchiR.HoshiS.KimuraS.2006Thyroid-specific enhancer-binding protein/NKX2.1 is required for the maintenance of ordered architecture and function of the differentiated thyroid. Mol Endocrinol. 201796809'},{id:"B79",body:'TakasuN.OhnoS.KomiyaI.YamadaT.1992Synthesis; cytoskeletons and iodine metabolism in polarized monolayer cells on collagen gel and in double layered, follicle-forming cells. Endocrinology 131311438'},{id:"B80",body:'MassartC.GibassierJ.RaoulMl.Le GallF.LancienG.GenetetN.et al.1997Action of peripheral or intrathyroidal lymphocytes on autologous thyrocytes cultured in follicles in collagen gel. J Mol Endocrinol. 1813745'},{id:"B81",body:'EliaN.Lippincott-SchwartzJ.2009Culturing three dimensional MDCK cells for analyzing intracellular dynamics. Curr Protoc Cell Biol. Available: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786266/'},{id:"B82",body:'SpinelC.1987Aspects morphologiques et fonctionnels des follicules thyroïdiens en culture. [PhD thesis]. Brussels: Univ Catholique de Louvain. Brussels.'},{id:"B83",body:'SpinelC.ColinI.van den-Fhove. M.Denef-FJ.1990Correlated morphological and functional study of isolated rat thyroid follicles in culture. Mol Cell Endocrinol. 2134758'},{id:"B84",body:'HerreraM.OndoA.SpinelC.2007Culture of follicles isolated and closed in the long term of pig thyroid and its morphologic study. Acta Microsc. 16(1-2) Supp 23156\n\t\t\t'},{id:"B85",body:'CabezasR.OndoA.AriasJ.SpinelC.2005Isolation and culture of closed thyroid follicles in mouse, pig and man. Thyroid 15 (Suppl. 15):84-5.'},{id:"B86",body:'HerreraM.OndoA.SpinelC.2008Estudio morfológico del cultivo a largo plazo de foloculos aislados y cerrados de tiroides de cerdo. Acta biol Colom. 1334960'},{id:"B87",body:'Phillips HS1973Dye exclusion tests for cell viability. In: Kruse PF, Patterson MK, editors. Tissue culture methods and applications. New York: Academic Press. 406408'},{id:"B88",body:'Schneider WC1957Determination of nucleic acids in tissues by pentose analysis. Meth Enz. 36804'},{id:"B89",body:'van denHove. M. F.PaternosterS.CouvreurM.1986Hormonogenic peptides of thyroglobulin aer released during the coupling of iodotyrosines. Ann Endocrinol. 47: 62A.'},{id:"B90",body:'HerreraM.RiveraJ.SpinelC.2010Swine follicles in three-dimensional morph-functional culture are homologous to the thyroid in vivo and by morphometric analyze can determinate the functional status. Paris: Abstract Book 14th International Thyroid Congress. OC-125.'},{id:"B91",body:'TachettiC.ZurzoloC.MonticelliA.NitschL.1986Funtional properties of normal and inverted rat thyroid follicles in suspension culture. J Cell Physiol. 1261938'},{id:"B92",body:'ManyM. C.MestdaghC.van den-FHove. M.Denef-FJ.1992In vitro study of acute toxic effects of high iodide doses in human thyroid follicles. Endocrinology 13162130'},{id:"B93",body:'SatoK. T.SatohT. K.ShizumeK. M.OzawaM. D.HanD. H.ImamuraH.et al.1990Inhibition of 125I-organification and thyroid hormone release by interleukin-l, tumor necrosis factor-α, and interferon-γ in human thyrocytes in suspension culture. J Clin Endocrinol Metab. 70173543'},{id:"B94",body:'LangerR.BurzlerC.BechtnerG.GärtnerR.2003Influence of iodide and iodolactones on thyiroid apoptosis. Exp Clin Endocrinol Diabetes 1113259'},{id:"B95",body:'YamasakiK.SuzukiK.YamadaE.YamadaT.TakeshitaF.Matsumotoet.al2007Suppression of iodide uptake and thyroid hormone synthesis with stimulation of the type I interferon system by double-stranded ribonucleic acid in cultured human thyroid follicles. Endocrinology 1487322635'},{id:"B96",body:'YamazakiK.YamadaE.KanajiY.ShizumeK.DSWangMaruo. N.et al.1996Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles. Endocrinology 137485763'},{id:"B97",body:'Hilfer SR, Searls RL1980Differentiation of the thyroid in the hypophysectomized chick embryo. Dev Biol. 79110718'},{id:"B98",body:'SpinelC.YildizI.1991High doses of iodide are not toxic for rat thyroid follicles in culture. Netherlands: Proceeding of the 10th International Thyroid Conference. 259\n\t\t\t'},{id:"B99",body:'Bastomsky CH1974Thyroid iodide transport. In: Greer MA, Solomon DH, editors. Handbook of Physiology Sec. 7, 3Washington DC: American Physiological Society, 1974, 8199'},{id:"B100",body:'BernalE.SpinelC.OndoA.2010Amiodarone and NIS in the mice thyroid. Paris: Book of Abstracts from the 14th International Thyroid Congress. 0294\n\t\t\t'},{id:"B101",body:'FollisR. H.Jr 1964Further observations on thyroiditis and colloid accumulation in hyperplastic thyroid glands of hamsters receiving excess iodine. Lab Invest 13159099'},{id:"B102",body:'HashizumeK.AkasuF.TakazawaK.EndoW.OnayaT.1976The inhibitory effect of acute administproportion n of excess iodide on the formation of adenosine 3’, 5’-monophosphate induced by thyrotropin in mouse thyroid lobes. Endocrinology 9914638'},{id:"B103",body:'MahmoudI.ColinI.ManyM. C.DenefJ. F.1986Direct toxic effect of iodide in excess on iodine-deficient thyroid glands: epithelial necrosis and inflammation associated with lipofuscin accumulation. Exp Mol Pathol. 4425971'},{id:"B104",body:'RiveraJ.HerreraM.OchoaC.SpinelC.2010Swine and mice closed follicular thyroid in a three-dimensional culture reproduce the Wolff-Chaikoff effect. Paris: Book of Abstracts from the 14th International Thyroid Congress. 0053\n\t\t\t'},{id:"B105",body:'UyttersprotN.PelgrimsN.CarrascoN.GervyC.MaenhautC.DumontJ. E.et al.1997Moderate doses of iodide in vivo inhibit cell proliferation and the expression of thyroperoxidase and NaI/I- symporter mRNAs in dog thyroid. Mol Cell Endocrinol. 131195203'},{id:"B106",body:'RapoportB.WestM. N.IngbarS. H.1975Inhibitory effect of dietary iodine on the thyroid adenylate cyclase response to thyrotropin in the hypophysectomized rat. J Clin Invest 56516519'},{id:"B107",body:'CorvilainB.LaurentE.LecomteM.VansandeJ.DumontJ. E.1994Role of the cyclic adenosine 3’,5’-monophosphate and the phosphatidylinositol-Ca2+ cascades in mediating the effects of thyrotropin and iodide on hormone synthesis and secretion in human thyroid slices. J Clin Endocrinol Metab. 791529'},{id:"B108",body:'HashizumeK.IchikawaK.KomiyaI.OnayaT.1984Thyrotropin-induced acceleration of calcium efflux from mouse thyroid: evidence for inhibition by excess iodide. Endocrinology 11416727'},{id:"B109",body:'RiedelC.LevyO.CarrascoN.2001Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J Biol Chem. 2762145863'},{id:"B110",body:'HodgesR.EvansT.BradburyJ.KeettelW.1955The Accumulation of radioactive iodine by human fetal thyroids. J Clin Endocrinol Metab. 1566617\n\t\t\t'},{id:"B111",body:'SherwinJ. R.TongW.1974The actions of iodide and TSH on thyroid cells showing a dual control system for the iodide pump. Endocrinology 9414651474'},{id:"B112",body:'SaitoT.EndoT.KawaguchiA.IkedaM.NakazatoM.KogaiT.OnayaT.1997Increased expression of the Na+/I- symporter in cultured human thyroid cells exposed to thyrotropin and in Grave’s thyroid tissue. J Clin Endocrinol Metab. 8233316'},{id:"B113",body:'Weiss SJ, Philp NJ, Ambesi-Impiombato FS, Grollman EF1984Thyrotropin- stimulated iodide transport mediated by adenosine 3’ 5’-monophosphate and dependent on protein synthesis. Endocrinology 1141099107\n\t\t\t'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Clara Spinel",address:null,affiliation:'
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1. Introduction
Oryza sativa is an essential food crop contributing 30% of calories for at least 62.8% of the world population. Presently, rice was cultivated over 167.13 m ha within 114 countries with annual production of 486.62 mt (on milled basis) [1]. Majority of population in Asian consumed rice as a basic staple crop with over than 90% of rice were produced in Asia [2]. Revolution in Green technology played an important role in adoption of major increasing in rice production over last four decades. Approximately 450 million tons to 650 million tons of world rice consumption is projected to surge by 2011 and 2050, respectively [3].
Scientist and plant breeder are facing a new challenge in crop improvement due to climate change issue including limitation of water and cultivated land, new emerging pathogen and pest resulted serious yield losses. Among the major constraints, blast disease, bacterial blight, sheath blight, tungro viruses, brown spot and a number of grain discoloration diseases [4] accounted for serious rice losses. Several amendments can be implemented to overcome these limitations such as breeding high yielding varieties, durable resistance to diseases and tolerance to abiotic stresses [5]. Rice yield potential can be enhanced by using numerous approaches such as conventional hybridization, ideotype breeding, heterosis breeding, wide hybridization and molecular breeding [6]. In molecular rice breeding, there are two main strategies can be used by applying the biotechnology concept which are MAS and Genetically Engineering. MAS like genetic engineering is a technique used to introgress targeted gene however its offers tools for specific selection for further breeding in existing plant material. The field of plant genetics and breeding has irreversibly changed with the development of DNA (or molecular markers) [7].
Since past two decade, revolutionized in molecular marker to enhance the effectiveness in breeding and to significantly shorten the development time of varieties bring about the concept of molecular marker assisted selection (MAS) as an efficient plant selection [8, 9, 10]. A genetic marker is any noticeable character or otherwise assayable phenotype, for which alleles at individual loci segregate in a Mendelian manner. The genetic markers covered include (1) morphological markers (2) biochemical markers (alloenzymes and other protein markers) and (3) molecular markers (based on DNA-DNA hybridization) [11, 12]. In this chapter, we present the latest DNA-based markers in a few studies related to abiotic and biotic stress genes using the MAS application in rice breeding programs. These markers have proven significantly useful from different findings published in screening, fine mapping and gene pyramiding approaches.
2. Molecular marker
Rediscovery Mendelian theory in the early part of 20th century, reveal that inheritance (genes) linked together in chromosome. Molecular marker defines as individual genes flanking within a defined close interval. It can be found at a specific location of the genome and linked with inheritance of a trait or gene [13]. Thottappilly et al. [14] refers molecular marker as polymorphism identified between species including proteins and nucleic acids. Collard et al. [15] had divided molecular marker into 3 major groups which are morphological marker, biochemical marker and DNA marker (Figure 1). However, Xu [16] had categorized markers into two broad groups based on detecting method; (i) classical marker and (ii) DNA marker. Classical marker was initiated with the mutations in the genomic loci controlling plant morphology [17] (Table 1). Advent in polymorphism detecting methods such as Polymerase Chain Reaction, Southern Blotting and Sequencing had invented the development of DNA markers (Table 2). Variation were detected at DNA level such as nucleotides changes ie; substitution (point) mutation, rearrangement (insertion and deletions) or errors in replication of tandemly repeated DNA [18].
Figure 1.
Different types of molecular marker available.
Types of marker
Morphological marker
Biochemical marker
Advantages
Readily available
Highly reproducible
Usually require only simple equipment
Applicable for measure a wide range of population genetic parameter
The most direct measure of phenotype
Inexpensive
Disadvantages
Requires expertise on crop or species
Limited biochemical assay for detection
Subjected to environmental influences
Phenotype-based analysis
Limited in number
Application
Conventional plant breeding program
Genetic diversity Map construction
Table 1.
Comparison between classical markers.
PCR based markers
Advantages
Disadvantages
Application
RFLP
Reliable and rigid
Harmful and tedious
Map construction Hybrid fixation Genetic diversity
Easy to conduct across laboratory
Required a larger amount of DNA
RAPD
A small amount of DNA required
Not transferable across laboratory
Genetic diversity Saturation mapping
Simple, rapid and lowest cost
Irreproducibility
Single primer generates multiple loci
SSR
Reliable, powerful and easy
Tedious and costly for initial establishment
Linkage and QTL mapping Marker-assisted selection Hybrids fixation
Transferable between populations
Required polyacrylamide gel electrophoresis
AFLP
Multiple loci
Required several DNA
Saturation mapping Genetic diversity
Complicated
SNP
Multiplexing large number of markers
Costly to assay
Fine mapping Map-based cloning
Degraded DNA can be used
Marker has less allele
Table 2.
Comparison of different PCR based marker.
Although there are many characteristics determine the suitable markers to be considered in MAS development; there are five main characteristics need to be considered [13, [19, 20, 21, 22].
Reliability: Attribute of the desirable marker need to close proximity with the target loci, preferably less than 5 cm genetic distance or one marker every 10 cm [23]
Repeatability and reproducible: In plant breeding, a lot number of plants are usually screened for desirable marker pattern and result is required instantly. Therefore, level of simplicity in terms of time required and quick detection method are highly desirable for rapid selection. Statistical and bioinformatics software are significant for this purpose.
Cost effective: Marker assay should be not expensive feasible for selection process.
Level of polymorphism: Marker should discriminate between two similar accessions of species especially in parent breeding material.
DNA quality and quantity: A few markers require a complex DNA extraction technique and it too laboratories. In addition, it adds cost to the process.
3. Successful application of molecular marker in rice
Recently, MAB has become a well-known approach develop a superior genotype in rice. The aims of the breeding programs were to develop a rice variety with high yield potential, resistant to diseases and insects, tolerance to adverse environments, and acceptable grain quality. Since the first successful experiment stated for rice by Chen et al. [24] with introducing resistance to bacterial blight (BB) disease into Chinese hybrid parents. Till date, many varieties had been developed thru the application of molecular marker with backcrossing method. Varieties developed were improved from the wild type by incorporate desirable gene depending for biotic and abiotic stresses. Recently, in a few years there is a trend in producing more rice varieties that resistant in abiotic stress such as drought tolerance, submergence tolerance and salt tolerance. This demand comes from the periodic natural disaster in many regions of the rice producing country specifically in Asian country [25]. Resistant varieties against pathogenic disease were continuously developed. This scenario can be seen in blast disease where there are increasing number of resistant varieties were developed due to instability of the fungus pathogen.
Numerous molecular markers have been utilized in marker-assisted breeding however microsatellites are the preferable markers for plant breeding applications. The characteristic as codominant markers which inherited in a Mendelian fashion have emerged as a best choice compare to other markers. Additionally, microsatellite identify as preferable markers used in plant breeding programs due to ubiquitous, high polymorphism rates and wide range of distribution throughout the genome [26, 27]. Other markers possess major drawbacks including Restriction fragment length polymorphism (RFLP) markers which are not integrated with high-throughput technique; Random amplification of polymorphic DNA (RAPD) assays which are often not reproducible or immobile. AFLP analysis is not straightforward and commonly produces multiple fragments with the use of large genomic templates [28]. Application of molecular marker in rice breeding was demonstrated in Table 3 to develop superior varieties in rice against abiotic and biotic stresses.
Four QTL (chromosome 1, 2, 7 and 9) was introgressed to NIL developed from crossed between Azucena (donor parent) and IR64 (recipient parent) [29]. Twenty-nine NIL successfully developed in which three NIL carrying target 1 demonstrated significantly increased trait over IR64 while 3 out of 8 NIL consist combination target 1 and 7 exhibited higher root mass and depth. Four NIL carrying targets 9 showed increased maximum number of root length and 2 NIL showed no significant difference on root phenotypic compare to IR64. Results suggest the introgression of genes linked to root improvement is an ideal system compared to change the morphological characters of plant.
4.2 Quality
Waxy locus (wx-MH) from Mighui 63 was successfully introgressed into Zhenshan 97 which having poor agronomically quality including amylose content (AC), gel consistency (GC), gelatinization temperature (GT) and endosperm [30]. MABC scheme had been used to develop 3 backcross and 1 selfing generation. Improved variety of Minghui 63 (Shanyou 63) carrying wx-MH gene at 6.1 cm length have similar agronomy characteristic as Minghui 63 with low AC, high GC and GT with low grain opacity. Result indicated that waxy region gives significant effect on quality trait.
4.3 Root trait and aroma
MABC strategy had adopted by high yielding India upland rice variety, Kalinga III to insert 5 segments of QTL including 4 QTL carrying root length and thickness and 1 QTL control recessive aroma trait from Azucena donor variety which come from Philippines japonica rice [31]. This experiment was performed until 8 generation which using 3000 SSR markers for almost 8 years to develop NIL. Twenty-two NIL successfully develop which carrying QTL on root length at chromosome number 9 linked with RM242-RM201 and RM248 on chromosome 7 control for delayed flowering.
4.4 Submergence tolerance
Ten improves lines from OM 1490/IR 64 -Sub1 was developed from Marker-assisted backcrossing and having 90–99% survival under field submergence treatment [78]. Foreground selection was carried out using microsatellite, RM23805 marker which identified allele as 240 and 230 bp bands, respectively from OM1490, susceptible parent and IR64-Sub1, tolerant parent. Entire lines of OM1490/IR64-Sub1 introgression possess an increase rate of survival from original parent. In India, high yielding mega variety, Swarna had been incorporated with Sub1 gene by using three selections namely foreground, recombinant, and background selection, respectively. Findings indicated that superior variety Swarna successfully transformed into submergence tolerant variety within 2–3 years with three backcross generations [33]. Popular rice variety, AS996 successful incorporated sub1 gene from rice variety IR64 as a donor parent through MABC. Insertion of sub1 was confirmed with foreground SSR marker, ART5 and SC3 [36]. Background analysis was screened with 53 polymorphic SSR markers to assess BC2F1 and BC3F1 generations. The highest recurrent background was achieved 100% donor parent. Results suggest the development a new submergence tolerant rice variety ASS996-SUB1 to stand with climate change. Superior varieties from India (Samba Mahsuri and CR1009), IRRI, Philippines (IR64), Laos (Thadokkham 1) and Bangladesh (BR11) successfully introgress sub1 gene exhibited an improve survival rate compare to parental variety by applying MABC approach. Septiningsih et al. [79] studied the expression level of sub genes revealed that sub1C-1 was dominant over the sub1A allele however no reduction of expression observed when intolerant sub1C allele merged with the tolerant sub1A-1 allele. Survival rates of heterozygous plant with sub1A expression higher compared to homozygous tolerant parent. Existence of sub1A-2 will not significantly function in intolerant plant with turn off expression of sub1C-1. Study by Ahmad et al. [38] developed 30 newly lines of BC2F3 population from backcross of MR219 and Swarna-Sub1 with 95.37% recurrent parent genome (RPG) recovery. Rahman et al. [39] stated a marker-assisted backcross breeding (MABB) approach to incorporate the Sub1 locus into popular rice varieties of southern India, CO 43, from tolerant FR13A variety. An elite NILs of CO 43 harboring Sub1 locus possessing 95.78% of the recurrent parental CO 43 genome produced from genotyping and phenotyping analysis.
4.5 Salt tolerance
ASS996 a high yielding and widely grown cultivar in Vietnam had been cross with FL478, a highly salt tolerant rice variety using MABC strategy [40]. Four Saltol-AS996 introgression lines, QF3–1, QF3–2, QF 4–3-3, QF6–4 was developed with 3 foreground SSR markers, AP3206, RM3412 and RM10793 and 63 polymorphic markers were applied across rice genome for background analysis. The promising lines showed significant salt tolerance or similar compare to recurrent parent, FL478. MABC approach had been applied for introgressed Saltol QTL from donor parent, FL478 into Bacthom 7 recipient rice cultivar. Eighty-nine polymorphic SSR markers including 8 foreground markers to screen heterozygous plant carrying Saltol locus in each backcrossed generation. Background analysis demonstrated the restoration of recipient genome up to 96.8–100% in the best plant of BC3F1 generation. Finding concluded that improve version of salt tolerance-Bacthom 7 is relevant for cultivation in coastal areas of the Vietnamese Deltas [41]. De leon et al. [43] reported the utilization of SSR and SNP markers to characterize the ILs (Pokkali x ‘Bengal) and identify 14 QTLs traits related to salinity tolerance.
4.6 Early maturation
In Indonesia, Hd2 gene located at chromosome 7 from Nipponbare was transferred to Code variety [44]. Two markers linked with early maturation trait, RM1362 and RM7601 was used to identify heterozygous carrying this gene in 2 backcross and 1 selfing generation. Improved lines showed a similar agronomic characteristic with code variety except for heading time which 73–85 days in improved version compared to recipient parents, 89 days. Maturation days for improved version of Code carrying Hd2 gene were 103–104 day which is at considerable maturation days. Result concluded the availability to use these improved lines in Indonesia for increasing rice production.
4.7 Heading time
Koshihikari and Kasalath were crossed by using MABC strategy to introgress 3 QTL segment which code for Qdth3, Qdth6 and Qdth8 [47]. Genotyping analysis was performed using five different types of DNA marker including RFLP, STS, SSR, CAPS and dCAPs. Three NIL successfully produced namely Wakei 367, Wakei 371 and Kanto IL1 which carrying Hd2 gene coded for heading time. All NIL exhibited same morphological trait with Koshihikari with early heading time for Kanto IL1 (12 days), Wakei 367 (10 days) and Wakei 371 (11 days) compare to 95 days on Koshihikari.
4.8 Phosphorus tolerance
MABC approach had been successfully adapted to introgress a major quantitative trait locus (QTL), Phosphorus uptake1 (Pup1), positioned on rice (Oryza sativa) chromosome 12 into two types of Indonesian rice varieties, irrigated and upland. SNP and CAPs had been used as foreground marker to identify desirable traits and background selection had been conducted using 47 to 61 polymorphic SSRs markers and restored 89–93% of the recipient genome in BC2F3 population. Phenotyping finding suggests that Pup1 is effective to enhance grain yield under field treatment [48].
4.9 Drought tolerance
Crossing by hand emasculation and artificial pollination was performed between IR20 (japonica) and lowland (indica) cultivar to develop near isogenic lines (NILs) [42]. Foreground and background selection were conducted using 100 SSR markers that distributed entire genome. Five NILs exhibited high yield performance compare to IR20 in rain-fed and irrigated conditions. NIL-212 and 297 successfully incorporated with three and two root QTLs with NIL-297 demonstrated maximum value of nodal root and surface area whereas increase number of nodal roots than recurrent parent, IR20 observed in NIL 212. Backcrossed population (BC2F3) was developed through Marker Assisted Selection (MAS) to transfer QTLs for drought tolerant characters into Thai famous variety KMDL105 using 3 different donor parents, DH212, DH103 and DH126, respectively. Hundred and three KMDL 105 introgression lines were developed carrying selected target [80]. Genome analysis claimed that donor segment was found in non-carrier chromosomes and minimum proportion of recurrent genome was determined in carrier chromosome. Results suggest that background analysis is required to restore the recurrent genome segments. Sandhu et al. [46] enhance the grain yield of Samba Mahsuri under reproductive-stage drought (RS) through marker-assisted selection (MAS) and marker-assisted recurrent selection (MARS). Findings recorded four qDTYs (qDTY1.1 + qDTY2.1 + qDTY3.1 + qDTY11.1) and two QTLs (qDTY1.1 + qDTY11.1). Presence of gene MQTL1.1 responsible for the drought tolerance was identified from the cross (Sarjoo52 × Nagina- 22) in the study reported by Awasthi and Lal [49]. Ramchander et al. [50] study 101 Backcross Inbred Lines (BILs) of rice (BC1F5) for drought tolerance by develop a cross between Norungan//TKM9/Norungan. Marker aided selection used in this study efficiently pyramiding favorable QTL alleles to enhance drought tolerance in elite background of famous rice varieties using 15 polymorphic SSR markers. Muthu et al. [51] successfully developed abiotic stress-tolerant rice genotypes of the famous rice variety, Improved White Ponni (IWP) through a marker assisted backcross breeding approach by introgression major effect quantitative trait loci (QTLs) conferring tolerance against drought (qDTY1.1, qDTY2.1), salinity (Saltol), and submergence (Sub1). Backcrossed inbred lines (BILs) developed in this study showed minor effect caused by drought, salinity, and submergence. Root trait genes were introgressed through marker-assisted backcross breeding (MABC) between aerobic rice variety, AERON1 and MRQ74, a high yielding rice variety of Malaysia, [52]. Six best rice lines in BC2F1 with highest recovery percentage and resemble similar phenotypic appearance were identified with RM242 and RM263 foreground and 57 background markers.
4.10 Grain fragrance
Ahn et al. [81] was first discover the molecular marker linked with grain aroma. Lorieux [82] was used RFLP marker, RG28, which linked with aromatic located at chromosome 8 with genetic distance of 4.5 cm. The uniqueness of RG28 was interchangeable into STS marker and can be used to distinguish between fragrance and non-fragrance rice cultivars [83, 84, 85]. Improved version Pusa Bashmati and improved versions of PRR78 was successfully introgressed with quality trait from Basmati 370 as a donor with Manawthukha cultivar as a recipient using MABC scheme [86, 87].
4.11 Semi-dwarfing
Sd1 gene coded for semi-dwarf trait creates an interest among breeder as it enhances crop production. Advent in MAB had made it possible to track the introgression of sd1 gene by using SSR markers in two traditional japonica varieties, ‘IR36’ and ‘Allorio’ (IRGC284) [88].
5. Biotic stresses
5.1 Bacterial blight resistance
Marker assisted backcrossing coupled with stringent phenotypic screening was adapted to transfer BB resistance gene, Xa23 into Minghui63, YR293 and Y1671 which famous recipient lines. Foreground selection was screened with RM206 located at 1.9 cm near to the Xa23 locus [89]. The improved version of BB resistant was identical with original recipient for agronomic traits [30]. NSIC Rc142 and NSIC Rc154 donor cultivar for Xa4 + Xa5 + Xa21 genes were incorporated into susceptible cultivar IR64 in Philippines [61]. Currently, Xa13 and Xa21 had been successfully inserted into recurrent line PRR78 through MABC [90]. In Thailand, three backcrossed generation required to insert Xa21 (BB resistance gene) from IR1188 variety into famous KDML105 variety using MABC approach together with phenotypic selection. Similar agronomic characteristic was observed between developed introgressed lines having BB resistant gene (Xa21) with local variety KDML105 [91]. Foreground selection was screened with six SSR markers namely RM302, RM212, RM210, RM149, RM287, RM224 at different chromosome while 66 rice SSR markers was applied for background genotype. In China, popular restorer line, Yihui1577 was incorporated with Xa7 and Xa21 from Huahui20 thru MABC strategy [92]. Foreground marker were applied using RM20582 in existence of Xa7 to map a 0.14 cM interval at chromosome 6 between RM20582 and RM20593 [93]. Huang et al. [40] in his study successfully employed of Xa7, Xa21, Xa22 and Xa23 from Jinke 1 a cytoplasmic male sterile line into Huahui 1035 line using MABC. High yielding F1 hybrid was used to develop more hybrid rice in China. Samba Mahsuri a mega high-yielding, fine grain-type variety was selected as donor for Xa21and Xa13 to incorporate into susceptible varieties, Taraori Basmati and Basmati 386 through MABC scheme [94]. Molecular backcross breeding was used to pyramided Xa21 + xa13 and Xa21 + xa5 combinations genes in a background of lowland cultivar, Jalmagna [65]. Twenty pyramided lines with targeted gene combinations showed resistant against BB pathogen. Balachiranjeevi et al. [66] reported the development of durable BB resistance through marker-assisted backcross breeding (MABB) into background of elite rice, DRR17B by introgress Xa21 and Xa33 dominant genes.Six promising pyramiding lines of DRR17B with durable resistance and similar or superior agro-morphological attributes to DRR17B have been identified.
5.2 Blast resistance
C101A51 and Tetep contribute for Piz-5 and Pi54 blast gene was introgressed into PRR78 variety [69]. Two improved lines were developed, Pusa1602 (Piz5) and Pusa1603 (Piz54) through MABC breeding strategy by two backcross series. Foreground selection was performed by using AP5930 and RM206 while background analysis using SSR polymorphic markers revealed 89.01 and 87.88% in Pusa1602 and Pusa1603 lines recovery of RPG, respectively. Hybrid develop carrying blast resistance gene produced exhibited similar performance with parental Pusa RH10 in terms of yield, grain and cooking quality traits. Multiple resistance gene against leaf blast (Pi1), neck blast (Pi2) (donor BL122) and bacterial (Xa23) (donor CBB23) was successfully introgressed into Ronfeng B, a mega variety with early maturating line thru MABC scheme [70]. Improved lines D521, D524 was developed using three foreground markers (MRG4766, AP22 and RM206) and 131 polymorphic background markers. Proportional recipient genome for D521 and D524 were 96.18 and 96.56%, respectively after 4 backcrossed generations. The size of lesions exhibited from 0.77 to 1.18 cm with 96.7–100% resistance frequencies values. Rongfeng 3A was developed as an improve version of cytoplasmic male sterile line from Rongfeng by a series of conducive backcross breeding with Pi1, Pi2 and Xa23 gene. A series of superior lines with resistance to bacterial blight and blast diseases were developed by using MAB [30, 90, 95]. Hybrid Pusa RH10 is likely susceptible to BB and blast therefore by employing the bacterial and blast resistance gene, it will be improved adjustability to disease endemic parts and also retain the rice production. In Iranian, two crosses were made between susceptible, Tarom Mahali (TAM) and resistance cultivar, Khazar (KHZ) to increase blast resistance. Screening with 74 polymorphic markers on 192 F2:3 families demonstrated both parental exhibited maximum genetic variation between the two varieties on entire chromosomes except chromosome 6, 7, 8 and 12 [96]. In Malaysia, improved version of MR219 was developed by introgression of Pi-z and Pikh and Pi-b by using two different donor, Pongsu Seribu 1 and 2 respectively [10, 97]. Hasan et al. [98] had successfully introgressed Pi7(t) and Pikh in MR264 from Pongsu Seribu 2 by using MABC approach. Thailand rice, the popular Sakon Nakhon (SKN) rice had been introgressed with QTLs on chromosomes 1, 2, 11 and 12 by using marker-assisted backcrossing. Srichant et al. [72] in his study presented 2 lines (SKN 39-10-19-29-12 and SKN 39-10-19-29-13) with high resistance to leaf and neck blast; and having similar agronomic traits as the SKN.
5.3 Brown plant hopper resistance
Bph3, gene coded for resistance against brown plant hopper had been introgressed into the famous Thai variety ‘Khao Dawk Mali 105’ [99]. Backcrossed population had been screened with RM589 and RM190, highly linked to Bph3 and Wx-RH loci. Fifty NIL were developed from background selection using 75 polymorphic SSR markers disperse 12 rice chromosomes. Cross had been made between Junambyeo, an elite japonica cultivar with IR65482-7-216-1-2 a donor indica rice by a series of backcrossing through MAB [75]. Bph18 gene was transferred and 7312T4A, the most closely polymorphic STS marker was applied to screen the existence of the Bph18 gene in BPH-introgress backcrossed lines [100]. Two hundred and sixty SSR marker were used to screen the RPG percentage in the four NIL breeding lines.
5.4 Gall midge resistance
Gm8 resistance gene code for rice gall midge had been employed from donor parent Aganni into Samba Mashuri having gene Xa21 using MABC. Four plants identified in selfing population (BC2F2) carrying Gm8 and Xa21 genes and further tested for phenotypic selection against BB and gall midge [76]. Exploration of gm gene had been a great achievement in modern crop however resistant variety carrying 1 major gene easily lost the resistancy [101]. Till date, 11 Gm genes in the plant [102] and 7 pathogens have been identified [103].
6. Conclusion
Advent in marker technology has offer a remarkable progress in crop improvement specifically in rice. Present paper reviews the application of DNA markers in abiotic and biotic stresses in rice using MAS. Interval between molecular markers and gene/QTLs linked with target traits play a significant role in efficiency of MAS technique. Generally, marker will be validated in fine mapping study. Thus, the development of important marker associated with abiotic and biotic stresses resistance is proficient by QTL mapping experiments. MAB greatly increase the effectiveness and efficiency of breeding. Desirable individual in germplasm collection with genes or QTLs can be characterized in genotyping analysis by using the DNA markers for target genomic regions rather than phenotyping analysis. Pyramiding tolerance genes and QTLs will develop a resistant cultivar against abiotic and biotic stresses. MAS refers to selection by DNA markers linked to QTLs or target genes. Nowadays, DNA-based genetic markers play a significant part in the forthcoming of MAB and molecular genetics analysis for establishment of stress-tolerance and resistance in plants through molecular linkage maps. Knowledge of markers for abiotic and biotic tolerant traits presented here will serve a fundamental guide and help rice breeders in their MAB.
Acknowledgments
The authors are grateful to Universiti Teknologi MARA, Universiti Putra Malaysia and Malaysian Nuclear Agency for the facilities, support and financial aids during the research work. The author also would like to extend appreciation to the Ministry of Higher Education (MOHE) for providing financial aids during this research.
\n',keywords:"abiotic stress, biotic stress, marker-assisted selection, rice, resistant",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/72661.pdf",chapterXML:"https://mts.intechopen.com/source/xml/72661.xml",downloadPdfUrl:"/chapter/pdf-download/72661",previewPdfUrl:"/chapter/pdf-preview/72661",totalDownloads:133,totalViews:0,totalCrossrefCites:0,dateSubmitted:"May 27th 2020",dateReviewed:"June 5th 2020",datePrePublished:"July 15th 2020",datePublished:null,dateFinished:null,readingETA:"0",abstract:"Rice, (Oryza sativa L.) account as the second cereal most cultivated in the world. Unfortunately, global rice production is rendered by significant number abiotic and biotic stresses. Breeding for resistant variety through conventional breeding is an economical method; generally, it takes at least 10 years to release a new rice variety. Advance technology in molecular marker had revolutionized and irreversibly changes the disciplines of plant genetic and breeding. Integration of DNA-based markers in selection process enhances the effectiveness and accuracy of conventional plant breeding. It offers a novel tool for discovering and tagging alleles and genes specifically in plant. Ubiquitous of DNA marker-trait associations for diverse crops species are available with the findings of many quantitative trait loci (QTLs) mapping studies. The linkage drags, and time-consuming in conventional breeding can minimize with the application of DNA markers in plant breeding. The utilization of DNA marker in QTL mapping, MAS and gene pyramiding has been investigated. In this chapter, we discussed the recent utilizing markers in rice breeding program against abiotic and biotic stresses. In a few decades, molecular marker assisted breeding (MAB) provide a boundless task for breeders in attaining an important impact on crop development.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/72661",risUrl:"/chapter/ris/72661",signatures:"Nor’Aishah Hasan, Mohd Rafii Yusoff, Abdul Rahim Harun and Faiz Ahmad",book:{id:"9669",title:"Recent Advances in Rice Research",subtitle:null,fullTitle:"Recent Advances in Rice Research",slug:null,publishedDate:null,bookSignature:"Dr. Mahmood-Ur- Rahman",coverURL:"https://cdn.intechopen.com/books/images_new/9669.jpg",licenceType:"CC BY 3.0",editedByType:null,editors:[{id:"185476",title:"Dr.",name:"Mahmood-Ur-",middleName:null,surname:"Rahman",slug:"mahmood-ur-rahman",fullName:"Mahmood-Ur- Rahman"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Molecular marker",level:"1"},{id:"sec_3",title:"3. Successful application of molecular marker in rice",level:"1"},{id:"sec_4",title:"4. Abiotic stresses",level:"1"},{id:"sec_4_2",title:"4.1 Deep root",level:"2"},{id:"sec_5_2",title:"4.2 Quality",level:"2"},{id:"sec_6_2",title:"4.3 Root trait and aroma",level:"2"},{id:"sec_7_2",title:"4.4 Submergence tolerance",level:"2"},{id:"sec_8_2",title:"4.5 Salt tolerance",level:"2"},{id:"sec_9_2",title:"4.6 Early maturation",level:"2"},{id:"sec_10_2",title:"4.7 Heading time",level:"2"},{id:"sec_11_2",title:"4.8 Phosphorus tolerance",level:"2"},{id:"sec_12_2",title:"4.9 Drought tolerance",level:"2"},{id:"sec_13_2",title:"4.10 Grain fragrance",level:"2"},{id:"sec_14_2",title:"4.11 Semi-dwarfing",level:"2"},{id:"sec_16",title:"5. Biotic stresses",level:"1"},{id:"sec_16_2",title:"5.1 Bacterial blight resistance",level:"2"},{id:"sec_17_2",title:"5.2 Blast resistance",level:"2"},{id:"sec_18_2",title:"5.3 Brown plant hopper resistance",level:"2"},{id:"sec_19_2",title:"5.4 Gall midge resistance",level:"2"},{id:"sec_21",title:"6. Conclusion",level:"1"},{id:"sec_22",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Shahbandeh M. World rice acreage 2010-2018. 2020. Available from: https://www.statista.com/statistics/271969/world-rice-acreage-since-2008/'},{id:"B2",body:'Bandumula N. Rice production in Asia: Key to global food security. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences. 2017;88:1323-1328. DOI: 10.1007/s40011-017-0867-7'},{id:"B3",body:'Rejesus RM, Mohanty S, Balagtas JV. Forecasting Global Rice Consumption. Working Paper. North Carolina State University; 2012'},{id:"B4",body:'Singh PK, Nag A, Arya P, Kapoor R, Singh A, Jaswal R, et al. 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Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Malaysia
Faculty of Applied Science, Universiti Teknologi MARA, Cawangan Negeri Sembilan, Kampus Kuala Pilah, Malaysia
Agrotechnology and Bioscience Division, Malaysian Nuclear Agency, Malaysia
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At IntechOpen, we’re laying the foundations for the future by publishing the best research by women in STEM – Open Access and available to all. Our Women in Science program already includes six books in progress by award-winning women scientists on topics ranging from physics to robotics, medicine to environmental science. Our editors come from all over the globe and include L’Oreal–UNESCO For Women in Science award-winners and National Science Foundation and European Commission grant recipients.
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