Estrus Physiology and Potential of Extracellular Vesicular miRNA as Biomarkers: A Theoretical Review

Abstract

Timely estrus detection is one of the critical factors for increasing reproductive efficiency in animals. Estrus physiology is under the influence of the endocrine signals that include a network of miRNAs. EV miRNAs are more stable than the other cell free miRNAs as they are doubly protected from endogenous RNase activity by means of cellular packing within the membrane-enclosed structures. Review of literature indicated the differential expression of miRNA at the estrus stage and other stages of the estrous cycle in various biological fluids, the role of miRNAs in oviductal function as well as their relation to the dynamics of preovulatory sex-steroid concentration or vice-versa by influencing the genes of miRNA biogenesis pathway. Interestingly, overlapping expression of miRNAs between tissues and EVs released from tissue fluids, as well as unique and differential expression of miRNA between bodily fluids and EV fractions of biological fluids has been identified. Studies focusing on the miRNA secreted in easily accessible urinary extracellular vesicles during the estrus stage in relation to the endocrine profile may pay the way for the identification of biomarkers for detecting estrus.

1. Introduction

Estrus is the stage of the estrous cycle when the animal shows sexual receptivity to the male, which is termed standing estrus [1]. Identifying females in estrus aids in either natural service by males [2] or artificial insemination (AI) [3]. During the estrus stage, the animals can facilitate the transport of spermatozoa through the uterus. The reproductive system of estrus animals will be in optimal environmental conditions, close to ovulation which follows the estrus stage, such that the animal can become pregnant. Detection of estrus in animals is also considered as an important prerequisite in the process of adoption of several protocols of estrus and ovulation synchronization, use of sexed semen, which are attempted to improve reproductive efficiency in the recent years [4, 5].

Estrus is dynamic, and the estrus cycle is regulated by interplay of the different organs and hormones inclusive of complex biological systems [6]. The onset of estrous cycles associated with the cyclic ovarian activity in animals occurs at the time of puberty, during which period, via the hypothalamic-pituitary-gonadal (HPG) axis, gonadotrophin releasing hormone (GnRH) stimulates the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) to induce steroid production and oogenesis in ovary. Multidirectional interactions between the oocyte, granulosa cells (GCs), theca cells and the HPG axis are crucial for all the complex developmental transitions of folliculogenesis [7, 8]. Ovarian cyclic activity/estrous cycle is a dynamic period between two consecutive ovulations and consists of a luteal phase (metestrus and diestrus) and a follicular phase (proestrus and estrus). Additionally, there are differences across species in the length of the heat and the estrous cycle [9].

Estrus physiology is influenced by the endocrine signals that also include a network of miRNAs [10, 11, 12]. Studies have indicated that, the miRNA present in biological fluids can indicate physio-pathological states. Extracellular vesicles (EVs) are found to be associated with cellular and molecular processes of ovarian cyclic activity [13]. Additional protection of miRNAs or proteins of EVs from degradation by endogenous RNase and protease activity [14, 15, 16, 17, 18] make them a better basis for identifying physio-pathological biomarkers, when compared to the cell free miRNA and/proteins. Further, a deeper comprehension of the patterns of follicle development in various species is necessary to create more efficient methods to manage domestic animal fertility. Hence, this chapter attempts to cover in detail the physiology of estrus, role of miRNA in estrus associated events, and the potential of extracellular vesicular miRNA as biomarkers to identify animals in estrus stage.

2. Physiology of estrus

Ovaries are critical organs of the female reproductive system associated with follicular development, ovulation, formation, function and subsequent regression of corpus luteum (CL). An adult ovary contains follicles at various developmental stages [19] with intensely coordinated cellular processes controlled by timely and spatially expressed genes and gene products [10, 12]. These cellular processes not only drive the folliculogenesis [7, 8, 20, 21, 22, 23], but also the synthesis of ovarian steroid hormones which are important for estrus behavior [24, 25, 26].

2.1 Cellular processes associated with ovarian cyclic activity and endocrinology of estrous cycle

Studies on estrus cyclicity [27], cellular interactions during folliculogenesis [7, 8], follicular development in a wave-like fashion [28, 29], cellular and molecular processes occurring during folliculogenesis [30] have been put forth. FSH (Follicle stimulating hormone) and LH (Leutinizing hormone) signaling in the follicular cells are not only crucial for steroidogenesis [26, 31] and cellular signaling mechanisms for the ovulation to occur [21, 23, 24, 32, 33, 34, 35, 36, 37], but also for driving estrus behavior in animals [38, 39] by guiding the biosynthesis of E₂ and P₄ at the estrus stage [27, 40, 41].

2.1.1 Ovarian follicular development and estrous cycle

Follicular development occurs in a wave-like fashion with two waves [28] or three waves [29] as the most common pattern [32, 33]. GCs of small growing follicles secrete inhibin and acquire receptors for FSH (FSHR). Further, it has been put forth that, each follicular wave begins with a transient FSH surge in the circulation, and the loss of dominance and the end of the follicular wave that does not result in ovulation are followed by a decrease in LH secretion [34, 35]. FSH controls the development of GCs by stimulating their proliferation and differentiation, and promoting the formation of the follicular antrum [21]. Subsequently, the dominant follicle acquires luteinizing hormone/choriogonadotropin receptor (LH/CGR) in its GCs, secrete more E2 than the subordinate follicles [24], triggering the LH surge that allows them to develop into the preovulatory follicle [23].

Various cellular processes like angiogenesis, steroidogenesis, basement membrane turnover, oocyte growth and maturation and follicular atresia [30] occur during folliculogenesis. Also, antioxidant system/reactive oxygen species (ROS) is found to modulate the events of folliculogenesis, ovulation, formation and activity of the CL and luteolysis [36]. Further, within the preovulatory GCs, initiation of the transcriptional upregulation and downregulation of genes, including cytokines, transcription factors (TFs) and matrix-remodeling proteins (MMPs) is caused by the LH surge [37]. However, the exact molecular mechanisms involved in the initiation of the growth of the primordial follicle and the further development of primary follicles up to the pre-ovulatory stage remains unknown [42, 43].

2.1.2 Biosynthesis of ovarian steroid hormones

The ovarian steroid hormones, progesterone (P₄) and estradiol 17β (E₂) are synthesized from cholesterol through the cooperative interactions of theca and GCs [26, 31]. LH binds to LH/CGR on the theca cell surface and stimulates the expression of the steroidogenic enzymes [steroidogenic acute regulatory protein (STAR), cholesterol side chain cleavage enzyme (CYP11A1), 3β-hydroxysteroid dehydrogenase (3BHSD), 17α-hydroxylase/17,20desmolase (CYP17A1)] necessary for androgen production. Pregnenolone synthesized from cholesterol gets converted to P₄ by the action of 3BHSD. P₄ then gets converted to androstenedione by the action of CYP17A1. Androstenedione produced by theca cells diffuses into GCs and gets converted to E₂via FSH signaling through FSHR and stimulates the expression of enzymes [17βhydroxysteroid dehydrogenase (HSD17B), aromatase (CYP19A1)] in GCs.

Though the gonadal hormones (P₄ and E₂) are important for estrus behavior, their circulatory levels are actually under the control of GnRH, FSH, LH and inhibins which have an important role in determining the expression of heat signs. Further, circulatory concentrations of FSH and LH were highest on the day of estrus compared to the other days of the estrous cycle [27, 40, 41].

2.2 Differential expression of mRNA and proteins during various stages of estrous cycle

Studies on differential expression of mRNA transcripts [44, 45, 46], heat shock proteins [47, 48, 49, 50] and other proteins [44, 51, 52, 53, 54] in various biological fluids and/or tissues collected at different stages of estrous cycle have been reported and the use of the same as biomarkers for the identification of estrus has been opined.

2.2.1 Differential expression of mRNA transcripts at estrus stage

The transcripts of lactoferrin (LF) and glutamate receptor-interacting protein 1 (GRIP1) were highly expressed in the uterine tissue of cattle during the estrus when compared to other stages of the estrous cycle [44]. Likewise, higher expression of mRNA transcripts of HSD17B1 and heat shock 70-kDa protein 1A (HSPA1A) in saliva during the estrus stage in both cyclic heifers and pluriparous buffaloes was reported [45]. In yet another study, it was reported that the mRNA transcript of tissue inhibitors of metalloproteinase 1 (TIMP1) was significantly over-expressed in cell free saliva at the estrus stage when compared to the diestrus stage in buffaloes [46].

2.2.2 Heat shock proteins (HSPs) as estrus indicators

Higher expression of heat shock protein-27 (HSP-27) in porcine endometrium during the estrus stage compared to other estrous cycle stages was demonstrated [47]. Likewise, higher expression of heat shock protein-70 (HSP-70) in cervico-vaginal fluid (CVF) was identified during the estrus stage when compared to the diestrus stage in buffaloes [48]. In yet another study conducted in sheep, higher expression of several HSP family proteins was identified in the luminal fluid samples collected from inner cervix, uterus, and oviduct during estrus stage when compared to other stages of estrous cycle [49], among which, heat shock protein-105 (HSP-105), heat shock protein-90-β (HSP-90-β) were abundant in cervical mucus, while heat shock protein-90-α (HSP-90-α) was expressed abundantly in both the uterine fluid as well as the cervical mucus. Further, the heat shock protein (HSP) family proteins (HSPH1, HSPAA1, HSP90AB1, HSPB1, HSPA4, and HSPA8) were found to be highly expressed during the estrus stage [50].

2.2.3 Differential expression of other proteins at estrus stage

Specific expression of β-enolase (ENO3) and TLR 4 (Toll like receptor 4) proteins in the saliva of buffaloes at the estrus stage was identified [51]. So also, some of the proteins that were discovered to be overexpressed in the saliva of buffaloes during the estrus stage included heat shock 70-kDa protein 1A (HSPA1A), lipocalin 1 (LCN1), odorant-binding protein (OBP), leukocyte elastase inhibitor (SERPINB1), vitelline membrane outer layer 1 (VMO1), 45-kDa calcium-binding protein (SDF4), and ENO3 proteins [54]. Likewise, LF and GRIP1 proteins were highly expressed in bovine cervical mucus during the estrus stage compared to other stages of the estrous cycle in cattle [44].

Further, the proteins, cullin-associated NEDD8-dissociated protein 1, HSP701A, HSD17B type 1, inhibin beta A chain, and testin were identified to be estrus specific by proteomic analysis of buffalo saliva using in-solution digestion and nano-Liquid Chromatography-Mass Spectrometry (LC-MS/MS) [52]. In yet another study, higher expression of LH in saliva was reported during the estrus stage compared to other estrous cycle stages in buffaloes [53].

The above studies on differential expression of mRNA transcripts, HSPs and other proteins during estrus, when compared to other stages of estrous cycle when compared to other different stages of the estrous cycle as well as unraveling their relationship to the cellular processes associated with ovarian cyclic activity and/or the regulatory mechanisms involved in the differential expression of the same, may pave the way for the identification of a biomarker for estrus in animals.

2.3 Role of microRNA (miRNA) in estrus associated events

Studies on the role of non-coding RNA, especially the miRNA [55, 56, 57, 58, 59] associated with ovarian function as well as female reproduction [11, 60, 61, 62, 63, 64], regulatory mechanisms of miRNA on expression of genes associated with ovarian function [65, 66, 67, 68, 69] have been postulated.

MiRNA, circular RNA (circRNA), long non-coding RNA (lncRNA), small interfering RNA (siRNA) and PIWI-interacting RNAs (piRNA) constitute the non-coding RNAs [11, 70]. Among the non-coding RNAs, the role of miRNAs in female reproduction, primarily associated with the function of the ovary has been explored widely in the last decade [11, 62, 63, 64]. The role of miRNA on ovarian functions, the regulatory role of miRNA in maintaining the ovarian function, cyclicity and oocyte maturation has been postulated [11].

Earlier, miRNA was termed small temporal RNAs (stRNAs) for their sequential expression at specific times and regulation of various developmental events in Caenorhabditis elegans [71, 72]. In 2001, these small non-coding RNAs were given the term miRNAs [73, 74]. MiRNAs are found to be strongly conserved between vertebrates, invertebrates, and plants [65]. The miRNAs are 20–22 nucleotide long single stranded non-coding RNA molecules which have an important role in regulating gene expression by promoting mRNA degradation (via direct cleavage or by mRNA deadenylation) or preventing translation [66]. Further, the miRNAs are transcribed from individual genes, sometimes clustered and located intergenic or in introns or exons of protein-coding genes [67].

Various in vitro and/or in vivo studies have been conducted to identify the miRNA associated with follicular/luteal development in the mammalian ovary. Further, the analysis of whole ovaries was very useful for comprehensively identifying miRNA sequences. Study of small RNA population in the ovaries of humans and other species using molecular techniques like microarrays, high-throughput quantitative polymerase chain reaction (PCR) and next-generation sequencing (NGS) [55, 56, 57, 58] has revealed that miRNAs constitute the most abundant class of small RNAs in the ovary.

Regardless of the species, it was discovered that the let-7 family miRNAs miR-21, miR-99a, miR-125b, miR-126, miR-143, miR-145, and miR-199b were the most prevalent miRNA populations in the ovary [61]. Likewise, the study of the expression profiling of miRNA in pigs [58], mice [57], cows [75] and sheep [76] has confirmed the role of miRNAs in the different functions of ovary. In yet another study, miR-21, miR-143, let-7 family, miR-26a and miR-125b were identified as highly abundant miRNAs in mammalian ovaries as revealed by cloning based technologies or NGS, while the miR-21 has been identified to promote follicular cell survival during ovulation and the miR-17-5p and let-7b were identified as pro-angiogenic and therefore opined to be essential for the development of CL [60].

Above all, specific sets of miRNAs were found to be expressed within the ovary and are found to strictly regulate the gene expression patterns in ovary spatiotemporally [59]. Further, various genes involved in follicular development, oocyte maturation and implantation have been reported to be regulated post-transcriptionally by miRNAs [68, 69]. In yet another study, it was reported that gene ontology (GO) analysis and bioinformatic screening revealed that the targets genes of predominantly expressed miRNA in the mammalian ovaries were associated with a number of biological pathways or molecular networks, including cellular growth, development, and proliferation, cell to cell signaling, cell cycle regulation, cell death, endocrine system disorder, and various pathways underlying ovarian functions [56].

Therefore, miRNA appears to be involved in regulating estrus-associated events. Hence, the literature on the miRNA associated with ovarian cyclic activity, biogenesis and nomenclature of miRNA is being reviewed below.

2.3.1 Biogenesis of miRNA

MiRNAs are generated from its precursors, i.e., long strands of RNA called primary RNA (pri-miRNA). Pri-miRNA upon cleavage by the enzyme Drosha, a nuclear RNAse III endonuclease and simultaneous binding of RNA-binding cofactor, DGCR8 (DiGeorge syndrome critical region gene 8) leads to the formation of hairpin shaped precursor miRNA (pre-miRNA) within the nucleus which is of 70–100-nucleotide (nt) in length [77].

This Pre-miRNAs get transported out of the nucleus into the cytoplasm by exportin 5 [78]. The RNAse III endonuclease Dicer breaks down the pre-miRNAs in the cytoplasm, decreasing the hairpin loop and creating a duplex miRNA. The final active miRNA is created when the miRNA-induced silencing complex (miRISC), which is composed of proteins from the Argonaute (AGO) family among others, attaches to one of the strands of this duplex miRNA. This complex then binds to the complementary 3′ or 5′-untranslated region (UTR) of the target mRNA [79, 80], open reading frames (ORF) or promoter regions [81] and exert their effects. This miRNA biogenesis pathway involving Drosha/DGCR8 is universal to all mammalian miRNAs and is known as the “canonical” pathway of miRNA biogenesis which is depicted in Figure 1.

Figure 1.

The canonical pathway of miRNA biogenesis (adapted from [82]).

2.3.2 Nomenclature of miRNA

Based on the discovery sequence, the initial nomenclature was followed for miRNA [83]. For example, miR-3 was the third miRNA to be discovered using the guidelines. The let-7 and lin-4 are exceptions to the numerical naming rule, whose original names were given based on their historical significance (www.mirbase.org). Lin-4 and let-7 were the first two miRNAs originally discovered in the nematode, Caenorhabditis elegans and control the timing of stem-cell division and differentiation [71, 84]. With the increasing research on the role of miRNAs in disease and health, the list of miRNAs is becoming large and many other publicly available miRNA databases are now available [85].

The prefix added to the specific miR signifies the species (hsa, human; mmu, murine; bta, cow, etc.). For example, bta-miR-21 refers to cow miR-21. Suffixes have also evolved over time. A number placed after the miRNA name (i.e., miR-218-1 or miR-218-2) indicates the exact same mature miRNA sequence. Still, it suggests that it was derived from independent gene loci (in this case, human chromosomes 4 and 5, respectively).

The passenger strand of a miRNA duplex, which is less frequent and is thought to have no biological purpose, was previously mentioned and was denoted by an asterisk (*). However, it has been established that the guide miRNA strand may not always be present or as physiologically active as the miRNA* in some cells or tissues. The * has been replaced by a -5p or -3p mark. The -5p/-3p suffix is a critical differentiator since these miRNA target very diverse groups of mRNAs based on their various seed sequences (i.e., bases 2–8 from the 5′ end of the mature miRNA).

The bases used by the majority of algorithms to find possible targets are assumed to be the main bases that direct the mi-RISC to certain mRNA targets. A note (e.g., miR-34a, miR-34b, etc.) can be included after the miRNA name to identify two miRNAs with closely comparable sequences and 100% homology in the seed sequence.

2.3.3 MicroRNA associated with ovarian cyclic activity

The role of miRNA related to endocrine regulation of ovarian cyclic events has been postulated [12, 26, 60, 86, 87, 88, 89, 90, 91, 92].

The miRNA regulating aromatase expression (encoded by CYP19A1 gene) during follicle development include miR-224, miR-378 and miR-383 [60]. So also, miR-378 was found to suppress the E₂ release in swine GCs by targeting the CYP19A1 gene [87]. In yet another study, it was revealed that, in vivo injection of miR-320 in mouse was associated with decreased E₂ levels by targeting the transcription factor (TFs) genes, elongation factor 1 (E2F1) and steroidogenic factor 1(SF-1) [90]. While, miR-764-3p was found to target the gene SF-1, thereby suppressing E₂ release in mouse [92]. In contrary to the above findings, various studies conducted in mouse have revealed that miR-224, miR-383, miR-133b and miR-132 can suppress the E₂ release in cultured GCs by targeting the genes SMAD4, RBMS1, FOXL2 and NURR1 respectively [86, 88, 89, 91].

Therefore, the biosynthesis of the two main ovarian steroid hormones, E₂ and P₄ is regulated in the ovary by miRNA at all levels that includes (1) transcription of genes (STAR, CYP11A1, 3B-HSD-II, SR-BI, 17B-HSD-I, CYP19A1, RBMS1, FOXL2, RUNX2) coding for essential enzymes (2) direct post-transcriptional regulation of the enzymes involved in hormone biosynthesis and (3) expression of enzymes that catalyze the conversion of these hormones to inactive metabolites [26].

Further, the role of miRNA in follicular atresia [93] and the miRNA transcriptome dynamics of different stages of CL during an estrous cycle [94] has been postulated. Notable miRNA families and clusters that have been functional during the process of follicular atresia, which is mainly indicated by GC apoptosis include the let-7 family, miR-23-27-24 cluster, miR-183-96-182 cluster and miR-17-92 cluster [93]. Many researchers have applied the in vitro gain- and loss-of-function studies to explore the effective functions of miRNAs during atresia. The use of small RNA sequencing technology for studying the miRNA transcriptome dynamics at different timely defined CL classes using ovaries collected from cycling German Fleckvieh cows covering the entire physiological estrous cycle revealed that, bta-miR-143 expression reached its peak in the regressed CL (rCL, days >18 of the estrous cycle), whose expression was significantly downregulated in the early CL (eCL, day 5–7 of the estrous cycle). In addition, regardless of CL developmental or functional status, bta-miR-21-5p and bta-miR-143 were found to be abundantly expressed [94].

Above all, differential expression of miRNA in GCs of preovulatory dominant and subordinate follicles [95], the housekeeping role of miRNA in the ovary during anestrus stage [56, 95, 96, 97, 98, 99, 100] was documented. When compared to the subordinate follicles, the preovulatory dominant follicles significantly downregulated the miR-17-92 cluster, bta-miR-409a, and bta-miR-378, according to a study of the expression pattern of miRNAs in GCs of bovine preovulatory dominant and subordinate follicles during the late follicular phase of bovine estrous cycle [95]. This suggests that miRNAs may play a role in the post-transcriptional control of genes involved in bovine follicular development during the late follicular phase of the estrous cycle. This is supported by the unique sets of miRNAs found in the GCs of preovulatory dominant and subordinate follicles.

Further, among the highly abundant miRNAs expressed in the ovary across the estrous cycle in various species [56, 96, 98, 99], the miRNAs, viz., bta-miR-10b, bta-miR-26a, bta-miR-27b, bta-miR-30d, bta-miR-30a-5p bta-miR-92a, bta-miR-99b, bta-miR-125a, bta-miR-143, bta-miR-148a, bta-miR-186, bta-miR-191, bta-let-7a-5p, bta-let-7f and bta-let-7i, were found to be expressed irrespective of the stage of follicular development [95, 97]. Therefore, the housekeeping role of the afore mentioned miRNAs in maintaining the normal physiological processes in mammalian female reproduction was opined [100].

In yet another study, the acquisition and combinatorial analysis of the databases of miRNA, circRNA, lncRNA, and mRNA obtained from the ovary of estrus synchronized Xinong Sannen goats in estrus and diestrus stages of the estrous cycle revealed the following [98]: (a) Differential expression of miRNA, circRNA, lncRNA (non-coding RNAs) and mRNA (coding RNA) at estrus and diestrus stages was evident. (b) Screening of differentially expressed (DE) non-coding RNAs and coding RNAs illustrated their regulatory role in ovary to maintain the homeostasis. (c) As taken from the network, differentially expressed miRNAs and mRNAs that are important in controlling the estrous cycle include miR-21-3p, miR-202-3p, and miR-223-3p. (d) Tissue inhibitors of metalloproteinases (TIMP1), matrix metallopeptidase 9 (MMP9), 3-hydroxysteroid dehydrogenase (3BHSD), and prostaglandin I2 synthase (PTGIS) were chosen from the differentially expressed mRNAs in order to screen the differentially expressed miRNAs and circRNAs that may have regulated their expressions by creating circRNA-miRNA-mRNA networks. (e) Differentially expressed miRNAs were found to target the mRNA of TIMP1, 3BHSD and PTGIS, but no miRNA that can target the mRNA of MMP9 was identified.

It is important to note that, most of the research has been focused on studying the regulatory role of miRNA in ovary by various in vitro studies. As a result of the complexity of the physiological mechanisms underlying inter- and intracellular signaling, in vitro investigations are still crucial for unraveling the secrets of cellular communication [101]. However, in vivo studies have reported the role of miRNAs, let-7, let-7b, 17-5p, 21, 125b, 181a, 224 and 430a in specific cells of the ovary.

2.3.4 Physiological stage specific miRNAs in biological fluids

It is apparent from the above that miRNA is involved regulates the molecular events associated with the ovarian cyclic activity. Accordingly, the literature indicating the differential expression of miRNA in biological fluids for identifying physiological states is being reviewed below.

Differential expression of miRNA at estrus stage and other stages of estrous cycle in various biological fluids [46, 102, 103, 104], the role of miRNAs in oviductal function as well as their relation to the dynamics of preovulatory sex-steroid concentration or vice-versa by influencing the genes of miRNA biogenesis pathway [105, 106], overlapping expression of miRNAs between tissues and EVs released from tissue fluids [102, 105, 106, 107] as well as unique and differential expression of miRNA between biological fluids and EV fractions of biological fluids [102, 107] and differential expression of miRNA in urine in accordance with the season of breeding [108] has been postulated.

Study on plasma miRNA profiles during the estrous cycle in estrus synchronized Holstein-Friesian heifers using NGS and PCR-based platforms (PCR array analysis and RT-qPCR analysis) [103] revealed the differential expression (up to 2.2-fold increase, P < 0.05) of let-7f, miR-125b, miR-145 and miR-99a-5p in the plasma on the day of estrus (Day 0) when compared to the days 8 and 16 of the estrous cycle and therefore the feasibility of using circulating miRNAs as biomarkers of reproductive function were opined. Likewise, differential expression of bta-miR-99a-5p in buffalo urine between estrus and diestrus stages of estrous cycle was reported [104]. In yet another study, higher expression of miR-141 was found in cell free saliva at the estrus stage compared to the diestrus stage in buffaloes [46].

Further, the circulatory steroidal concentration of E₂ and P₄ was found to influence on the miRNA expression in the oviduct of estrus synchronized Nellore cow animal models [106]. Interestingly, the periovulatory sex-steroid milieu was also found to affect the miRNA processing machinery (by influencing the expression of genes, DROSHA, DICER1 and AGO4 involved in miRNA processing pathway-components) and the expression of specific miRNA levels (miR-125b, miR-200b, miR-30d, miR-375, miR-92a) in bovine oviductal tissues. The fact that the previously mentioned differentially expressed oviductal miRNAs were also discovered in the EVs of the bovine oviductal fluid suggests that the miRNAs discovered in the oviduct may be charged into EVs and released into the oviductal fluid and have an impact on embryonic survival and development [105]. It was apparent from the findings of [106] that the miRNAs have a role in the oviductal function and their role on the post-transcriptional control is subjected to the dynamics of periovulatory sex-steroid concentration which can vary widely between individual animals.

Likewise, controlled ovarian hyperstimulation (COH) of estrus synchronized Simmental heifers intended to stimulate the growth of multiple dominant follicles with ovulating capability [107] by maintaining an elevated level of circulating FSH gonadotrophin was found to induce the differential expression of circulatory miRNA in bovine follicular fluid (FF) and blood plasma when compared to the unstimulated heifers. Further, bioinformatics analysis of the differentially expressed circulating miRNAs indicated that their potential target genes are associated with various pathways including transforming growth factor-beta (TGF-β) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, pathways in cancer and oocyte meiosis. Above all, it was discovered that the majority of these miRNAs could be located in the FF and blood plasma’s exosomal and Ago2 protein complex fractions. According to the results of qRT-PCR, there are variations in the expression patterns of miR-103, miR-127-3p, miR-134, miR-147, miR-221 and let-7 g depending on the stage of the estrous cycle. The exosomal component of the miRNAs, which were found in both blood plasma and FF, had a higher abundance. However, it was discovered that only the exosomal fraction could detect miR-182 from FF and miR-221, miR-103, and miR-127-3p from blood plasma [102].

Interestingly, obtaining the urinary miRNA profile by NGS in male goats has revealed differential expression of 40 miRNAs in urine during the breeding season compared to the non-breeding season. Among the differentially expressed miRNAs, miR-1246 was the most downregulated microRNA during the breeding season characterized by elevated testosterone levels. Further, testosterone through androgen receptors (AR) was found to be involved in the regulation of miR-1246 expression and other miRNA genes, whose expression differed between breeding and non-breeding season, indicating that miRNAs could serve as intermediaries of testosterone preparation of the male urogenital tract for high metabolic demands of the breeding season [108].

The preceding studies on differential expression of miRNA in biological fluids at various stages of the estrous cycle, the role of miRNAs in oviductal function as well as their relation to the dynamics of preovulatory sex-steroid concentration or vice-versa by influencing the genes of miRNA biogenesis pathway, overlapping expression of miRNAs between tissues and EVs released from tissue fluids as well as unique and differential expression of miRNA between biological fluids and EV fractions of biological fluids, differential expression of miRNA in urine in accordance with the season of breeding speculate the presence of EVs in biological fluids like urine as well as the differential expression of EV miRNA, if any in animals at the estrus stage and other stages of estrus cycle.

2.3.5 Extracellular vesicular miRNAs as indicators of physio-pathological states

EVs are cell-derived vesicles of 30–1000 nm in diameter, includes both exosomes and microvesicles and carry protein, lipid, RNA and miRNA [109]. EVs can be detected in all biological fluids as they are being shed by any cell type in the organism [110]. The recipient cells can take up EVs released into the extracellular space and modulate their biology [111].

Studies on various means of extracellular release of miRNA, reasons for the use of extracellular miRNA especially, EV miRNA &/protein as an alternative source for the search of biomarkers [15, 16, 18, 112, 113, 114, 115] and differential expression of miRNA in EVs of biological fluids and/or reproductive cells associated with various reproductive diseases has been postulated [113, 116, 117].

The tissue/organ specific miRNAs find their way into serum or plasma by several cellular release mechanisms [112, 115]. For instance, mature miRNA synthesized in the cell can either bind to RNA-binding proteins or lipoproteins, or get loaded inside microvesicles or exosomes when they are to be released. Since the miRNAs secreted in the plasma can mediate the uptake of miRNA at distant sites in the body, miRNA in body fluids has been regarded as hormones by some authors [113]. Various means of extracellular release of miRNA from a cell is depicted in Figure 2. Additionally, it was discovered that extracellular miRNAs serve as novel biological tools for intercellular cross-talks across cells in several organs, including the female reproductive system [114].

Figure 2.

Different forms of extracellular miRNA (adapted from [118]).

Above all, extracellular miRNAs could be used as the novel potential biomarkers of various physio-pathological conditions for the following reasons: (a) Stability of the miRNA in the extracellular environment against the activity of RNases, extremes of pH and/or high temperature. (b) Distinct expression profiles of extracellular miRNA in different body fluids such as blood, urine and follicular fluid and even in cell culture media. (c) Protection of extracellular miRNAs from degradation either by packaging in the lipid vesicles as EVs/exosomes or forming complexes with the RNA-binding protein, Argonaute 2 protein (Ago2) [14, 17, 18]. (d) Circulating miRNA with in the microvesicles and the miRNAs associated with Ago2, high density lipoprotein (HDL) and nucleophosmin 1 (NPM1) [119, 120] are protected from endogenous RNase activity [15, 16].

Therefore, cell free miRNA (Extracellular miRNA) found in various biological fluids like serum, plasma, urine and saliva can be broadly of two types, miRNA found in EVs and miRNA associated with proteins. The existence of primarily exosomal or vesicle-free miRNA can depend on the miRNA itself, the cell type from which they arise, and/or other factors impacting the secretion of miRNA in a particular individual.

Exosomes from biological fluids and/or exosomes from particular reproductive cells have also been proposed as biomarkers for a variety of reproductive diseases, including uterine fibroids, preeclampsia, polycystic ovary syndrome (PCOS), endometriosis, ovarian cancer, and Asherman’s syndrome. The need for research to specify the functions and mechanisms of exosomes has been highlighted by many researchers [113, 116, 117].

2.4 Extracellular vesicular miRNA associated with physiological states

Proteins and miRNAs carried within the EVs are found to be the major regulatory components and are associated with the alteration of the cell biology of the recipient cells up taking the EVs [121, 122]. MiRNAs of exosomes can be delivered to distant target cells, which is recognized as an important mode of cell-cell communication [123]. Further, EV miRNAs are more stable than the other cell free urinary miRNAs as they are doubly protected from endogenous RNase activity by means of cellular packing within the membrane-enclosed structures [124].

Exosomes, microvesicles and apoptotic bodies are the three main subtypes of EVs. The EV subtypes are distinguished according to their biogenesis, size, release pathways, cargo and functions [125, 126]. It is evident that EVs present in various biological fluids can be used to identify physiological stage specific biomarkers. Therefore, the literature pertaining to the studies on EV miRNA and/or proteins related to physio-pathological states along with their biogenesis is being reviewed below.

2.4.1 Biogenesis of EVs

All types of cells secrete exosomes, which are EVs. Exosomes are created as intraluminal vesicles (ILVs) and range in size from 30 to 150 nm. They originate from the endosomal compartment. ILVs are created by the early endosomal membrane budding inward, encasing proteins, lipids, and cytosol for later degradation, recycling, or exocytosis. After early endosomes develop into late endosomes, multivesicular bodies (MVBs) are created as a result of the buildup of several ILVs. These MVBs may either fuse with lysosomes and get degraded or fuse with the plasma membrane (PM) to release their sequester ILVs as exosomes in the extracellular space [127].

Microvesicles are larger EVs than exosomes and are 100 nm to 1 μm in diameter and bud directly from the plasma membrane (PM). The EVs involved in cellular communication are characterized by protein markers such as tetraspanins (CD9, CD63, and CD81) and adhesion integrins. Both exosomes and microvesicles form a source of cellular clearance and are involved in cell–cell communication both between local and distant cells [128].

The size of apoptotic bodies varies from 50 to 5000 nm in diameter and are released into the extracellular space as blebs of cells undergoing apoptosis [125] and therefore contain nuclear fragments and cell debris. Apoptotic bodies do not play a part in cellular communication or exhibit surface markers characteristic for exosomes or microvesicles [129].

2.4.2 Studies on physiological stage-specific EV miRNA in biological fluids

Studies on the presence of EVs in biological fluids of female reproductive tract [130, 131, 132, 133, 134, 135], role of EV miRNA of biological fluids [136], differential expression of uterosomal EV miRNA during pregnancy [137], the impact of estrous cycle on the miRNA content of FF EVs [138] and the influence of lactation and energy status on miRNA content of follicular fluid [139] has been postulated.

Oviductal luminal fluid (OLF) is one of the reproductive secretions which is rich in EVs (Oviductal EVs or Oviductosomes, OVs) containing both exosomes (50–100 nm) and microvesicles (100–1000 nm) which are known to play a role in inter cellular communication [130]. Likewise, EVs isolated from human uterine flushing’s and in vitro cultured human endometrial epithelial cells contained the miRNA [131]. EVs found in the uterine fluid are termed uterosomes and EVs present in the uterine lumen flush (ULF) of pregnant animals contain EVs emanated from both the endometrial epithelia of the uterus and trophectoderm of the conceptus [135]. In particular, OVs have been found in media of cultured bovine epithelial cells [132, 133], bovine oviductal secretions (in vivo), human oviductal fluid [134] and in mouse [134].

The following was discovered as a result of the research done to determine whether OVs contain miRNAs during the estrous cycle and whether particular miRNAs in the estrus oviductosomal cargo can be transferred to sperm during their communication inside the oviduct in mice: (a) MiRNAs in OVs of the mouse were found to have similar expression levels for the majority throughout the murine estrous cycle (b) OVs can transfer miRNAs to sperm, notably miR-34c-5p, which is only produced from sperm in the zygote and is necessary for the initial cleavage. (c) Particularly the miR-34c-5p, which is strongly concentrated at the centrosome where it is known to function, miRNAs transported by OVs to sperm are mostly localized in specific head compartments [136].

Further, the uterosomal miRNA cargoes of pregnant and non-pregnant sheep were found to be significantly different [137]. Likewise, EVs present in the uterine lumen flush (ULF) of early pregnant sheep were found to modulate trophectoderm cell growth by decreasing the proliferation of ovine trophectoderm cells and promoting the secretion of interferon tau (IFNT) without affecting gene expression. EVs have been reported to decrease early in pregnancy in the ovine uterus, to carry lipid and protein cargo, and to control trophectoderm cell proliferation. Hence, EVs’ protein and/or lipid cargo along the nucleic acid cargo in the lumen of the pregnant ovine uterus in conceptus development has been highlighted [135].

In yet another in vitro study conducted with the ovaries obtained from crossbred Nellore cows to know the impact of the estrous cycle on the microRNA content in EVs of FF that modulate bovine cumulus cell transcripts that in turn influence the oocyte maturation revealed the following: (a) 161 miRNAs were significantly upregulated in the small EVs of FF with low P₄ levels when compared to the ones with high P₄ levels, (b) the uptake of small EVs by cumulus cells started after 1 h of in vitro maturation (IVM) as evident from live cell imaging, (c) within just 9 hours of IVM, small EVs derived from low and high P4 FF can induce a specific RNA profile in the cumulus cells and (d) cumulus cells supplemented with EVs obtained from FF of low P₄ group presented a large number of genes that are upregulated that modulate biological processes involved in reproduction and immune responses [138].

Further, a study conducted to know the association between postpartum negative energy balance (NEB) and EV-coupled miRNA signatures in follicular fluid (FF) of cows revealed that NEB in postpartum cows is mainly associated with downregulation of EV-coupled miRNAs in FF and vice-versa. Moreover, regardless of the energy status, lactation induced changes in FF EV-coupled miRNA profiles in dairy cows compared to the heifers [139].

3. Conclusion

MiRNA are involved in regulating molecular events at the level of mRNA and proteins related to the biogenesis of E₂ and P₄. MiRNA are also identified to be involved in follicular atresia and have a housekeeping role in the function of the ovary across the estrous cycle. Differential expression of mRNA transcripts, HSPs and other proteins connected to the cellular processes of ovarian cyclic activity has also been identified in various species of animals. Differential expression of miRNA in GCs of preovulatory dominant and subordinate follicles have been detected. Differential expression of miRNA in biological fluids at various stages of estrous cycle, overlapping expression of miRNAs between tissues and EVs identified in tissue fluids as well as biological fluids, unique and differential expression of miRNA between biological fluids and EV fractions of biological fluids was demonstrated. Importantly, EV miRNAs are more stable than the cell free miRNAs making them promising biomarkers for the identification of physiological states. Estrus being dynamic and EVs identified as associated with cellular and molecular processes of ovarian cyclic activity around the estrus stage, disclose the potential of EV miRNAs in easily accessible biological fluids to act as biomarkers.