Our Clear-Cut Improvement to the Impact of Mouse and Rat Models in the Research Involving Female Reproduction

Abstract

In most research involving female reproductive function, female animals particularly mice and rats are usually employed. This may perhaps be due to their well-defined reproductive cycle (estrous cycle) as well as the ability to breed and handle them easily. The short and precise length of estrus cycle usually 4–5 days make mice models the choicest mammal when it comes to human related research. Also, they possess very short reproductive age typically 7–8 months reaching sexual maturity at weeks 4–7 following their birth. Although many similarities exist between this model and humans, however, there also exist obvious distinctions between the human female reproductive system and that of mice. Humans have average length of their reproductive or menstrual cycle of about 28–29 days with their reproductive ages between 10–40 years. These relevant differences between mice and human reproductive system constitute the limitations to the use of this models. Therefore, the scope of this chapter will be to explore the basic knowledge of laboratory mice by examining their reproductive system anatomy and physiology, the fertilization process, estrous cycle and genetic make-up. We hope that this will provide many insights to the use of animal models in female reproductive research.

1. Introduction

The female reproductive system and processes are dynamic in both humans and rodents, undergoing morphological and cytological changes in response to hormonal signals throughout the estrus (for rodent) or menstrual (for human) cycle. These changes are also observed during pregnancy as well as during menopause or ovarian aging. Mice and rats are animals belonging to the Rodentia order and the Muridae family, and so are referred to as murine. Mice and rat models have been established in preclinical research involving investigations and assessments of reproductive physiological and pathological processes in humans. Their little size as well as the simplicity of their housing and maintenance allow them to be commonly employed in research. Mice and rats are employed as good models of mammalian pathology and physiology given that the use of people and food animals in studies is hampered by economic and ethical reasons.

In experiments relating to human development, the mice models have been mostly utilized to examine the pathogenesis as well as the general attributes in developmental processes. The ample justification for their relevance to human reproductive tract development and functions lies in their hormone action. Just like in humans, their reproductive cycle oscillates periodically through fluctuations in ovarian steroid concentrations (estrogens and progesterone). There also exist these steroid hormone receptors (ER, PR) systems in some of their reproductive organs like the uterus and mammary glands which act as controls to check hormone levels and translate the info into suitable developmental effects. They possess similar reproductive cycle phases as humans. Their reproductive cycle is divided into two developmental phases: the preparatory phase, which is evidenced by high estrogen and progesterone concentrations and occurs throughout proestrus stage. During estrus, this phase leaves the ovarian target tissues prepared for gestation. The second phase occurs in diestrus when progesterone levels drop, and it is characterized by the apoptotic elimination of previous arrangements.

In order to uncover human developmental pathways and gain insight into female reproductive behaviors, developmental scientists have used a number of animals to explore the morphogenetic and molecular mechanisms involved in vertebrate development [1]. Organogenesis and differentiation have been studied extensively in embryonic, fetal, and neonatal mice and rats, with the unspoken assumption that morphogenetic and molecular processes in mice are equivalent to those in humans. This argument is supported by the fact that mice and humans share many developmental features. With a few exceptions, both species share the same range of organs and developmental phases that appear to be equivalent, if not identical [2, 3].

Whereas the data above illustrate that mice research is important to human reproductive biology, the mouse and human reproductive systems have distinct morphogenetic, physiologic, and anatomical distinctions. The lack of information on human organogenesis, particularly in terms of molecular pathways, makes human/mouse comparisons challenging.

1.1 Comparison of the basic anatomy of the reproductive organs of the female mice to humans

The female mice reproductive systems are made up of two ovaries, two small tightly coiled uterine tubes, two uterine horns, the cervix and the vaginal canal (Figure 1). The vagina is a tiny gray tube that connects the bladder to the uterus. In mice, the Mullerian ducts start at the opening of the uterine tube and develop into the female genital tract while in the rats, the ovarian bursa is formed when the ostium forms a complete capsule around the ovary. The uterine horns divide the vagina and reach toward the kidneys. Both the mice and rats possess bicornuate uterus which may accommodate several embryos known as litters. On the other hand, women have simple uterus which have only one compartment for the development of one or two embryo(s) (Figure 1). Ovaries in humans are little lumpy glands that sit at the terminals of the uterine horns and are connected to the uterine horns by oviducts.

Figure 1.

Anatomical feature of the reproductive system of a female rat and human.

1.1.1 Mice uterus and cervix in comparison with humans

The human uterus is simplex and appears in form of a large pear-like shape with a unique cavity which measures about 8 cm when assessing it from one cornu to the other cornu and 5 cm in width (Figure 1). Nevertheless, steroid hormone levels as well as pregnancy status extensively determines the thickness of the uterus. Other factors that affect uterus thickness include the existence of leiomyomata. On the other hand, mouse uterus is bicornuate having two cornua that unite distally to create a singular body (corpus), but in rats, the uterus is duplex, with twin uterine horns that are partially linked at the caudal end. A part of the broad ligament known as mesometria suspend the mouse uterus horns from the dorsal wall and hold them in place. These ligaments encircle the blood and lymphatic vessels as well as many nerves. Similarly, from the lateral pelvic side walls, the human uterus is also suspended by comparable ligaments. The body of the uterus both in humans and mice is made up of the caudal-cervical and cranial-fundal sections. The caudal-cervical and cranial-fundal regions of the uterus are also found in mice. However, the cranial portion of the body of mouse uterus is separated by a median septum into two chambers. On the other hand, rat uterus is partly connected and has no complete body. Nonetheless, it has a unique cervix with two uterine horn canals that are distinct. Both dorsally and ventrally, mice and rats have a continuous cervix and vaginal walls but not laterally. The lateral vaginal walls continue on both sides forming deep fornices. But then, in humans, the cervix (ectocervical portion) protrudes into the vaginal canal, having wide posterior and anterior fornices and thinner lateral fornices.

1.1.2 Comparison of the oviduct (uterine tube)

The oviduct, generally termed the uterine tube in mice and rats is a complex tiny-curled tube that links to the periovarian area. The mesotubarium helps to suspend the oviduct to the dorsal body wall, and is connected to the mesovarium, uterine mesometrium and ovarian bursa. Whereas the human oviduct is a muscle-lined straight tube that is about 12 cm long. The human oviduct, like that of rodents, is connected to the respective ovary by a broad ligament that runs from the uterine cornu (horn) (Table 1). In both the mice, rat and human oviducts are found the intramuscular segment, the isthmus, the ampulla, the infundibulum, and a fimbriated terminal [4].

Feature	Rodent (rat and mice)	Human
Gross anatomy
Ovary	Sphere-shaped and contained in bursa	Oval-shaped and opened to peritoneal cavity
Uterine tube	Thin tightly coiled tube, 18–30 mm in length	Muscle-lined straight tube, 12 cm in length
Uterine tube association to ovary	Open immediately into the ovarian bursa	Fimbriated end of the ampulla opens immediately into peritoneal cavity near the ovary
Segments of uterine tube	Ampulla, infundibulum and intramural	Like rodents
Type of uterus	Rat: has duplex and bicornuate uterus having double lateral horns.; Mouse: Bicornuate uterus possessing dual lateral horns and a one body.	Simplex uterus
Histology
Type of epithelium in the oviduct	Ampulla segment diffusely ciliated	Scattered peg cells and simple ciliated columnar cells
Number of ciliated cells	Reduces as the uterine tube move towards the uterus	The ampulla and infundibulum have a large number of ciliated cells, but the intramural portion has a smaller number
Myometrium	Outer longitudinal and Inner circular smooth muscle coats	Single smooth muscular layer
Myometrial glands	Mouse: incidence differs by strain rat: absent	Absent

Table 1.

Comparison of the female reproductive system of rat and human.

Inside the uterine wall is found the oviduct’s intramuscular section which continues into the dorsolateral side of uterus wall in rodents, forming the colliculus tubarius, a modest extension into the uterine cavity. The dimension of this extension differs although it is usually a smaller number (1mm in length). Depending on uterine size, the human oviduct’s intramuscular section length varies and reaches laterally from the superior corner of the cornu until it arises from the uterus wall. A brief segment of the oviduct, the isthmus runs laterally from the uterus wall to the ampulla (Figure 1). This segment is significantly more firmly twisted in mice than in humans [5]. The ampulla is a dilated section that connects the oviduct’s infundibulum to the isthmus. From the infundibulum, the oviduct of mice grows a fimbriated side that emerges into periovarian area and the peritoneal cavity close to the ovary. In humans, one of these fimbriae joins the ovary to the oviduct.

1.2 Sexual maturation in female murine (rat and mice) compared to humans

In adult female mice, reproduction is comprised of a sequence of neurological and hormonal changes that interact to allow the creation of offspring. The connection between the anterior pituitary and ovarian hormones, as well as placental hormones, is involved in this process (during pregnancy). The hypothalamus, in particular, plays a key role in controlling the anterior pituitary’s ability to manufacture and secrete gonadotropin hormones (Follicle stimulating hormone, FSH and Luteinizing hormone, LH). FSH enhances the development of gametes in both male and female animals, whereas LH stimulates the release of estrogen and progesterone (gonadal hormones) in females as well as androgen in males. Lactation is regulated by prolactin, an anterior pituitary hormone.

Secondary sexual traits and the correct functioning of the genital tract are maintained by gonadal hormones, that also work somewhat on central nervous system to ensure effective coupling [6]. Each part develops at a varying rate, and normal sexual maturity in rats relies on a complex and poorly known interaction among them all.

Sexual maturity normally correlates with increasing circulating gonadotropin titers after 4 weeks of age. In contrast to humans, the specific moment at which maturity occurs is quite varied, therefore such a comment must be interpreted in the context of the measurement used to determine when “sexual maturity” has been attained (Tables 2 and 3). Vaginal introitus and a cornified vaginal smear, both estrogen-dependent, are the earliest detectable indications of puberty in mice and rats. Other markers of sexual maturity, such as mate readiness, the capacity to conceive and carry a litter to term, and maybe sexual maturity as measured by the ability to produce weanling-age young, have a more convoluted hormonal foundation. The vaginal opening can happen as early as day 26 and is usually complete by the 7th weeks (Tables 2 and 3). The first vaginal cornification occurs between 24 to 120 hours after the vaginal opening has been created, however this is very variable. Furthermore, estrus (the urge to mate) does not always occur on a regular timetable, which can contribute to unpredictability and make it difficult to use as an experimental tool.

Feature	Rodent (rat and mice)	Human
Puberty
Age at onset	Mouse: Approximately 4 weeks rat: 26–49 days	Almost 10–11 years, continuing for 4 years
Estradiol production by ovaries	Effect varies with strains	Causes breast and uterus development as well as pubertal developmental surge and closure of epiphyseal plate
Exposure of immature females to male urine	Can hasten puberty	Not studied
Exposure of immature females to urine from group-housed females	Mouse: be able to slow down puberty; Rat: undetermined	Not studied
Day length and temperature	Be able to affect puberty	No effect

Table 2.

Sexual maturity and ovulation in rat and human.

Feature	Endometrial phase	Mice and rats	Human
Proestrus	Proliferative	Distended and hyperemic with mitotic epithelium and presence of leukocytic stroma	Glands of tubular shapes with columnar epithelial cells. Mitotic glands and stroma
Estrus	Secretory	Full mitoses, little or no leukocytes	Columnar epithelium; more tortuous and distended glands, subnuclear vacuoles in cells at the beginning of secretory phase, after which supranuclear vacuoles followed by ultimate disappearance of the vacuoles. Under the effect of progesterone, gradually increasing epithelioid stroma ensues through the last part of the secretory phase
Metestrus	Shedding	Degenerated epithelium, uterine wall collapse, elevated amounts of leukocytes	Condensed stroma; neutrophilic infiltration and epithelial disintegration
Diestrus	Early proliferative	Uterus collapses with high leukocytes and redevelopment	Lacking

Table 3.

Difference between human endometrial cycle and mouse estrous cycle.

Regardless of how it is measured, the process of achieving sexual maturity is a very varied one. According to the timing of vaginal opening, inbred mice strains generally mature by 7 weeks of age, however some researchers have observed a median age of 35 days and a range of 26 to 49 days in C57BL/6 and BALB/c strains [7]. Vaginal opening has been discovered as early as 24 days in certain experimental animals. Two key drivers to such variety are genetic background and season. Many studies have discovered that there is some inter-strain variability. For example, Drake et al. [8] discovered that vaginal introitus occurs at a younger age in the summer than in the winter. He was able to account for a considerable percentage of the linked variation by matching a sine curve to the data and accounting for seasonal fluctuations. The season of the year also has a huge impact. Experimental exposure to cold delays vaginal opening, the first cornified smear, and the first typical estrus.

1.3 Relationship between menstrual cycle and estrous cycle

The menstrual cycle, as well as the estrus cycle, is controlled by the regular intermittent fluctuations in the mean value of estradiol (E2) and progesterone (P4) gonadal steroid hormones in endocrine fashion (Figure 2). In tissues such as the uterus and ovaries, E2 and P4 receptors (ER, PR) track the concentrations of these hormones and convert the information into timed developmental feedbacks.

Figure 2.

The 4–6 day mouse reproductive cycle (left panel) is compared to the 28-day human menstrual cycle (right panel). In this diagram, average fluctuations of estradiol, progesterone, LH, and FSH (Maeda et al., 2000).

The key integrator of both the menstrual and estrous cycles is gonadotropin releasing hormone (GnRH), a neurohormone discharged in pulsatile manner by the hypothalamus. The GnRH, when released is transported via the hypophyseal tract to the anterior pituitary gland that causes the production of FSH and LH gonadotropic hormones (Figure 3). These hormones are conveyed in the blood vessels to the ovaries to cause development of follicles and resultant production of E2. In this regard, FSH has stronger effects. During the pre-ovulatory stage, E2 confers a positive feedback effect on GnRH, FSH and LH production thereby increasing its own concentration, while it elicits a negative feedback effect during the post-ovulation period [9].

Figure 3.

Activities of reproductive hormones on the ovaries in estrus phase. The hypothalamus through E₂ positive feedback loop secretes GnRH which consequently increases FSH concentration. The FSH reaches the ovaries to cause the development of the follicles which secrets E₂. The increased E₂ levels cause the proliferation of endometrial lining and inhibition of DA. The inhibition of DA releases PRL from DA suppression. PRL maintains CL formed after ovulation. After ovulation, E₂ confer a negative feedback effect on the secretion of GnRH, FSH and LH. CL formed produces high concentrations of P₄ and low E₂ levels leading to secretory activity in oviduct and endometrium. The low E₂ concentration releases the GnRH and DA from inhibition. DA suppresses PRL leading to CL regression and consequent low levels of P₄. GnRH = gonadotropin releasing hormone, LH = luteinizing hormone, FSH = follicle stimulating hormone, P₄ = progesterone, E₂ = estrogen, PRL = prolactin, DA = dopamine and CL = corpus luteum.

Two contrasting developmental phases exist in both menstrual and estrous cycles. The first is the follicular phase (in humans) also known as pro-estrus phase (in animals). It is a preparatory stage evident by the raised E2 and P4 levels (Figures 2 and 4). These steroid hormones initiate the proliferation of the endometrium and growth of blood vessels and prime the reproductive tissues for pregnancy [10]. The second is the luteal phase (in humans) also identified as metestrus phase (in animals). It begins as E2 and P4 concentrations decline and manifests as apoptotic breaking up of the former preparations in addition to resorption of the uterine endometrium. The most outstanding peculiarity of the estrous cycle is the steady E2 surge in late diestrus to pro-estrus. This event marks the border between the two developmental phases of the cycle and signifies the beginning of a fresh cycle [11].

Figure 4.

The hormonal levels in each phase of mouse reproductive cycle. Diestrus is the end of luteal phase; pro-estrus is the beginning of follicular phase. DE = diestrus, PE = pro-estrus, E = estrus, ME = metestrus, GnRH = gonadotropin releasing hormone, E₂ = estradiol, P₄ = progesterone, PRL = prolactin, DA = Dopamine, LH = luteinizing hormone, FSH = Follicle stimulating hormone.

1.4 Difference between menstrual cycle and estrous cycle

Mice and rats go through estrous cycles, of which if fertilization failed to occur during the cycle, resorption of endometrium occurs. Humans on the other hand, experience menstrual cycle of which their endometrium is shed during the course of menstruation if fertilization and conception failed to occur. One more difference is in their sexual behavior during ovulation [12]. Female mice and rats are normally sexually receptive and active solely at some point in their estrous cycle such as during late proestrus and in estrus phase which are the phases where ovulation occurs. This is known and referred to as “heat period.” Females of species with menstrual cycle, such as humans, on the other hand, can be sexually receptive and active all over the course of their cycle. Humans also undergo covert ovulation with no visible outside displays of sexual receptivity unlike the mice. Rats, on the contrary frequently display unmistakable outward receptivity to demonstrate estral receptivity at ovulation [13].

A classic menstrual cycle in humans lasts about 28 days, with ovulation taking place at 14th day (Figure 2). The estrus cycle is considerably shorter than the menstrual cycle, occurring within 4 to 6 days [14, 15].

Additional key difference between the cycles, aside the overall period necessary for a full cycle, remains that the E2 and P4 peaks are classically disconnected in human menstrual cycle, while they intersect in rodent estrous cycle at the pro-estrus phase [16]. Rodents are furthermore disposed to estrous cycle interference from sensitivity to external or environmental cues such as light, temperature, stress and other factors than the humans are [17].

1.5 The mouse estrous cycle and hormonal changes

Hormonal changes always and frequently manifest as regular alterations in the morphology and cytology of the animal’s reproductive tract.

The estrus cycle in mice is divided into 4 distinctive phases – pro-estrus, estrus, metestrus and diestrus. The pro-estrus phase corresponds to follicular phase in humans. At about midday of the beginning of the pro-estrus phase, there exists a significant surge of estradiol (E2) triggering a fast peaking of the LH and FSH in the evening of pro-estrus and an increased progesterone (P4) secretion [11]. As in humans, the gonadotropin surge prompts ovulation and subsequent formation of corpus luteum. This high concentration of E2 inhibits dopamine (DA) and simultaneously increases the concentration of prolactin (PRL) by relieving it from dopamine inhibition (Figure 3). All hormones come back to starting levels when ovulation occurs in estrus.

The PRL and P4 levels increase markedly at the early post-ovulation phase (i.e. late estrus) and drops abruptly in metestrus phase (Figure 4). The PRL is responsible in maintaining the corpus luteum. During the estrus and metestrus phases, the corpus luteum secretes P4 and to a lesser extent E2 as well as inhibin [14]. All these hormones have combined negative feedback on the GnRH, FSH and LH [9]. By the late diestrus phase, the corpus luteum regresses due to decrease in PRL levels. This effect consequently leads to a decline in E2, P4 and inhibin levels. All these releases the hypothalamus and anterior pituitary from the negative feedback effects of these hormones [11] and initiates the start of a new phase

1.6 Characterization of menstrual cycle using different phases of estrous cycle

1.6.1 Pro-estrus phase

Pro-estrus phase lasts for 24 hours (approximately 1 day) in mice and rats [15]. This phase parallels the pre-ovulatory day of menstrual cycle. For example, E2 concentration rises and confers a positive feedback mechanism on GnRH release. At mid pro-estrus phase, E2 concentration reaches its peak to induce the LH and the FSH surge resulting in ovulation while it confers an inhibitory effect on DA to release PRL suppression [17]. During this phase, P4 and PRL levels begin to rise (Figure 4). One or several follicles of the ovaries start(s) to grow and mature (Figure 3).

The main characteristics of vaginal cytology of this phase is the existence of large or small, rounded epithelial cells that may be nucleated or enucleated. The cells are fairly of uniform appearance and size. They are usually seen in cohesive sheets, clusters, or strands. The appearance might sometimes not be observed particularly in hypocellularity samples, therefore should not be taken as a yardstick in determining pro-estrus. Sometimes no leucocytes will be seen, however, leucocytes can be found in early pro-estrus.

Moderately low amounts of large epithelial cells plus cornified cells may as well be detected. As the cycle nears estrus, abundant cornified cells will be present (Table 4). The presence of high amounts of cornified cells or low numbers of leucocytes should not impede the identification of pro-estrus especially when the usual features of the smear are the small, round epithelial cell population. There might be presence of the secretion in the smear. Visual observation of the vulva will reveal wide opened vagina which will be moist, and the tissues appears deep pink or red. Striations will be seen in both the ventral and dorsal lips of the vulva. The lips may appear swollen.

Cycle phase	Duration (h)	Behavior	Vaginal smear morphology
Proestrus	14	At the end of this phase male acceptance start	Mostly nucleated and non-nucleated epithelial cells (75%) present
Estrus	25–27	Lordosis; sexually receptive	75% cornified cells; 25% nucleated and non-nucleated epithelial cells
Metestrus	21	Not sexually receptive	Many leukocytes (50%) with nucleated and cornified cells
Diestrus	55–57	No male acceptance	Leukocytes (85–100%)
Cycle phases

Table 4.

Vaginal smear cytology and mice behavior during estrous.

1.6.2 Estrus phase

Estrous phase lasts between 12 - 48 hours (approximately 2 day) and signifies the beginning of the luteal period in humans [18, 19]. Estrus is the stage when the female mice remain sexually receptive (“on heat” or “in heat”). Under regulation of gonadotropic hormones, estrogen secretions exert their biggest influence. Throughout the morning of estrus E2 levels remains elevated conferring inhibitory effect on DA. The action of high E2 on DA releases PRL from DA suppression. PRL is important in maintenance of CL after ovulation. During the afternoon, E2 falls back to basal levels and the P4 concentration on the other hand rises and peaks (Figures 3 and 4). The P4 concentration is important for the secretory activities in the endometrium. The high level of P4 with corresponding low concentrations of E2 inhibits GnRH and FSH and releases the DA from suppression (Figure 4). DA then inhibits the PRL causing CL to regress which in turn decreases the concentration of P4 at late estrus.

This phase is typically recognized by the presence of abundant cornified squamous cells. These cells appear in clusters and are of irregular shapes. They have no visible nucleus, and their cytoplasm looks granular. Abundant bacterial cells may be seen. The bacterial cells may stick to the cells or appear freely in the background of the smear. During early and mid-estrus phase, no leucocytes are observed however, can be seen during late phase. There will be predominance of cornified cells (75%) with epithelial cells (<25%) (Table 4). Cells can appear in clusters or scattered. During physical visual inspection, the vagina will appear similar to pro-estrus, but it will be less pink or red, less moist but striations might be very prominent for some animals. The vulva lips will be swollen.

1.6.3 Metestrus phase

Metestrus phase marks the mid of luteal phase (post-ovulation) in humans. During this phase, the signs of E2 and P4 stimulation subside. The decline in the plasma levels of P4 stimulates the hypothalamus to secrete GnRH and consequently the gonadotropic hormone, FSH. The FSH in the ovaries starts the development of preantral follicle which secretes E2 (Figures 3 and 4). The Low levels of P4 also lead to resorption of endometrial lining. This phase typically is brief occurring within 24 hours [18] or may last for 1–3 days [16], personal experience in our lab, data not published. In this stage, there is presence of leucocytes in combination with few cornified squamous epithelial cells (Table 4). In early metestrus, leucocytes are sometimes interspersed or may be tightly clumped together around the cornified cells; the leukocyte cells may equal the quantity of the cornified cells or may be less. At mid-metestrus, leucocytes become higher in number than the cornified cells [14] and the smear might be extremely dense and cellular. By late metestrus, the number of cornified cells decreases with a correspondingly reduction in smear cellularity displaying conversion to diestrus phase. Visual observation of the vulva is characterized by pale coloration. The vaginal canal will be completely sealed and dry. It may additionally be sloughed with white cellular debris.

1.6.4 Diestrus phase

This phase marks the boundary between luteal phase and follicular phase and lasts between 55–57 hours (approximately 3 days) (Table 4). If pregnancy fails to occur, the diestrus stage ends with the recession of the CL. In the late stage of diestrus, the hypothalamus through E2 positive feedback loop secretes GnRH and consequently FSH (Figures 3 and 4). The FSH in the ovaries causes the development of the follicles from preantral follicle to early Graffian follicle which secretes E2. The increased E2 levels cause the endometrial lining to begin to proliferate. The vaginal cells consist predominantly of leucocytes at the early phase and with nucleated cells but no cornified cells at the late phase. Visual observation will reveal vaginal opening which may be actually moist. The orifice will be slightly opened or totally closed in some animal. There will be no tissue swelling or striation.

1.7 Importance of rodent (mice and rats) in research

Because better molecular tools to modify the mouse genome were available in the past, the mouse was frequently used instead of the rat. Recent improvements in genetic methods for creating knockout rat models promise to break down these obstacles, potentially allowing rats to be used in a wider range of scientific studies. In the end, the rodent model of choice is determined by which species best mimics the symptoms and illness process seen in humans. It’s evident that rats aren’t just big mice, and that each species has advantages and disadvantages that vary depending on the process or gene under investigation. It is very important to adopt the right paradigm for translational medicine because a lot of money is spent exploring medications and cures that ultimately fail at various levels of pre-clinical and clinical trials. One explanation for this is that animal trial results do not always correctly represent human outcomes.

1.8 Limitation of mice as an ideal model of human physiology and disease

When comparing mice and humans, there are several variances in developmental timing and reproductive organ shape, as well as discrepancies in their metabolism. Poly-ovulation and a brief period of gestation period in mice, for example, are important variations from humans. Unlike humans, mice are multiparous species such that exposure to endocrine disrupting compounds during gestation has complicated effects on the offspring. Morley-Fletcher and colleagues [20] showed that during sexual differentiation, exposure to sex steroids gave varied results relative to hormone exchange between the female and male fetuses in mice uterine horns.

There are further differences in reproductive function to consider when comparing humans to other mammals like mice. Human oogenesis determinants, for example, are far more intricate than those of most other mammalian species, including mice. When it comes to studying human reproductive disorders, rodent species/strains have several important limitations. Except in rare strains with certain genetic backgrounds, there are still no germ cell tumors (primary ovarian tumors) in the ovaries of mice or rats; yet it is a common human malignancy in young adult females [21]. It’s also worth noting that the outcomes responsive to the effect of repro-toxicants differ amongst mice and rat strains even within the same species. In responding to in utero exposure to a particular phthalate (dibutyl phthalate (DBP)), Wistar rats show elevated incidence of cryptorchidism and reduced rates of epididymal hypoplasia than Sprague-Dawley rats [21].

1.9 Criteria for estrous cycle staging

Changes in estrous cycle can only be effectively predicted and identified if the investigator is adequately acquainted of the continuous variations and appearance in the vaginal cytology throughout the several stages of the animal’s reproductive cycle (Table 5). There are numerous reports on the appearance of normal reproductive tract of all the different phases of the estrous cycle [14, 16], nevertheless, to attain consistency in method of staging the cycle, the following criteria should be followed.

1. To clarify the interval as well as the stages of the reproductive cycle, cytological samples of the vaginal cells should be collected for a minimum of 14 successive days (can also be done for up to 19 or 21 days). It has to be prepared at a particular time of the day preferably in the early periods of the morning between 7 am and 12 noon [15]. This is vital as Certain short phases may be “missed” particularly if samples are not consistently gathered for instance, in smears harvested too late or very early in the day, pro-estrus phase might be missed. Irrespective of what time the samples are harvested, they must be prepared at about same matching period of the day. This must be maintained throughout the collection period to diminish inconsistency. Vaginal lavage method is ideal for collection of the samples.

2. The typical interval of the estrous cycle is commonly between 4–6 days for 60%–70% of the sample population. However, some animals may present longer regular or irregular cycles.

3. A mouse is considered as cycling regularly, when it presents at least two repeated cycles of the same interval within the 14 days. In other cases, cycling can be determined by checking if estrus immediately follows pro-estrus [22]. This should be the case when ascertaining cycling in mice as they exhibit more varied cycling patterns and can lead to erroneous conclusions.

4. Two repetitive cycles with 1–2 days of estrus, 1 day of metestrus, 1 day of pro-estrus or any with only a day of estrus and 2 to 3 successive days of diestrus in each cycle will also be considered regular.

5. Cycles will be considered ‘extended’ if there is 4–5 days of estrus or 4–5 days of diestrus.

6. Irregularities in cycle length or cycle contiguity is greater in mice than rats i.e., 4 days followed by 5 days instead of 4 days and vice versa [23, 24] although irregular cycles are predominant with aged animals (6 months and above).

7. Cycles, in which the alternations between the phases do not follow the sequence pro-estrus to estrous specifically, will be considered irregular. They may not necessarily follow the consistent pattern of Pro-estrus, estrous, metestrus and diestrus to conclude as regular. This position is especially advised for mice. Irregular cycles may be characterized by placing them in one group (or they may be excluded from the experiment).

8. Animals that are pseudo-pregnant before administration should be eliminated from the experimental protocol. Such animals will exhibit persistent estrous or diestrus. Constant light causes persistent estrus and failure to cycle while housing in large groups causes persistent diestrus [25]. Nonetheless, mice in persistent diestrus will synchronize upon exposure to male urine.

9. If the outcome of drug or extract/compound expression on the cycle is even in a set (e.g., consequential persistent diestrus or estrous), so this may provide a valuable summary of the data. Conversely, if some animals exposed to the compound or extract display persistent diestrus, while others exhibit prolonged estrous, then such outcomes may possibly not reveal the alterations caused by the test compound. Therefore, the discrepancy in collection may perhaps result in a general belief that reproductive cycle remained unaffected. The procedure should be repeated in such cases.

	Mice and rats	Humans
Length	Roughly 4–6 days of estrous cycles except when disrupted by pregnancy	About 28 days
Onset	Starts between 21–26 days after birth	Starts at puberty (10–13 years old) until menopause (40–50 years)
Phases	Metestrus, diestrus, proestrus and estrus,	Proliferative (follicular) Menstruation, and secretory (luteal)
Follicle development/cycle	Numerous follicles mature concurrently	Several follicles (1–12) start developing, only one or two follicle(s) become(s) dominant
Ova released at ovulation	Multiple	Normally one or two ova per menstrual cycle

Table 5.

Similarities and differences in estrous cycle in mice and ovarian cycle in humans.

1.9.1 Vaginal lavage or smear protocol

The vaginal smear technique for describing the phases of the estrous cycle was initially probed in 1917 by Stockard and Papanicolaou [26, 27]. A slight adjustment of the method is implemented for this procedure, but it essentially involves the recognition of the cell categories and their relative amounts existing in the sample gotten from flushing walls of the vagina. Ascriptions of the features to explicit phases of the estrous cycle were attributed and recorded by Elvis-Offiah and Bafor [28].

1.9.1.1 Preparations

1. Normal saline: use normal saline for intravenous drips or weigh 0.9 g of NaCl (MW 58.44) powder and dissolve in 100 mL distilled water. Thereafter, stocked in a securely air- and watertight vessel at room temperature till required.

2. Slides: clean and label the glass slides according to animal’s number and groups.

3. Methanol: Cold 100% methanol is recommended for fixing cells. Though 95% ethanol from absolute ethanol (≥99.8% GC) can also be used. It can be prepared by measuring 95 ml of absolute ethanol into a calibrated cylinder and adding 5 mL of distilled water to make it up to 100 mL.

4. Stains: monochromatic stains are recommended. These include Wright-Giemsa stain, methylene blue, eosin, and crystal violet (5%). For RHERG, we will be using methylene blue/eosin staining. Prepare 0.1% methylene blue in a separate bottle and 0.1% eosin is also prepared separately.

1.9.1.2 Collecting vaginal cells (vaginal lavage)

1. Vaginal smears should be steadily collected daily between 9 am and 12 noon by washing out the vaginal epithelium with normal saline using a disposable Pasteur pipette. The tips of the pipette to be used must compulsorily be even and pointed with an endorsed inner tip bore of ≥1.5 mm for pipette tips (OECD n.d.) [29]. The practice of using a new dropper or tip for each animal is best. Should a lone dropper or tip be employed on different animals, it must be meticulously cleaned with fresh sterile or distilled water. Thorough cleaning is appropriate to avoid sample contamination between animals which may result in erroneousness staging or, occasionally, reproductive tract infection.

2. Draw around 0.2 mL (rats) or 0.1 mL (mice) of saline water into the dropper or pipette.

3. Pick up the animal from the cage to the cage lid (hopper) with its rear end placed in your direction. Grasp the tail firmly and raise the hind feet. The animal will use merely her forelimbs to grasp the hopper. On the spot, urination may ensue. If so, pause till urination ends. It is advised to clean the vaginal orifice with the saline and with a different tip should urine is left at the vaginal canal opening.

4. The dropper tip should be carefully and softly inserted without penetration into the vaginal opening. At that point, the vaginal wall should be flushed with the saline 2 or 3 times. Draw little amount of vaginal fluid out. If the fluid is milky at the initial draw, then successive flushing is not needed (OECD n.d.) [29]. Inserting the tip too deep into the vaginal orifice may stimulate the cervix to induce pseudo pregnancy therefore care must be taken. Pseudo pregnancy may appear as persistent diestrus or estrus for up to 14 days [11].

5. Gently transfer the fluid comprising the cells on a clean, pre-labelled glass slide bearing the appropriate animal’s identity.

6. Allow the smears to air dry then fix with 100% cold methanol for 5–10 minutes on the laboratory bench and allow to dry. Drying can be done by air-drying or by placing on heated surface set at 50°C.

7. Add one-two drops of methylene blue (0.1%) on the slide for 3 minutes. Remove surplus stain by placing a piece of filter paper, cotton wool or any absorbent for a second. Afterward, add 2 drops of eosin (1%) on the slide for 2 minutes. All work must be carried out at room temperature. Avoid heavy staining.

8. Rinse the slide briefly and appropriately with small amount of tap water and keep in standing position to dry.

9. View the slides under light microscope starting from the least eyepiece magnification. Koehler illumination: this gives optimal resolution and contrast [30].

2. Conclusion

Both in humans and rats, the female reproductive system and processes are dynamic, changing morphologically and cytologically in response to hormonal cues during the course of the estrus or monthly cycle. Along with menopause and ovarian age, these changes are also seen during pregnancy. For preclinical evaluations of human reproductive physiological and pathological processes, mice and rats are ideal models. They are frequently used in research due to their small size, simplicity of housing, and maintenance requirements. Their reproductive cycle oscillates periodically because to variations in ovarian steroid concentrations, just like in humans (estrogens and progesterone). However, there are morphogenetic, physiological, and anatomical differences between the mouse and human reproductive systems.