Roles of microRNAs in mammalian reproduction: from the commitment of germ cells to peri‐implantation embryos

ABSTRACT MicroRNAs (miRNAs) are active regulators of numerous biological and physiological processes including most of the events of mammalian reproduction. Understanding the biological functions of miRNAs in the context of mammalian reproduction will allow a better and comparative understanding of fertility and sterility in male and female mammals. Herein, we summarize recent progress in miRNA‐mediated regulation of mammalian reproduction and highlight the significance of miRNAs in different aspects of mammalian reproduction including the biogenesis of germ cells, the functionality of reproductive organs, and the development of early embryos. Furthermore, we focus on the gene expression regulatory feedback loops involving hormones and miRNA expression to increase our understanding of germ cell commitment and the functioning of reproductive organs. Finally, we discuss the influence of miRNAs on male and female reproductive failure, and provide perspectives for future studies on this topic.


I. INTRODUCTION
Mammalian microRNAs (miRNAs) are non-coding RNAs that can regulate post-transcriptional gene expression by targeting the 3 -untranslated region and/or coding region of messenger RNAs (mRNAs) (Hausser et al., 2013), and are involved in the regulation of approximately one-third of all mammalian genes (Lewis, Burge, & Bartel, 2005). While a single miRNA may target many genes, the expression of a particular gene can be modulated by multiple miRNAs (Sood et al., 2006). Hence, miRNAs are involved in the active regulation of biological and physiological processes in both normal and disease conditions (Reza et al., 2016(Reza et al., , 2017(Reza et al., , 2018aReza, Choi, & Kim, 2018b). In mammalian reproduction, miRNAs are widely involved in sex differentiation (Cook & Blelloch, 2013), gametogenesis , fertilization , zygotic genome activation (ZGA) and early development (Suh et al., 2010), implantation , germ layer specification (Vidigal & Ventura, 2012), and pregnancy (Otsuka et al., 2008). Understanding the functions of miRNAs in mammalian reproduction will therefore improve our understanding of the biological challenges of mammalian reproduction. Several reviews have focused on the event-specific involvement of non-coding RNAs (miRNAs, endogenous siRNAs, piwi-interacting RNAs) in the reproduction of different species. However, those reviews included all types of non-coding RNAs and a wide range of species (insects, non-vertebrates, fishes, and birds), in order to develop an understanding of their roles in reproductive biology. To comprehend specifically the roles of miRNAs in mammalian reproduction requires an in-depth review focusing on mammalian species, as mammals are relatively unique in their structure, physiology, and psychology. Furthermore, the available reviews mainly focused on particular aspects of reproduction such as oogenesis (Donadeu, Schauer, & Sontakke, 2012;Hossain et al., 2012a), spermatogenesis (Kotaja, 2014;Yadav & Kotaja, 2014;Hilz et al., 2016;Chen et al., 2017), early embryonic development (Suh & Blelloch, 2011;Cook & Blelloch, 2013;Dallaire & Simard, 2016), or implantation (Liu & Yang, 2011;Dior et al., 2014;Galliano & Pellicer, 2014;Liu et al., 2016), with a holistic review focusing on all processes of mammalian reproduction lacking. Such a holistic review focusing on miRNA involvement in all aspects of mammalian reproduction is necessary to improve our understanding of reproductive biology and to mitigate mammalian reproductive challenges.
Herein, we summarize recent progress in miRNAmediated regulation of mammalian reproduction, and highlight the significance of miRNAs in different aspects of mammalian reproduction including the biogenesis of germ cells, functionality of reproductive organs, and development of early embryos, as well as covering infertility and sterility derived from miRNA deregulation in males and females. First, we discuss impairments and abnormalities caused by depletion of miRNA-processing machinery during germ cell commitment and the functioning of reproductive organs. Second, we discuss how miRNAs interfere at different stages of male and female reproduction. Furthermore, we focus on the distribution and dynamics of miRNAs during gametogenesis, fertilization, embryo development, and implantation, as well as specification to three different germ layers. Finally, we discuss the influence of miRNAs on male and female reproductive failure, and provide a set of conclusive remarks to guide future studies in this topic.

II. LOSS OF miRNA-PROCESSING MACHINERY IMPAIRS MAMMALIAN REPRODUCTION
Loss of one or both of the components of the miRNAprocessing machinery (Dicer and Drosha) causes severe impairment during gametogenesis, leading to infertility in both males and females (Fig. 1). Especially, Dicer and Drosha are indispensable during the early stages of development such as before sex differentiation (Bernstein et al., 2003), while in the later stages, their loss causes impairment to different extents depending on the stage at which Dicer or Drosha is deleted Liu et al., 2010;Liu et al., 2012). However, it is clear that this impairment does not arise from the absence of the proteins themselves, but rather from the associated failure of biogenesis of small-RNAs (miRNAs  (Papaioannou et al., 2009;Hossain et al., 2012b). The severity of Dicer1 deletion is highly stage and organ specific, with other organs or processes often remaining functional. Interestingly, sexual behaviour, mounting ability, external genitalia, and oestrous are not affected by the depletion of Dicer (Papaioannou et al., 2009). In the male, primordial germ cell (PGC)-specific deletion of Dicer and Drosha restricts the propagation, differentiation, and maturation of male PGCs  and causes defective spermatozoa and infertility (Maatouk et al., 2008). Spermatogonia-specific ablation of Dicer1 impairs the leptotene to zygotene/pachytene transition during prophase I with an increase in apoptotic pachytene spermatocytes, deformed chromatin compaction, acrosome formation, and sperm-head structure . Round spermatid-specific deletion of Dicer1 deregulated actin related protein 2/3 complex subunit 5 (Arpc5), leading to abnormal chromatin compaction and causing deformed acrosomes and sperm heads, as well as apoptosis of spermatids in mice . Sertoli cell-specific deletion of Dicer1 caused apoptosis of Sertoli cells after birth leading to degenerated testes, defective prepubertal spermatogenesis, and infertility (Papaioannou et al., 2009). Epididymis-specific deletion of Dicer1 resulted in imbalanced lipid homeostasis and instability of the sperm membrane (Bjorkgren et al., 2015) as well as leakage of immature spermatocytes to the epididymis and a thick blood-testis barrier with a multilayered organization (Korhonen et al., 2015). Hence, Dicer/Drosha safeguard meiotic progression and the production of spermatozoa, as well as the development of accessory organs, by supplying functional miRNAs.
The replacement of Dicer1-ablated ovaries with wild-type (WT) ovaries enable the mice to produce viable offspring, while implantation of Dicer1-ablated ovaries into WT mice results in failure to produce offspring (Otsuka et al., 2008). The failure is prominent during post-implantation development, while ovulation, fertilization, and first embryonic cell division appear normal (Otsuka et al., 2008) and may be due to disintegration of the corpus luteum (CL) and lower progesterone levels (Otsuka et al., 2008), which may derive from impairment to miRNA regulation required for the angiogenesis, maintenance, and functioning of the CL (Yang et al., 2005;Otsuka et al., 2008). Moreover, depletion of Dicer1 causes disintegration of the structural, mechanical, and functional ability of the fallopian tube including loss of the smooth muscle layer, disorganized epithelium, and deregulation of signalling networks (Luense, Carletti & Christenson, 2009): this might also cause failures in the transportation and implantation of embryos to the uterus (Luense, Carletti, & Christenson, 2009), and the resulting offspring may have impaired reproductive organs because of maternal loss of oviductal Dicer1 . Fallopian tube-specific loss of Dicer1 results in shorter tubule length, abnormal coil formation and the presence of fluid-filled sacs Gonzalez & Behringer, 2009). Taken together, the observations suggest that Dicer1 in the female safeguards molecular and signalling networks related to gametogenesis, steroidogenesis, the genomic and structural architecture of the oocytes as well as the development and implantation of embryos.

III. miRNAs ARE INVOLVED IN SEX-SPECIFICATION AND COMMITMENT OF MAMMALIAN GONADS
Prior to sex determination PGCs carry a unique miRNA profile  that does not match sex-specific PGCs (Fig. 2), and PGCs undergoing sex determination display dimorphic expression patterns of miRNAs (see online Supporting information, Table S1). For example, the let-7 family, miR-125a, and miR-9 are overexpressed in PGCs for male germline commitment . By contrast, female germline-committing PGCs show overexpression of miR-29b, which targets DNA methyltransferase 3 alpha (Dnmt3a) and DNA methyltransferase 3 beta (Dnmt3b), and epigenetically regulates gene expression during oogenesis in mice (Takada et al., 2009). Embryonic loss of miRNAs resulting from Dicer1 deletion is associated with embryonic lethality in mice near the time of PGC specification (Bernstein et al., 2003). Dicer1 depletion just before PGC migration impaired the proliferation and gonad-specific colonization of PGCs in mice , and oocytes originating from Dicer1-ablated mouse germline cells failed to establish functional zygotes and commence first zygotic cell division (Tang et al., 2007). These findings indicate that sex differentiation and embryonic development require parentally derived miRNAs.
B-lymphocyte-induced maturation protein 1 (Blimp1) regulates PGC specification by repressing Hox genes (Ohinata et al., 2005;Vincent et al., 2005;Robertson et al., 2007) and is predominantly targeted by let-7 (West et al., 2009). It is thought that suppression of Blimp1 by let-7 upregulates Hox genes, leading totipotent PGCs towards male germline specification. Migration of PGCs to the female gonad is mediated by the miR-17-92 and miR-290-295 cluster, and the loss of this cluster disrupts PGC colonization in the female gonad, resulting in premature ovarian failure (POF) and sterility Medeiros et al., 2011). The impact of PGC loss is much more severe in females than in males as female germline stem cells cannot propagate and self-renew during adulthood, while spermatogonial stem cells (SSCs) successfully propagate and self-renew throughout the lifetime, thus the loss of these miRNAs results in infertility in females but only subfertility in males.

(1) The formation of male gonads
Most miRNAs that are highly expressed in male PGCs are also detected in spermatogonia cells. Validated targets of these miRNAs are involved in mitogen-activated protein kinase (MAPK ), janus kinase (JAK )-signal transducer and activator of transcription proteins (STAT ), and transforming growth factor beta (TGFβ) signalling pathways Garcia-Lopez et al., 2015). A panel of miRNAs was detected in human and mouse testes (Watanabe et al., 2006). Among these miRNAs, miR-449a is abundant in both mouse and human testes (Luo et al., 2015b). In boar testes, a subset of miRNAs are expressed differentially between mature and immature testes (Luo et al., 2010) and regulates stage-specific testicular gene expression. For example, miR-34b and miR-34c regulate the differential expression of AQN-1 protein (AQN-1), hyaluronan synthase 3 (HAS3), ring finger protein 4 (RNF4), sperm mitochondria-associated cysteine-rich protein (SMCP), and sperm adhesion molecule 1 (SPAM1) between mature and immature porcine testes (Luo et al., 2010), indicating that these miRNAs play important roles in species-and stage-specific male germ cell development. Similarly, the testicular somatic cells of fertile and infertile males express miRNAs differentially, for example miR-202-5p, a member of the let-7 family, is selectively expressed in Sertoli cells of fertile males with normal germ cells, but not in the Sertoli cells of infertile males (Dabaja et al., 2015). Previous studies also showed that miR-202-5p, which is a downstream mediator of the testis-determining factor SOX9 , is expressed in Sertoli cells in the early XY gonad (Rakoczy et al., 2013) and its expression is up-regulated again in the testis at later developmental stages (Ro et al., 2007). These data indicate that miR-202-5p is closely associated with male gonad development.
(2) The commitment and maintenance of SSCs Differential expression of miRNAs is observed at different stages of development and maturation of male germline cells ( Fig. 2)  . A group of these miRNAs regulating phosphatase and tensin homolog (Pten) and Stat signalling is predominantly downregulated in spermatogonia compared to PGCs, and thus enhances the expression of Pten and Stat levels in spermatogonia compared to PGCs . Likewise, members of the miR-290-295 cluster are pluripotency related, and are downregulated in spermatogonia compared to PGCs and gonocytes McIver et al., 2012b), the proliferation-and differentiation-regulatory miR-143-3p is upregulated in mice spermatogonia compared to in PGCs , and miR-293 is expressed differentially between gonocytes and spermatogonia (McIver et al., 2012b). This indicates that these miRNAs may specifically participate in the commitment of PGCs to SSCs. Among the miRNAs involved in SSC maintenance, miR-21 regulates the stemness, proliferation, and survival of SSCs by regulating ETS variant 5 (ETV5)/glial cell-derived neurotrophic factor (GDNF ) signalling (Niu et al., 2011). GDNF is a well-defined paracrine factor that promotes SSCs self-renewal (Yang & Oatley, 2014), and is closely connected to the ret proto-oncogene (RET ) that is required for GDNF family receptor alpha 1 (GFRα1) signalling in mouse SSCs (Jing et al., 1996;Naughton et al., 2006). With the assistance of the phosphoinositide 3-kinase (PI3K )/AKT serine/threonine kinase (AKT )-dependent pathway or sarcoma (Src) family kinase pathway, GDNF regulates the expression of ETV5, inhibitor of DNA binding 4 (ID4), LIM homeobox 1 (LHX1), and POU class 3 homeobox 1 (POU3F1) to drive SSC self-renewal Oatley, Avarbock, & Brinster, 2007). Thus, miR-21 appears to have a very important role in PGCs self-renewal and maintenance.
Similarly, miR-221/222 promotes the stemness characteristics of SSCs by downregulating KIT proto-oncogene receptor tyrosine kinase (c-Kit) expression in mice (Yang et al., 2013b), and both miR-20 and miR-106a directly target signal transducer and activator of transcription 3 (Stat3) and cyclin D1 (Ccnd1), and play critical roles in maintenance and renewal of SSCs by increasing proliferating cell nuclear antigen (Pcna) and promyelocytic leukaemia zinc finger (Plzf ), and reducing c-Kit expression in mice . miR-290 and miR-302 are also involved in the maintenance and pluripotency of SSCs in mice (Zovoilis et al., 2008). It is well known that miR-135a and miR-544 are essential factors for the regulation of self-renewal of SSCs in rat and goat, respectively (Moritoki et al., 2014;Song et al., 2015). In addition, miR-184 and miR-204 are involved in mouse and goat SSC proliferation , and protocadherins, which are also important cell-adhesion molecules, are also targeted by miR-320 during germ cell biogenesis (Marcon et al., 2008). Differential expression of numerous miRNAs occurs between early and late stages of mammalian spermatogonia; these miRNAs are involved in either the maintenance or differentiation of SSCs (Niu et al., 2011;Tong et al., 2011Tong et al., , 2012Yang et al., 2013b). Thus, growing evidence strongly suggests that many miRNAs are closely linked to the post-transcriptional regulation of SSCs self-renewal and maintenance.

(4) Meiotic sex chromosome inactivation (MSCI) and chromosomal stability during spermatogenesis
MSCI refers to the transcriptional silencing of genes located on the X-chromosome at the pachytene spermatocyte stage. While most X-chromosomal genes are silenced, X-chromosome linked miRNAs including miR-718-3p, miR-883a-3p, and miR-883a-5p can escape MSCI-mediated silencing in mice (Song et al., 2009). This exceptional event allows these miRNAs to remain active throughout spermatogenesis and play a crucial role in the differentiation of SSCs (Song et al., 2009). miRNAs are fine-tuners of many important genes, and thus their absence can cause overexpression of X-linked genes that are normally silenced by MSCI (Greenlee et al., 2012), ultimately leading to the impairment of meiotic progress, acrosomal defects, abnormal chromatin condensation and nuclear shaping, degenerated seminiferous tubules, mixing of different stages of haploid spermatogonia, and the presence of exfoliated and immature germ cells in the epididymis (Korhonen et al., 2011), followed by infertility in males (Greenlee et al., 2012). Although miRNAs such as miR-871-3p, miR-743-3p, and miR-883b-3p may regulate MSCI-mediated gene expression (Song et al., 2009), but there is not yet direct evidence for this. miRNAs are also important for maintaining chromosomal stability; the loss of several miRNAs such as miR-16, miR-18, miR-183, and miR-762 caused abundant activation of ATM serine/threonine kinase (ATM) and deregulation of genes including mediator of DNA damage checkpoint 1 (Mdc1), Cdk2, trimethylation of lysine 9 on histone H3 (H3K9me3), and ring finger protein 8 (Rnf8), which are involved in chromosomal stability and DNA repair during spermatogenesis in mice (Modzelewski et al., 2015). miR-762 downregulates DNA damage-related gene H2A histone family member X (γ -H2AX ) and upregulates the DNA damage repair-related genes RNF8, ring finger protein 168 (RNF168), DNA repair associated (BRCA1), tumor protein P53 binding protein 1 (TP53BP1), and poly (ADP-ribose) polymerase 1 (PARP), as well as promoting the growth of immature Sertoli cells in pigs (Ma et al., 2016a), indicating that both miRNA-mediated gene regulation and fine-tuning of gene expression are critical factors for MSCI and chromosomal stability during spermatogenesis.

(5) miRNAs as regulators of spermiogenesis and chromatin body compaction
A wide range of miRNAs are expressed differentially during the transition of pachytene spermatocytes to round spermatids and might be important in the spermiogenesis process. For example, round spermatid basic protein 1 (Rsbn1) is an important molecule for the transcriptional regulation of haploid germ cell formation and is a target gene of miR-355, miR-181b, and miR-181c McIver et al., 2012a). In addition, miR-122 downregulates transition protein 2 (Tnp2) (Yu, Raabe, & Hecht, 2005), which replaces protamine, an essential component for the compaction and patterning of chromatin bodies into small sperm heads (Krawetz et al., 2011); testis-specific miR-469 also plays an important role in chromatin remodelling and elongation of spermatids by repressing Tnp2 and protamine 2 (Prm2) (de Mateo & Sassone-Corsi, 2014). Similarly, loss of miRNA-dependent regulation of Arpc5 impairs translational activation of protamine 1 (Prm1)/Prm2 by blocking 80S ribosome formation and facilitating mRNA transportation to chromatoid/P bodies in mice . miRNA-dependent regulation of tudor domain containing 6 (Tdrd6) during spermiogenesis of mice is essential for chromatin body formation (Vasileva et al., 2009). However, unlike the early stages of spermatogenesis, most of these miRNAs disappear during spermiogenesis, leaving a few sperm-borne miRNAs such as miR-34 to regulate fertilization and early zygote development . Therefore, some of the sperm-borne miRNAs are important epigenetic contributors to the embryo. However, the roles of miRNAs in chromatin compaction require further investigation to determine whether the impairments caused by deregulated miRNAs are permanent or recoverable.

(6) miRNAs regulate maturation and viability of spermatozoa during storage and transportation through the epididymis
Epididymal spermatozoa are enriched with distinctive miR-NAs (Fig. 2), which are uncommon in other spermatozoal stages (Nixon et al., 2015b;Reilly et al., 2016). Additionally, epididymal epithelial cell-derived miRNAs may influence the post-testicular progression of sperm to their destination and mediate cell-to-cell communication (Belleannee et al., 2013a;Belleannee, 2015;Qing et al., 2017); vasectomy results in alteration of miRNAs in both epididymal and seminal microvesicles (Belleannee et al., 2013b). Differential expression of miRNAs is also found between epididymal and freshly ejaculated sperm, as well as among the segments (caput, corpus, cauda) of the epididymis in different species including human (Belleannee et al., 2012), horse (Twenter et al., 2017), mouse (Nixon et al., 2015a,b), pig (Chang et al., 2016) and bull (Belleannee et al., 2013a). These miRNAs might be involved in spermatozoa maturation (Twenter et al., 2017), intracellular trafficking signalling (Nixon et al., 2015a), death signalling (Chu et al., 2015), and epididymal cell proliferation . The differential expression of miRNAs among the epididymal segments is common to various mammalian species, thus it could be assumed that the contribution of epididymal miRNAs is an essential part of mammalian sperm maturation and concentration, and that these epididymal miRNAs potentially contribute to the motility of spermatozoa as well as to the content of the seminal fluid.
In oestradiol benzoate-treated rats, the miR-29 family resulted in extensive apoptosis of male germ cells by downregulating Dnmt and myeloid cell leukemia 1 (Mcl-1) (Meunier et al., 2012). Ochratoxin-A treatment induces extensive apoptosis of male germ cells, while inhibition of miR-122 minimizes this Ochratoxin-A-mediated toxicity (Chen et al., 2015a), indicating that miR-122 is a critical regulator for the germ cell apoptosis-induction pathway. In pigs, expression of miR-16 and miR-34 regulates the cell cycle progression during spermatogenesis and senescence of sperm (Luo et al., 2015b). miR-16 promotes the apoptosis of immortalized GC-1 spermatogonia cells (GC-1 SPG) by downregulating Ccnd1 . Deletion of the miR-17-92 cluster, which is potentially regulated by transcriptional regulation of c-MYC and E2F1 (Novotny et al., 2007), results in testicular atrophy and empty seminiferous tubules by reducing SSCs and increasing germ cell apoptosis (Xie et al., 2016). Taken together, these observations suggest that deregulation in miRNA expression is an important mechanism behind mammalian male germline cell apoptosis.

V. ROLES OF miRNAs IN FEMALE REPRODUCTION
Sex-specific and unique miRNA profiles are observed at every step of female reproduction (Fig. 2): female-specific miR-124 promotes the commitment of female gonads and prevents sex reversal by silencing Sox9 in the mouse ovary. Similarly, many miRNAs play substantial roles throughout folliculogenesis and oogenesis by regulating expression of important genes (see Table S3), and many are specific to particular developmental stages of the follicles, as well as segments of the reproductive tract.

(2) miRNA expressions varies in the granulosa cells (GCs) and thecal cells (TCs) of the dominant follicles (DFs) and subordinate follicles (SFs)
DFs and SFs show differential expression of some miRNAs, which may regulate gonadotropin-releasing hormone (GnRH) signalling (Salilew-Wondim et al., 2014). These miRNAs are expressed differentially among different subgroups of GCs ), TCs (Zielak-Steciwko et al., 2014, and between mural and cumulus cells (Velthut-Meikas et al., 2013). These miRNAs are also potential regulators of numerous signalling pathways including meiosis, wingless-type MMTV integration site family (WNT ), TGFβ, ErbB receptor tyrosine kinases (ERBB), insulin, P13K-AKT , and MAPK (Zielak-Steciwko et al., 2014). Another panel of miRNAs including miR-125b, miR-145, and miR-199a-3p target leukemia inhibitory factor (LIF ), cyclin dependent kinase inhibitor 1A (CDKN1A), and prostaglandin-endoperoxide synthase 2 (PTGS2), respectively, in GCs, but not in TCs of bovine follicles . Furthermore, a panel of miRNAs is highly expressed in atretic follicles compared to healthy follicles, with all miRNAs except miR-21-3p and miR-378 showing higher expression in TCs compared to GCs (Donadeu, Mohammed, & Ioannidis, 2017). This suggests that these miRNAs might regulate the phenotypic characters of different cell types by controlling the expression of important signalling molecules (Donadeu et al., 2017). Additionally, miR-708, miR-221, miR-21-3p, miR-335, and miR-214 are expressed abundantly in the follicular fluid of SFs compared to that of DFs (Gebremedhn et al., 2015) indicating a potential role in the selection of follicles. Hence, differential expression of miRNAs between TCs and GCs may play an important role in the selection and recruitment of DFs, as well as during the follicular wave (in different mammalian species the recruitment, growth and development of several waves of follicles is possible before follicle rupture and ovulation occur; during this process two or more follicles might develop, but only one follicle successfully releases the egg) and atresia.

(4) Regulation of luteinization and formation of the CL
The importance of miRNA signalling for regulation of the CL has been demonstrated using hypomorphic Dicer1 mutant mice, which fail to implant embryos and maintain pregnancy due to impaired growth of new capillary vessels in the CL (Otsuka et al., 2008). The study demonstrated that the impaired CL angiogenesis in Dicer1 (d/d) mice was associated with a lack of miR-17-5p and let-7b, which target tissue inhibitor of metalloproteinases 1 (Timp1). These data suggest that the pro-angiogenic miR-17-5p and let-7b are critical factors for the normal development of the CL. miRNAs are known to be involved in the process of CL formation (Fig. 2) such as during follicular to luteal transition (McBride et al., 2012), vascularization of luteal tissue (Otsuka et al., 2008), and in non-regressed CL , and regressed CL . In particular, reduction of miR-503 expression in pre-ovulatory follicles, signals the onset of luteinization, with its expression subsequently restored during CL development McBride et al., 2012). miR-503 directly regulates Gdf9, follicle stimulating hormone Receptor (Fshr), oestrogen receptor 2 (Esr2), activin A receptor type 2A (ActRIIa), Ccnd2, and activin A receptor type 2B (ActRIIb) and indirectly regulates inhibin alpha subunit (Inha), Inhba, Inhbb, Cyp19a1, luteinizing hormone/choriogonadotropin receptor (Lhcgr), Esr2, androgen receptor (Ar), Cdkn1b, and Cas3 . Thus, miR-503 could have a direct involvement in CL formation and maintenance. Another study showed that miR-378 plays an anti-apoptotic role in luteal cells of non-regressed CL by targeting interferon gamma receptor 1 (IFNGR1) . These observations provide evidence to explain the physiological requirement for miRNAs during luteal development.

(5) The prediction of oocyte quality
miRNAs are expressed abundantly in follicular fluid, cumulus cells, and oocytes. Therefore, it may be possible to use miRNAs to evaluate the quality of oocytes. Uhde et al. (2017) attempted to assess the quality of bovine oocytes based on the miRNA expression of the surrounding cumulus cells although they concluded that the quality of pre-fertilization-stage oocytes could not be determined in this way. As highlighted in Fig. 2, the functions of miRNAs are globally suppressed between matured oocytes and first zygotic cell division stages (Suh et al., 2010). As a result, the functional activity of miRNAs are generally abrogated (although the expression of miRNAs remains abundant) during those stages (Tang et al., 2007;Suh et al., 2010) representing an obstacle to the use of miRNAs as biomarkers to assess the quality of pre-fertilization-stage oocytes. However, the roles of maternal miRNAs (oocyte-derived miRNAs) are indispensable during the post-fertilization stages (Tang et al., 2007;Karakaya et al., 2015), especially during and after zygotic genome activation. It may be that the roles of oocyte-derived miRNAs will be understood only by monitoring the post-fertilization development potential of oocytes.

(6) miRNAs and extracellular vesicles within follicular fluid
Extracellular vesicles including exosomes are tiny particles of biological origin, contain RNAs, proteins, lipids and other small molecules, and are transported by the circulatory system to neighbouring or distant organs (da Silveira et al., 2012;Reza et al., 2016). These particles function as a cargo transport system and participate in cell-cell communication, embryo-maternal crosstalk (Saadeldin, Oh, & Lee, 2015) and communication among ovarian follicles (da Silveira et al., 2012). Vesicular miRNAs derived from follicular fluid are being investigated for their suitability as biomarkers for oocyte quality in matured and immature oocytes (Sohel et al., 2013;Santonocito et al., 2014;da Silveira et al., 2015), as well as in healthy and unhealthy follicles (Sang et al., 2013;Roth et al., 2014). For example, matured follicle-derived microvesicles are enriched with a panel of miRNAs including miR-99a, miR-100, miR-132 and miR-218 (Santonocito et al., 2014), among which miR-132, miR-212 and miR-214 negatively regulate the factors related to the inhibition of follicular maturation while miR-29a is involved in epigenetic changes (Santonocito et al., 2014). In addition, follicular-fluid-derived circulatory miRNAs are potentially involved in steroidogenesis processes (Sang et al., 2013), for example, miR-132, miR-320, miR-520c-3p, miR-24 and miR-222 regulate the concentration of oestradiol, while miR-24, miR-193b and miR-483-5p regulate the concentration of progesterone (Sang et al., 2013). These data indicate that follicular-fluid-derived extracellular miRNAs could be an important tool to assess oocyte quality and to differentiate between healthy and unhealthy follicles.

VI. miRNAs ARE POTENT REGULATORS OF THE STEROIDOGENIC PROCESS DURING GAMETOGENESIS
miRNAs are potent regulators of testicular, ovarian, hypothalamic and pituitary hormones (Figs 3 and 4) (Messina & Prevot, 2017), and regulate the expression and functions of numerous steroidogenic genes (see Table S4). Thus miRNAs act as an essential mediator to maintain interactions among the hypothalamus, pituitary, and gonad.
In human, miR-133b regulates Sertoli cell proliferation by directly targeting GLI family zinc finger 3 (GLI3) following the upregulation of CCNB1 and CCND1 .
steroidogenesis . miRNA-23b regulates Pten and epidermal growth factor receptor pathway substrate 15 (Eps15) in an FSH-dependent manner, and its function is implicated in endocytosis, focal adhesion, and actin cytoskeleton regulation during spermatogenesis (Nicholls et al., 2011). By contrast, numerous miRNAs in steroidogenic cells are expressed in an FSH-and androgen-dependent manner (Nicholls et al., 2011;Panneerdoss et al., 2012;Shih et al., 2015). This indicates that the expression of miRNAs and hormones are interdependent; a change in one of them can alter the expression of the other. It has been reported that patients suffering from azoospermia and oligospermia showed deregulated miRNA expression profiles (Ghorbian, 2012). Therefore, miRNAs and hormones potentially develop a feedback loop in the testes, and hormone-mediated gene expression in the testes is regulated in a miRNA-dependent manner.
By contrast, many miRNAs play anti-steroidogenic roles by inhibiting growth and proliferation, and increasing apoptosis of GCs and TCs: miR-145 attenuates mouse GC proliferation by targeting Acvr1b , miR-23a plays a pro-apoptotic role in luteinized human GCs by reducing expression of X-linked inhibitor of apoptosis (XIAP) and increasing CAS3 , and miR-23a and miR-27a also increase the apoptosis of human GCs by targeting SMAD5 and positively regulating Fas-induced death signals (Nie et al., 2015). Expression of miR-26b induces apoptosis of GCs in pigs by directly targeting ATM, a gene involved in DNA repair , and by suppressing SMAD4 signalling and BCL2 ; let-7 g promotes GC apoptosis in pigs by targeting TGFBR1 and downregulating the TGFβ signalling pathway  as well as by targeting CCND2 and BCL2L1 and regulating MAPK and p53 signalling (Cao et al., 2015). Additionally, a group of miRNAs including miR-15a, and miR-29a promote the accumulation of BAX , thus enhancing the apoptosis of human GCs (Sirotkin et al., 2010). In addition, the follicular to luteal transition process and ovulation are stimulated by the presence of steroidogenic genes that are also regulated by miRNAs Iwamune et al., 2014). Taken together, miRNAs control the biosynthesis of female steroidogenic hormones including oestrogen, progesterone, and androgens by regulating the proliferation, growth, and apoptosis of ovarian GCs and TCs.

VII. miRNAs AS REGULATORS OF ZYGOTIC AND EARLY EMBRYO REPROGRAMMING
Endometrial miRNAs along with parental miRNAs from the germline or uterine fluid could participate in maternal-embryo crosstalk (Saadeldin et al., 2015) and could play important roles during epigenetic reprogramming of the embryonic cells by regulating the expression and functions of genes related to early embryonic development (see Table S5).

(1) Roles during fertilization, ZGA, and epigenetic reprogramming of preimplantation embryos
Recent data suggested that sperm-borne miRNAs must be transferred into a fertilized embryo to maintain maternal and paternal communication. For example, Yuan et al. (2016) produced zygotes using Drosha conditional knockout (cKO) mouse-derived miRNA-depleted sperm and found the resulting zygotes to have reduced developmental potential. Of note, the developmental competence of these zygotes could be recovered through the injection of small miRNAs derived from WT sperm. Furthermore, they found 14 sperm-borne miRNAs to be present in both WT sperm and the zygote, but not in oocytes, indicating that sperm-borne miRNAs can affect the development potential of fertilized embryos. By contrast, most zygotic miRNAs are derived from maternal oocytes (Tang et al., 2007). Altered miRNA expression in cumulus cells could result in a poor ovarian response in women undergoing in vitro fertilization (Karakaya et al., 2015), and some miRNAs are highly specific to the blastocyst and are not expressed in the oocytes (Tulay et al., 2015). In addition, sperm-borne miR-34c-5p is thought to initiate the first cleavage divisions in the mouse , and is positively correlated with intracytoplasmic sperm injection outcomes, as well as high-quality embryos, implantation, pregnancy and live birth in humans , and the presence of miR-34c-5p indicates viability and high-quality spermatozoa in humans (Abu-Halima et al., 2014). Similarly, sperm-borne miR-34b/c and mir-449a/b/c play a critical role in the in vitro first cleavage division of mouse embryos Yuan et al., 2015). Based on the above findings, the presence of miR-34c-5p can be considered a non-invasive biomarker for viable sperm and embryos in the mouse and human. In future, miR-34c-5p could be a universal biomarker for viable mammalian sperm and embryos; however, it requires intensive validation using other species.
Another study suggested that miR-34b/c-and miR-449-null male mice were fertile, whereas miR-34b/c and miR-449 double-knockout (miR-dKO) male mice were infertile due to severe spermatogenic disruptions and oligoasthenoteratozoospermia . However, injection of miR-dKO round spermatids into fertile mouse oocyte successfully gave rise to healthy offspring : indicating that this miRNA family is essential for normal spermatogenesis, but not for fertilization and embryonic development .  are the most abundant miRNAs in early zygotes of mice , but their roles are not clear. Despite being silenced functionally, the biogenesis of miRNAs is not abrogated during fertilization to ZGA in mice (Tang et al., 2007;Suh et al., 2010), suggesting that miRNAs might have important roles during transformation of zygotes to pluripotent blastocysts (Tang et al., 2007;Suh et al., 2010). Some miRNAs are stage-specific: miR-125a and miR-496 are specific to four-cell bovine embryos, while miR-127, miR-145, and miR-496 are specific to eight-cell bovine embryos. However, most miRNAs showed fluctuating expression patterns according to the stage of bovine embryonic development . This switch in miRNA expression patterns between ZGA and pluripotent blastocyst involves massive molecular rewiring (Hemberger, Dean, & Reik, 2009), and thus miRNAs might play a vital role during the transition of fertilized oocytes to pluripotent blastocysts (Svoboda & Flemr, 2010). Based on these findings, we can conclude that parental miRNAs might have important but limited roles during fertilization and the ZGA; however, miRNAs might be a key regulatory player during the developmental transition from the ZGA to pluripotent blastocyst.
miR-29b has been suggested to target de novo DNA methyltransferases (Dnmt3a and Dnmt3b) transcripts in PGCs (Takada et al., 2009). The suppression of Dnmt by miRNAs safeguards proper DNA demethylation and establishes the new imprinting of genes. Similarly, Dnmt3a/3b expression was negatively regulated by miR-29b at the time of mouse ZGA . Given that DNA methylation plays a critical role in the morula-to-blastocyst transition, these data suggest a feedback relationship between the regulation of miRNA expression and DNA methylation. In addition, pluripotency markers including kruppel like factor 4 (Klf4) and nanog homeobox (Nanog) were downregulated in the morula stage upon miR-29b inhibition , indicating the involvement of miR-29b in the regulation of pluripotency. Similarly, the miR-290/302 cluster is involved with embryonic pluripotency, although an opposing role of this cluster was shown in one study . Again, differential expression of miRNAs subsets during trophectoderm specification  or severe preeclamptic placentas (Pineles et al., 2007) evidence a role for miRNAs during the implantation process. The endometrial miRNAs along with miRNAs from the uterine fluid also could participate during maternal-embryo crosstalk and play an important role during the epigenetic reprogramming of the embryonic cells (Fig. 5). Thus, accumulating evidence suggests that miRNAs are at least indirectly engaged in the epigenetic reprogramming process of early embryos.

miR-200 family:
Naturally expression decreases during implantation and forced expression reduced implantation rate by regulating Zeb1, Zeb2, Pten mediated proliferation and apoptosis of cells. Others: miR-451, miR-424, miR-125b, and miR-30b become downregukated during high progesterone level compared to the normal condition, and might have important role during hormonal regulation.  Mother to embryo  Based on their functions, miRNAs are categorized as anti-or pro-implantation miRNAs, pluripotency miRNAs, or miRNAs that promote differentiation and commitment of mesoderm, endoderm, ectoderm, and trophectoderm lineages. Parental miRNAs might be important regulators during embryonic germ layer specification; variation in the absorption of parental miRNAs could initiate variation in differentiation potential of the inner cell mass (ICM), and thus could direct the ICM towards different germ layers. For example, absorption of miRNA-A by an ICM cell could suppress the endoderm and ectoderm lineage in that particular cell and thus facilitate mesoderm commitment. Similarly, the absorption of miRNA-B by an ICM cell could suppress the mesoderm and ectoderm lineage in that respective cell and could direct the cell towards the endoderm lineage; the absorption of miRNA-C by an ICM cell might facilitate ectoderm commitment by suppressing the mesoderm and endoderm lineages. Muc1, mucin 1, cell surface associated; c-Myc, MYC proto-oncogene, BHLH transcription factor; TGFβ, transforming growth factor beta; Reck, reversion inducing cysteine-rich protein with kazal motifs; Mmp9, matrix metallopeptidase 9; Pten, phosphatase and tensin homolog; Ptgs2, prostaglandin-endoperoxide synthase 2; Zeb1, zinc finger E-box binding homeobox 1; Zeb2, zinc finger E-box binding homeobox 2; Tiam1, T-cell lymphoma invasion and metastasis 1; Rac1, Rac family small GTPase 1; Igf1, insulin like growth factor 1; Igf1r, insulin like growth factor 1 receptor; Oct4, octamer-binding protein 4; Nanog, nanog homeobox; Klf4, kruppel like factor 4; Smad5, SMAD family member 5; Id2, inhibitor of DNA binding 2; Lefty2, left-right determination factor 2; Acvr1b, activin A receptor type 1B; Smad2, SMAD family member 2; Hdac, histone deacetylase; TIMM8A, translocase of inner mitochondrial membrane 8A; EED, embryonic ectoderm development; SMAD4, SMAD family member 4.

Trophoblast cells
regulate early gastrulation-stage embryos (Berardi et al., 2012). In ESCs, miR-191 and miR-16-1 are abundant, and inhibit the formation of mesoderm by downregulating Smad2, an essential component of Activin-Nodal signalling (Hadjimichael et al., 2016), while miR-23a may suppress the differentiation of ESCs towards the endoderm and ectoderm lineages (Hadjimichael et al., 2016). By contrast, expression of miR-9, miR-124a, miR-155, and miR-708 facilitate the differentiation of mouse ESCs (Marson et al., 2008), and the overexpression of miR-21 facilitates the commitment of mesodermal tissues such as adipose and bone (Eguchi et al., 2013;Kang et al., 2013). Similarly, miR-303-367 and miR-290-295 clusters may play an important role during the patterning and specification of embryonic germ layers in humans and mice, respectively, by regulating Nodal signalling (Vidigal & Ventura, 2012). During this stage, BMPs, Activin/Nodal, and canonical Wnts signals play critical roles for the transition of inner cell mass (ICM) to mesoderm or endoderm, whereas non-canonical Wnts, fibroblast growth factors (FGFs), Notch, and hedgehog (Hh) signalling are closely involved in the transition of mesoderm posterior 1 homolog (Mesp1)-specified mesoderm to cardiac mesoderm. Embryonic mesoderm shows predominant expression of miR-145 and miR-302 in humans and miR-290, miR-93, and miR-17-5p in mice (Berardi et al., 2012). Similarly, the miR-200 family is known for its role in early mesoderm commitment by regulating snail family transcriptional repressor 1 (Snail) . miR-145 accelerates mesoderm and ectoderm commitment by targeting OCT4, sex determining region Y-box 2 (SOX2), and KLF4 in human ESCs (Xu et al., 2009), and miR-127 induces differentiation of mouse ESCs to the mesendoderm lineage by targeting left-right determination factor 2 (Lefty2) and regulating Nodal signalling (Ma et al., 2016b). let-7 and miR-18 target Acvr1b and Smad2, respectively, and may attenuate Nodal responsiveness as well as bias the blastomeres towards the ectoderm and mesoderm (Colas et al., 2012). Thus, in vivo blocking of let-7 and miR-18 activity may divert the presumptive mesoderm and ectoderm towards the endoderm (Colas et al., 2012).
Finally, early embryos may be enriched with a panel of parental miRNAs (Fig. 5), either through gametes (Tang et al., 2007;Yuan et al., 2016) or embryo-maternal crosstalk (Saadeldin et al., 2015;Bidarimath et al., 2017). Exosomes and cell-membrane-derived micro-and nano-vesicles could be the main medium for this embryo-maternal crosstalk (Diehl et al., 2012;Saadeldin et al., 2015), however, miRNAs could also be carried through the circulatory system by apoptotic bodies, RNA-binding lipoproteins and Argonaute protein complex (Ling, Bao-feng, & Jing, 2013;Sohel, 2016). These circulating miRNAs originating from one cell or organ could be transported to and absorbed by neighbouring or distant cells, for example, miRNAs originating from tumour cells could be absorbed by other healthy cells leading to their transformation to malignancy . Thus, it could be assumed that each cell of the embryonic ICM randomly absorbs different maternal miRNAs delivered through the circulatory system; this variation in miRNA intake among the cells of the embryonic ICM may lead the cells towards different lineages. For example, one cell may absorb a miRNA-A panel, leading to the mesoderm lineage, another may absorb a miRNA-B panel and commit to the endoderm lineage, while a third cell may absorb a miRNA-C panel to specify the ectoderm lineages (Fig. 5). However, further in-depth experiments, both in vitro and in vivo, are required to support these hypotheses.
(3) miRNAs regulate pregnancy by mediating adhesion molecules, vasculature and angiogenesis miRNAs involved in the implantation process can be categorized into two groups, anti-or pro-implantation miRNAs (Fig. 5); these miRNAs are potent regulators of many genes related to adhesion, angiogenesis, and vasculature (see Table S5), as well as foetal-maternal immune tolerance following endometrial reception of the conceptus (Bidarimath et al., 2014(Bidarimath et al., , 2015. Alterations in expression of these miRNAs in the uterine endometrium could cause implantation failure as well as disease (Hiroki et al., 2010). For example, the let-7 family regulates the expression of mucin 1, cell surface associated (Muc1) and facilitates implantation of mouse embryos (Inyawilert et al., 2015), the miR-30 family is generally upregulated at the implantation site and may participate in embryo-endometrium crosstalk and nourishment of the implanting embryo (Moreno-Moya et al., 2014), the miR-17-92 cluster is upregulated at the implantation site, and may regulate the decidualization (morphological and functional changes of the endometrium in preparation for pregnancy) and implantation process by targeting E2F and TGFβ signalling (Mogilyansky & Rigoutsos, 2013). Similarly, miR-21 is upregulated at the implantation site and regulates matrix metallopeptidase 2 (Mmp2) and matrix metallopeptidase 9 (Mmp9) expression by targeting reversion inducing cysteine-rich protein with kazal motifs (Reck) during implantation in mice (Hu et al., 2008;) and PTEN during the proliferation of endometrial epithelial cells (Qin et al., 2012). miR-29a is highly expressed in the uterus during implantation and inhibits apoptotic molecules , and miR-101a and miR-199a* regulate the implantation process by directly targeting Cox-2 (Ptgs), an important molecule for decidualization (Chakrabarty et al., 2007). A panel of miRNAs including miR-10a, miR-27a, miR-29c, miR-323, miR-331-5p, miR-339-3p, miR-374-5p, and miR-935 are reported to be associated with spontaneous foetal arrest in pigs (Wessels et al., 2013), while miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19 are associated with angiogenesis of the endometrial tissues in pigs (Bidarimath et al., 2015), and are differentially expressed between healthy and arresting conceptus attachment site (Bidarimath et al., 2015). Thus these miRNAs could be used to assess foetal health, although this requires validation with other mammalian species. Additionally, some miRNAs participate in the implantation process indirectly, such as by promoting the proliferation of trophoblast cells (Luo et al., 2012;Fu et al., 2013), by facilitating trophoblast migration Luo et al., 2012;Fu et al., 2013), by inhibiting the apoptosis of trophoblasts (Pineles et al., 2007), and by participating in conceptus-endometrial crosstalk (Bidarimath et al., 2017).
By contrast, the expression of oestradiol-and progesterone-regulatory miR-200 family members decreases naturally, while its targets Zeb1 and zinc finger E-box binding homeobox 2 (Zeb2) are upregulated during implantation in mice (Jimenez et al., 2016), and forced expression of miR-200a reduced the implantation rate of mouse embryos by regulating Pten-mediated proliferation and apoptosis of cells . Overexpression of miR-22 suppresses T-cell lymphoma invasion and metastasis 1 (Tiam1) and Rac family small GTPase 1 (Rac1), causing implantation failure , while overexpression of miR-145 also causes implantation failure in mice by blocking crosstalk between embryonic Igf1 and maternal insulin like growth factor 1 receptor (Igf1r) (Kang et al., 2015). Similarly, miR-451, miR-424, miR-125b, and miR-30b are downregulated when progesterone is elevated compared to that under normal conditions . Oestrogen-mediated inhibition of miR-705 facilitates Mmp9 translation ), which may be important for proper implantation of embryos and thus pregnancy. Some other miRNAs play anti-implantation roles by inhibiting proliferation (Dai et al., 2012;Gao et al., 2012), inducing apoptosis , and inhibiting migration Umemura et al., 2013) of the trophoblasts, as well as by suppressing vascularization and angiogenesis during implantation of embryos .
Active or pseudopregnancy could be confirmed by the expression of miR-21, which is expressed only in the presence of active embryos (Hu et al., 2008;Luense et al., 2009). As discussed earlier, miRNAs also participate in the maintenance of pregnancy by regulating angiogenesis and vasculogenesis of luteal tissues. In brief, miR-125b, miR-145, mir-31, miR-503 and miR-21 regulate interferon-mediated cell death, and vascularization and angiogenesis in ruminant CL , and miR-378 regulates the maintenance of the bovine CL by regulating its target IFNGR1 . miR-17-5p and let-7 play important roles in CL formation by targeting the anti-angiogenic gene Timp1 (Otsuka et al., 2008). In sheep, miR-199a-3p, miR-145 and miR-503 are expressed differentially at different stages of the CL and are involved in fibrosis, and immune and inflammatory responses . Taken together, miRNAs mediate implantation and pregnancy in mammals by regulating hormone levels throughout pregnancy, maintaining crosstalk between the embryo and mother, and facilitating angiogenesis and vascularization of the endometrium and luteal tissues.

VIII. CONCLUSIONS
(1) Loss of miRNA processing machinery could impair reproduction at the molecular level and causes infertility or sterility to different extents depending on sex, age and stage of loss, but the external sex characteristics along with mating behaviour remain normal in both males and females.
(2) miRNAs are mostly indispensable during pre-and post-fertilization stages (such as sex differentiation, germline establishment, gametogenesis, preimplantation development and implantation of embryos) but might have very limited roles during the fertilization event. miRNA-loss-mediated infertility therefore derives from the malformation of gametes, not because of its absence during the fertilization event itself.
(3) miRNAs could have a direct involvement in the global silencing of X-linked gene expression during the MSCI process of spermatogenesis and in the initiation of ZGA and the first embryonic cell division.
(4) Steroidogenesis in both sexes is regulated by potent feedback loops comprising miRNAs, genes, and hormones; deregulation in any of which could impair the whole process of mammalian reproduction.
(5) miRNAs could be used as fertility biomarkers for both gametes, and also to differentiate between pseudo and viable pregnancy.
(6) Parental miRNAs (derived from gametes or embryo-maternal crosstalk) could play very important roles in epigenetic modifications and germ-layer specification of embryos. They may also be important in the implantation and maintenance of pregnancy by regulating angiogenesis and vascularization of the endometrium tissues and CL.
(7) Much work remains to be done to understand the roles of miRNAs in mammalian reproduction, especially in MSCI, the first embryonic cell division and ZGA, germ-layer specification and embryo-maternal crosstalk.

XI. SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article. Table S1. Functions of microRNAs (miRNAs) in sex specification and commitment of the mammalian reproductive system. Table S2. Functions of microRNAs (miRNAs) in spermatogenesis and spermiogenesis.