MicroRNA in Ovarian Biology and Disease.

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MicroRNA in Ovarian Biology and Disease Lynda K. McGinnis1, Lacey J. Luense2, and Lane K. Christenson1 1

Department Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160

2

Epigenetics Program, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Correspondence: [email protected]

www.perspectivesinmedicine.org

MicroRNAs (miRNAs) are posttranscriptional gene regulatory molecules that show regulated expression within ovarian tissue. Most research investigating miRNAs in the ovary has relied exclusively on in vitro analyses. In this review, we highlight those few studies in which investigators have illustrated an in vivo effect of miRNAs on ovarian function. We also provide a synopsis of how these small noncoding RNAs can impact ovarian disease. miRNAs have great potential as novel diagnostic biomarkers for the detection of ovarian disease and in the assisted reproductive technologies (ART) for selection of healthy viable oocytes and embryos.

istorically, it was believed that the molecular biology of the cell was relatively simple: Messenger RNA was transcribed from DNA and formed the template from which proteins were produced. However, with the discovery of noncoding RNAs that often function as regulators of coding RNAs, we now know that these molecular processes are far more complicated with intricate interconnections and duplicated mechanisms of genetic regulation. In this review, we will focus on one family of these small noncoding RNAs known as microRNAs (miRNAs), explain their biogenesis, and explore their possible roles in ovarian development and diseases of the ovary.

H

miRNA BIOGENESIS

miRNAs are small RNA molecules consisting of short (17 – 25 nucleotides) noncoding RNAs

that repress expression of messenger RNAs (mRNAs). miRNAs act as posttranscriptional regulators of many processes in reproduction and development. They were originally termed small temporal RNAs (stRNAs) for their timed sequential expression and roles in the regulation of developmental events in Caenorhabditis elegans (Lee et al. 1993; Wightman et al. 1993; Banerjee and Slack 2002). In 2001, three papers published simultaneously in Science described the discovery of these tiny regulatory RNAs in human cells and it was agreed that these small RNAs would be renamed “microRNAs” (LagosQuintana et al. 2001; Lau et al. 2001; Lee and Ambros 2001). miRNAs commonly act by basepairing with the 30 untranslated region (30 UTR) of complementary messenger RNA targets, thus blocking the translation of select mRNAwith or without subsequent mRNA deg-

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L.K. McGinnis et al.

radation and effectively inhibiting gene expression (Bartel 2009). Most miRNAs are generated from long RNA strands termed primary RNA ( pri-RNA), that are cut by the enzyme nuclear RNAse III endonuclease, Drosha, and its RNA-binding cofactor DGCR8 (DiGeorge syndrome critical region gene 8) to form 70 – 100-nucleotide (nt) hairpin shaped precursor ( pre-) miRNA within the nucleus. Pre-miRNAs are transported out of the nucleus into the cytoplasm by exportin 5 (Yi et al. 2003) where they are cleaved by the RNAse III endonuclease Dicer, which removes the hairpin loop to yield duplex miRNA. One of the resulting strands of the duplex miRNA is then bound by the miRNA-induced silencing complex (miRISC) composed of, among other proteins, members of the Argonaute (AGO) family to yield the final active miRNA (reviewed in Christenson 2010). This pathway involving Drosha/DGCR8 is known as the “canonical” pathway of miRNA biogenesis. Noncanonical pathways also exist wherein miRNAs are created either without processing by Drosha/DGCR8 but still requiring Dicer or by passing directly from Drosha/DGCR8 to AGO2 thus bypassing Dicer (Castellano and Stebbing 2013). Because it was originally thought that all miRNAs required Dicer for expression, targeted deletion of Dicer in mice led to some initial erroneous conclusions (see section on miRNAs in the Oocyte, below). It is now known that other types of small RNAs (i.e., endogenous small interfering RNA) are also generated via Dicer, but these will not be covered in-depth here (reviewed in Christenson 2010). NOMENCLATURE OF miRNAs

Naming patterns for miRNA genes have been formalized (Griffiths-Jones et al. 2006). The initial nomenclature was based simply on the sequence of discovery. For instance, miR-1 was the first miRNA named under the established guidelines. A few notable exceptions to the numerical naming scheme included let-7 and lin-4, these have retained their original names owing to their historical significance (www .mirbase.org). Identifiers added to the front sig2

nify species (hsa, human; mmu, murine; etc.); for example, hsa-miR-21 refers to human miR21. Suffixes have also evolved over time. For example, a number placed after the miRNA name (i.e., miR-218-1 or miR-218-2) indicates the exact same mature miRNA sequence but informs the reader that it was derived from independent gene loci (in this case, human chromosomes 4 and 5, respectively). Previously, an asterisk ( ) was used to signify the less abundant strand of a miRNA duplex (i.e., passenger strand), which was thought to have no biologic activity. However, it is now recognized that, in certain tissues or cells, the miRNA can be more abundant and biologically active than the guide miRNA strand, and so the has now been replaced with a -5p or -3p designation. This -5p/-3p suffix is a very important distinction as these miRNA target very different sets of mRNA based on their different seed sequences (i.e., bases 2– 8 from the 50 end of the mature miRNA). The seed sequence are thought to be the primary bases that target the mi-RISC to specific mRNA targets, and are the bases used by most algorithms to identify potential targets. Lastly, a letter following the miRNA name (e.g., miR-378a, miR-378b, etc.) is used to indicate two miRNAs with closely related sequences, typically with 100% homology in the seed sequence. miRNA IN OVARIAN DEVELOPMENT

Ovarian development begins early in fetal life, although compartmental development (follicular growth and ovulation followed by luteal tissue development and death) continues throughout much of a female’s lifetime. Primordial germ cells (PGCs) enter the genital ridge by 8 wk (human) or 9 d (mouse) after fertilization (McLaren 1988). As the ovary forms, female germ cells multiply to form cysts of interconnected oogonia. Between 17 and 19 wk of gestation (human) or near the time of birth (mouse), oocyte cysts break down and each oocyte is enclosed by somatic cells termed pregranulosa cells (Byskov 1986; McLaren 1991). The new oocyte and companion cells have now formed a primordial follicle with direct cellto-cell contact between the oocyte and squa-

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MicroRNA in Ovarian Biology and Disease

mous pregranulosa cells. From this stage forward, select individual primordial follicles activate and initiate growth by mechanisms that are not fully understood. To elucidate the impact of miRNAs on ovarian development, multiple approaches have been undertaken. Targeted deletion of dicer and/or ago2 genes in mice has been performed to examine the net effect of miRNA on ovarian development. Complete knockout of either dicer or ago2 results in embryonic lethality between E7.5 and E9.5 (Bernstein et al. 2003; Liu et al. 2004). Therefore, tissue-specific conditional knockout mice (cKO) have been generated to study the function of these proteins during development. For instance, the effects of dicer on PGC development was examined by crossing mice with floxed-dicer alleles with alkaline phosphatase (tnap, now named alpl)-Cre mice. These mice termed Dicer-alpl-cKO mice express Cre-recombinase in the PGCs beginning at E10 (Lomeli et al. 2000; Hayashi et al. 2008). When crossed with a PGCspecific oct4-GFP transgenic mouse line, PGCs were isolated to produce a purified population of PGC cells for miRNA analysis. Fetuses ranging in age from E12.5 to E13.5—the gestational time period at which female PGCs undergo meiotic arrest and formation of primordial follicles begins—were examined for miRNA expression (Hayashi et al. 2008). Quantitative RT-PCR (qRT-PCR) amplification was used to determine age-specific changes in the expression levels of a panel of 214 miRNAs. miRNAs from the miR-17-92 cluster were highly expressed in PGCs, with the exception of miR-17-3p which was undetectable. Furthermore, Dicer-alpl-cKO animals had reduced numbers of germ cells, suggesting that both dicer and miRNAs are important for cell proliferation (Hayashi et al. 2008). The implication is that loss of dicer leads to a reduction or loss of the miR-17-92 cluster of miRNAs, which have known functions in cell proliferation (Mendell 2008). Targeted deletion of the miR-17-92 cluster in PGCs therefore provides conformational evidence for the importance of this cluster of miRNAs in early ovarian primordial follicle establishment. An alternate approach for determining specific miRNA involvement in early gonadal

development was accomplished by assessing changes in miRNA expression in whole fetal gonads rather than in PGCs. The timing of gonad collection spanned the critical period of sexual differentiation of the indifferent gonad to the ovary (E11.5 to E13.5 of mouse gestation). Several early studies on miRNA in gonadal tissue used this approach in an effort to identify either ovarian- or testis-specific miRNA (Ro et al. 2007; Hayashi et al. 2008). Unfortunately, most miRNAs that were originally designated as ovarian-specific have now also been detected in other tissues. However this approach has provided some leads to potentially important miRNA-regulated processes. The interactive regulation of sox9 and miR-124 is one example (Real et al. 2013). In this study, miRNAs that were significantly increased specifically in female gonads at E13.5 were considered to be involved in ovary differentiation. Fourteen miRNA were identified as such via microarray analysis and confirmed by qRT-PCR studies (Real et al. 2013). Bioinformatics analysis was then used to identify specific miRNA target sequences in the 30 UTR of genes associated with gene ontology (GO) terms related to sexual differentiation and yielded several potential candidate miRNAs. miRNA-124 was further investigated as it had previously been shown to bind to the mRNA of sox9, a gene known to be expressed in the undifferentiated gonad. Up-regulation of sox9 leads to testis formation, whereas low sox9 levels are associated with differentiation of the fetal gonad into an ovary. 30 UTR luciferase assays, in which the sox9 30 UTR is fused to the end of the luciferase coding sequence, followed by manipulation of miR-124 by either blockade or overexpression confirmed the ability of miR-124 to interact with sox9 30 UTR. To confirm a direct effect of miR-124 on ovarian development, an antagomir specific to miR-124 was transfected into primary germ cells and sox9 protein expression measured. In control XX germ cells, sox9 was absent from the nuclei; conversely, control XY germ cells had nuclear sox9 expression as expected. Antagomir inhibition of miR-124 activity resulted in increased sox9 mRNA and protein expression in the XX germ cell nuclei, similar to XY germ


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cells. The inverse experiment (i.e., overexpression of miR-124) similarly decreased sox9 mRNA in XY germ cells; however, sox9 protein levels were not significantly affected. The investigators concluded that sox9 expression levels in XY germ cells likely exceeded the suppressive capacity of miR-124 overexpression (Real et al. 2013). Confirmation of an in vivo effect for miR-124 in gonadal development remains to be completed to solidify this miRNA as a key regulator of ovarian development. This study of miR-124 is an excellent example of the methods typically used for the study of miRNAs that begin with a microarray screen comparing two tissues to identify specific miRNAs that are up- or down-regulated, followed by searches of 30 UTRs for potential target genes, and then followed by in vitro inhibition/overexpression studies. Often, as is in this case, subsequent in vivo studies to confirm the functional significance of a specific miRNA are not undertaken. A number of reasons may dictate why confirmatory in vivo experiments are the exception instead of the norm. The complexity and cost of designing appropriate in vivo experiments is likely an important factor that leads to the limited number of these types of studies. Redundancy of miRNAs (i.e., families of miRNA that modulate similar genes based on common seed sequences), as well as a lack of tissue specificity can also make it difficult to elucidate the function of a specific miRNA. Additionally, miRNAs are not expected to elicit all or none effects. For all these reasons, targeted gene deletion experiments of specific miRNAs often fail. Lastly, because research into miRNA function is in its infancy, such independent in vivo studies may still be ongoing. Examples of successful in vivo functional studies showing involvement of specific miRNAs in ovarian development include recent studies of the lin28/let-7 system defined through the use of transgenic and knockout mouse models. lin28 is an RNA-binding protein that down-regulates the biogenesis of let-7, a large family of tumor suppressor miRNAs. Recent studies have reported the involvement of this pathway in the control of PGC proliferation in both males and females, as lin28 knockout 4

mice produce fewer germ cells than control mice (West et al. 2009; Shinoda et al. 2013). In the adult male, spermatogonial stem cells compensate for this early reduction. However, because female germ cells do not proliferate in the adult, female lin28 knockout mice suffer a significant reduction in fertility owing to loss of PGCs, resulting in a reduction in the size of the germ cell pool of the adult female (Shinoda et al. 2013). Because let-7 miRNA are downstream targets of lin28, it was theorized that overexpression of let-7 would mimic loss of lin28. This was confirmed by examining inducible let-7 transgenic mice, which overexpress let-7 miRNA (Shinoda et al. 2013). Thus, expression of lin28 in the early ovary is critical for the down-regulation of miRNA let-7, which allows for the proliferation of PGCs necessary for normal adult ovarian function (Shinoda et al. 2013). To determine if lin28 was equally important in growing oocytes and/or early embryonic development, a transgenic RNAi mouse was generated in which lin28a and lin28b were selectively knockeddown in growing oocytes (Flemr et al. 2014). lin28a and lin28b are closely related genes whose mRNA is stored in high abundance in growing oocytes, but it was not known if they were critical for oocyte maturation and/or embryonic development. Using this transgenic RNAi approach, it was discovered that, in spite of lin28’s high abundance in growing oocytes, lin28 is not required for oocyte maturation or embryonic development before zygotic gene activation in the mouse; and loss of lin28 in growing oocytes had no effect on the levels of let-7 (Flemr et al. 2014). However, because recent studies have identified a novel isoform of dicer in mouse and rat oocytes (see section on miRNA in the Oocytes, below), lin28/let-7 may still play a critical role in oocyte maturation, fertilization, and embryonic development of other species (Flemr et al. 2013; Flemr et al. 2014). It is interesting to note that environmental factors and other maternal stressors can cause changes in gene expression and miRNA regulation in the fetus, resulting in abnormalities of ovarian development that can lead to disease in the adult (Barker 1990). For instance, prenatal





exposure to testosterone is known to cause polycystic ovarian syndrome (PCOS) in adult females as evidenced by increased follicle recruitment, disrupted cyclicity, and high estradiol secretion (Padmanabhan and Veiga-Lopez 2013). When pregnant ewes were exposed to high levels of androgens beginning at day 30 of gestation, the female offspring develop a PCOS phenotype as adults. Interestingly, this treatment also caused significant changes in gene and miRNA expression in fetal ovaries that persist into adulthood (Luense et al. 2011). Following 35 d of androgen exposure (E65– E100, term ¼ 147 d), this exposure of pregnant ewes to excess testosterone stimulated increased levels of Cyp19 and Srd5a1 (5-a-reductase) followed by a decrease in Cyp11a1 expression in the fetal ovaries (Luense et al. 2011). Such changes in steroidogenic gene expression would significantly alter steroid production in the developing ovaries. Similarly, significant changes were detected in miRNA expression, with 21 miRNAs up-regulated and 16 miRNAs down-regulated more than 1.5-fold. Two of these miRNAs (miR-15b and miR-497) were examined by qRT-PCR, which confirmed their up-regulation in the ovaries of testosterone-exposed fetuses. Bioinformatics analysis identified members of the insulin-signaling pathway as potential targets of miR-15b and miR-497, a pathway that is known to be dysregulated in PCOS patients (Luense et al. 2011). Subsequent in vitro and in vivo studies are needed to identify potential targets of these miRNA as well as define the functional importance of these miRNAs. miRNA IN FOLLICULAR DEVELOPMENT

The oocyte within the primordial follicle can remain arrested in prophase of meiosis-I for months to years, depending on the species. Cyclic signals of unknown origin then trigger the initiation of growth and development of a small subset of follicles. The oocyte then secretes a thick extracellular matrix forming the zona pellucida that creates a partial separation of the oocyte from the pregranulosa cell layer. As the cells are separating, the pregranulosa cells differentiate into cuboidal granulosa cells and begin

to multiply. Follicular growth continues with sequential growth of both the oocyte and the granulosa cells. As granulosa cells continue to proliferate and after several layers have been generated, a fluid-filled antrum forms between layers of granulosa cells (antral follicle). The synchronous growth of oocyte and granulosa continues, although the granulosa cells secrete the follicular fluid that forms the antral cavity of the enlarging follicle. This fluid is essential and is composed of a complex solution of proteins and other molecules that provides nutrition and molecular signaling between the various cells within the follicle. The formation of the antrum causes a separation and differentiation of the granulosa cells into cumulus (cells that border the oocyte) and mural cells (cells along the outer edges of the antral follicle). The layer of cumulus granulosa cells closest to the oocyte (corona radiata cells) also retain a direct communication with the oocyte through transzonal processes that stretch from the cumulus cells through the porous zona pellucida to contact the surface of the oocyte. Communication between the oocyte and cumulus granulosa is bidirectional through gap junctions, autocrine factors, and possibly through the transfer of small extracellular vesicles (exosomes/microvesicles). This sharing of molecules between oocyte and granulosa is essential to follicular development, as neither the oocyte nor the granulosa cells can survive alone. Because of this mutual dependence, regulation of gene expression is critical in both compartments to support the development of a healthy next generation. Several studies have characterized miRNA expression in different compartments of the adult ovary, including during periods of follicle development, oocyte maturation, and luteal function (Ro et al. 2007; Fiedler et al. 2008; Otsuka et al. 2008; Hossain et al. 2009; Yao et al. 2010; Ma et al. 2011; da Silveira et al. 2012; McBride et al. 2012; Yin et al. 2012). However, the majority of studies examining the role of miRNAs in the regulation of follicle growth have focused on granulosa cells (Christenson 2010). Numerous miRNAs have been identified with potential regulatory functions, either because of transcriptional up-regulation follow-


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ing the luteinizing hormone (LH) surge (miR21, miR-132, and miR-212) or through various bioinformatics and microarray techniques. In this section we will present some of this evidence (Fig. 1). As mentioned earlier, Dicer and/or AGO2 must both be expressed for a cell to generate mature miRNAs. To study the effects of miRNA pathways in the reproductive tract, our laboratory and others used conditional knockout mice by crossing the transgenic mouse line dicer fl/fl with the anti-Mullerian hormone receptor-2 – Cre (amhr2 cre/þ) mice. The amhr2 gene is expressed early in development in reproductive tissues that will form the ovary, oviduct, and uterus (Hong et al. 2008; Nagaraja et al. 2008; Gonzalez and Behringer 2009; Pastorelli et al. 2009). The Dicer-amhr2-cKO female mice lack Dicer expression in these reproductive tissues and suffer complete infertility due primarily to severe developmental defects of the oviduct and uterus. Within the ovary, amhr2 is specifi-

cally expressed in granulosa cells, thereby allowing the effect of loss of dicer to be examined in granulosa cells. Ovarian function within the Dicer-amhr2-cKO mice was compromised with both reduced natural and eCG/hCG-stimulated ovulation rates, an increased number of atretic follicles, and the presence of oocytes trapped within luteinizing follicles that failed to ovulate (Hong et al. 2008; Nagaraja et al. 2008). Overall, these studies indicate a requirement for Dicer and the resulting small RNAs within granulosa cells to support healthy development of the antral follicle and normal ovulation (Luense et al. 2009). miRNA IN SECONDARY AND EARLY ANTRAL FOLLICLES

Few studies have addressed the roles of miRNAs in small and growing follicles. This is due, at least in part, to the difficulty in isolating pure populations of small follicles and the limited

Antral miR-21 miR-132 miR-212 miR-513a-3p miR-125b

Secondary

Primary Primordial miR-224

Ovulation miR-17-92 cluster miR-124 Let-7

Corpus luteum

Let-7b miR-17-5p miR-122 miR-125 miR-378

miR-21 miR-224

Figure 1. MicroRNA involved in regulation of ovarian follicle development. Specific stages of follicular devel-

opment and those miRNA that have been confirmed through in vivo experiments to be involved in ovarian follicular development, ovulation, or luteal function are depicted.

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mechanisms available to study early folliculogenesis ex vivo. Additionally, although genetic and pharmacologic tools exist for selective knockdown of miRNA genes during early follicular development, these experiments are complex and often fail. As previously mentioned, this may be caused by the nature of miRNA regulation and temporal effects whereby a targeted knockout does not eliminate the previously synthesized miRISC, thereby failing to alter function. As a result, much of the current work examining miRNA effects on folliculogenesis are preliminary in vitro findings, which may explain some of the contradictory evidence found in the literature. Studies showing evidence of in vitro expression of miRNA involvement in varying aspects of granulosa cell functions are numerous and have been described in detail elsewhere (Luense et al. 2009; Donadeu et al. 2012; Nothnick 2012; Imbar and Eisenberg 2014). Unfortunately, most of the ovarian studies rely heavily on cultured granulosa cells or immortalized human granulosa cells lines treated with inhibitors or overexpression vectors, although critical in vivo functional validation studies are often lacking. In Table 1, we have summarized the current miRNA studies that have been linked to ovarian (follicular) function via in vitro assays as well as the putative pathways and functional impacts extrapolated from those in vitro analyses. Unfortunately, findings in these in vitro experiments may not reflect the endogenous in vivo situation. With no specific relationship to a documented in vivo effect, it is often difficult to interpret the overexpression and inhibitor studies. The roles of miRNAs are diverse and require careful validation and verification before they can be fully understood. The demonstration of specific in vivo regulation is an essential part of this validation. Research by early investigators provides the framework for future experimentation that can either confirm or refute the importance of these small RNAs in ovarian biology. It is important to acknowledge that many aspects of inter- and intracellular signaling are complex. For this reason, in vitro studies are important tools to help solve the mysteries of cellular communication. One example is the on-

going study of miRNA regulation of the transforming growth factor (TGF) signaling pathway. Proteins in the TGF-b1 superfamily play critical roles in follicular development, and miRNA regulation adds to the complexity of this signaling pathway. Yao et al. reported that miR-224 is up-regulated as one of 16 TGF-b1 responsive miRNAs identified in cultured murine preantral granulosa cells (Yao et al. 2010). Through bioinformatics investigation, smad4, an intracellular effector of TGF-b1 signaling (Moustakas et al. 2001), had been previously identified as a potential target of miR-224. Overexpression of miR-224 reduced the protein levels of smad4 in cultured granulosa cells, although it had an insignificant effect on mRNA expression (Christenson 2010; Yao et al. 2010). miR-224 is cotranscribed from within an intron of GABRE, which is itself a TGF-b1 responsive gene. GABRE and, thus miR-224, are negatively regulated by the tumor suppressor genes p53 and p65. Knockdown of p53 and/or p65 increased levels of miR-224 and enhanced TGF-b1-mediated granulosa cell proliferation (Liang et al. 2013). A recent study from this same research group indicated that miR-224 binds the 30 UTR of pentraxin 3 ( ptx3), a gene that is critical for cumulus expansion (Yao et al. 2014). For this to occur, one would anticipate that expression of miR-224 must decline following LH stimulation; however, the results of these studies showed highly variable and insignificant changes in miR-224. This study claimed that TGF-b1 caused a decrease in miR-224, thus enabling the necessary increase in ptx3 and cumulus expansion following LH/hCG treatment (Yao et al. 2014). Oddly, this is in contrast to their earlier studies that showed miR-224 increased in response to TGF-b1 in preantral granulosa cells of immature mice (Yao et al. 2010). It is important, therefore, that these studies be verified and extended to other model systems to confirm or refute the functional importance of this miRNA. With so many positive and negative regulators of the TGF pathway, it is always possible that other effectors could cause a differential response in different cell types. These studies provide a clear example of the complexity of studying miRNA in ovarian function.


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Table 1. miRNA expression in ovarian cells/tissue


miRNA

Tissue/cell

In vitro

Let-7 Let-7b 10a 15a 17-5p 21

Primordial germ cells Ovary Granulosa Granulosa Ovary Granulosa

X X X X X X

23a 29b 105 122 124 125b 132 133b 136-3p 143 145 145 151 181a

Granulosa Primordial germ cells Granulosa Ovaries Ovary cells Granulosa Granulosa Granulosa Ovaries Pre-granulosa Granulosa Primordial follicle Oocyte Granulosa Oocyte/embryo

X X X X X X X X X X X X X X

182 188 196a 212

Granulosa Granulosa Oocyte/embryo Oocyte/embryo Granulosa

X X X X

224

Granulosa

X

378 378-3p 383 430 513a-3p

Granulosa/corpus luteum Granulosa Granulosa/oocyte Oocyte/embryo (zebra fish) Granulosa

X X X X X

miRNA IN ANTRAL FOLLICLES

As the early antral follicles grow and mature, follicle stimulating hormone (FSH) and estradiol cause levels of luteinizing hormone receptor (LHCGR) to increase on the granulosa cells. This induction is essential for selection of the dominant follicle(s) as well as for the subsequent LH-responsive events within granulosa cells following the LH surge leading to ovulation. On a molecular level, the LH surge initiates major changes in gene activity within the preovulatory 8

In vivo

References

X X

Shinoda et al. 2013 Otsuka et al. 2008 Sirotkin et al. 2010 Sirotkin et al. 2010 Otsuka et al. 2008 Carletti et al. 2010; Mase et al. 2012 Yang et al. 2012 Takada et al. 2009 Sirotkin et al. 2010 Menon et al. 2013 Real et al. 2013 Sen et al. 2014 Fiedler et al. 2008 Dai et al. 2013 Kitahara et al. 2013 Zhang et al. 2013a Yan et al. 2012 Yang et al. 2013 Garcia-Lopez et al. 2013 Lingenfelter et al. 2011; Zhang et al. 2013b Sirotkin et al. 2010 Sirotkin et al. 2010 Tripurani et al. 2011 Fiedler et al. 2008; Tripurani et al. 2013 Yao et al. 2010; Liang et al. 2013; Yao et al. 2014 Ma et al. 2011; Xu et al. 2011b Toms et al. 2015 Yin et al. 2012 Giraldez et al. 2006 Troppmann et al. 2014

X X

X

X

X

X

granulosa cells resulting in alterations in many subcellular pathways, including transcription factors, matrix-remodeling proteins, and miRNAs (Richards 2007; Xu et al. 2011a). Following the LH surge, changes in miRNA expression are indicative of overall global changes in gene expression (Fiedler et al. 2008). Using a bioinformatics approach, Troppmann et al. examined the 30 UTR sequences of the LHCGR gene in search of miRNAs that might regulate its expression (John et al. 2004; Lewis et al. 2005; Troppmann et al. 2014; www.targetscan.org; 222.microrna





.org). Their analysis identified miR-513a-3p as a potential regulator of LHCGR. This miRNA was detectable in whole ovary lysates as well as human granulosa cells collected from large antral follicles of women undergoing assisted reproduction. Further examination of the gene sequence for miR-513a-3p identified it as an X-linked gene that is found only in primates. To determine if there was a relationship between expression levels of LHCGR and miR-513a-3p, the investigators measured the levels of both gene products in human luteinized granulosa cells collected during oocyte retrievals in assisted reproductive technologies (ART) throughout the time cultured in vitro (Troppmann et al. 2014). At the time of collection, LHCGR expression was low, but miR-513a-3p was high. Over time, as LHCGR levels decreased miR-513a-3p levels increased. Thus, they were able to show an inverse relationship between miR-513a-3p and the expression of LHCGR, supporting a role for miR-513a-3p in regulating LH receptor expression in granulosa cells (Troppmann et al. 2014). Future in vivo studies are needed to verify a direct regulatory function of miR-513a-3p on the human LHCGR. Our laboratory approached the study of miRNA regulation of post-LH gene expression using an in vivo mouse model. With this model, we identified rapid changes in granulosa cell miRNA following the LH surge in vivo, including increases in miR-21, miR-132, and miR-212

(Fiedler et al. 2008; Carletti et al. 2010). miR132 and miR-212 lie in tandem within the first intron of gene 11700016P03Rik and are therefore both part of the same pri-miRNA transcript. In neuronal cells, their expression is stimulated by the cAMP regulatory element-binding protein (creb) (Vo et al. 2005). Because creb is activated by LH signaling, this may explain the up-regulation of miR-132 and miR-212 following LH stimulation (Fiedler et al. 2008). Bioinformatics analysis of miR-132/212 sequences against the 30 UTR of existing ovarian mRNA databases identified 77 putative targets of which ctbp1 was of special interest. Ctbp1 (carboxyterminal binding protein 1) interacts with steroidogenic factor-1 (sf-1) thereby modulating its ability to stimulate promoter activity and regulating steroidogenesis in adrenal cells (Dammer and Sewer 2008). It is tempting to suggest a similar role for miR-132/212 in granulosa cell steroidogenesis, but this has not yet been proven. The LH/hCG stimulation of granulosa cells had no effect on mRNA levels of ctbp1, but significantly decreased ctbp1 protein levels supporting a role for miRNA posttranscriptional control (Fiedler et al. 2008). Our laboratory used a targeted gene deletion approach to test this hypothesis, but was unable to detect any effect of combined loss of miR-132/212 within granulosa cells on female fertility (Table 2). Interestingly, recent evidence in the bovine oocyte suggests that miR-212 controls the oocyte-spe-

Table 2. Fertility analysis in mice with miR-212/132 conditionally knocked out in granulosa cells Mouse line

miR-212/132-Amhr2-WT miR-212/132-Amhr2-cKO miR-212/132-Cyp19-WT miR-212/132-Cyp19-cKO

Number of litters

6 5 14 15

Pups/litter (mean + SEM)

8.2 8.4 7.9 6.6

+ 0.54 + 0.51 + 0.52 + 0.52

Ovulation rate (mean + SEM)

21.2 14.3 16.0 16.8

+ 4.5 + 2.5 + 3.4 + 1.6

Mice with the miR-212/132 allele flanked by loxP sites (miR-212/132fl/fl [Magill et al. 2010]) were crossed to Amrh2-Cre (Jorgez et al. 2004) mice to conditionally delete miR-212 and miR-132 in secondary and small antral follicles. Similarly, miR212/132fl/fl mice were crossed with Cyp19-Cre (Fan et al. 2008) to conditionally ablate miR-212 and miR-132 expression in granulosa cells of late-stage antral follicles and luteal cells. miR-212/132-Amhr2 wild type (WT) and conditional knockout (cKO) females (n ¼ 2/genotype) as well as miR-212/132-Cyp19-WT and cKO females (n ¼ 5/genotype) were bred to wildtype males of known fertility for 3 mo. The total number of litters and pups were recorded. To investigate ovulation in miR212/132 conditional knockout lines, juvenile females (n ¼ 6/genotype) were exposed to a low dose (2IU) of equine gonadotropin for 44 h followed by 2IU of human chorionic gonadotropin (hCG) to induce ovulation. Sixteen hours posthCG injection, ovulated cumulus oocyte complexes were dissected from the oviduct and total number was recorded to determine ovulation rate.


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cific expression of figla (see section on miRNA in the Oocytes, below) (Tripurani et al. 2013). Further studies may be warranted to assess the roles of miR-132/212 in the ovary. Another miRNA shown to be significantly and rapidly up-regulated in granulosa cells following in vivo hCG/LH stimulation was miR-21 (Fiedler et al. 2008; Carletti et al. 2010). miRNA21 is up-regulated in many cancers and tumors. It has been heavily studied for a possible role in carcinogenesis, and it is highly expressed in human granulosa cells (Mase et al. 2012). Knockdown of miR-21 using specific inhibitors causes granulosa cell apoptosis, suggesting a role for miR-21 in the maintenance of granulosa cells within the preovulatory follicle (Carletti et al. 2010). Most importantly, in vivo blockade of miR-21 action by administration of a blocking oligonucleotide into the ovarian bursa prevents ovulation (Hong et al. 2008; Carletti et al. 2010). Interestingly, the in vivo inhibition of miR-224 also disrupts ovulation (Yao et al. 2014). These studies show that loss of the activity associated with specific miRNAs in vivo can recapitulate the ovarian Dicer-knockout phenotype and imply critical functions for miRNAs in ovulation. Recently, miR-125b has been identified as an effector downstream from the androgen receptor in murine granulosa cells (Sen et al. 2014). In this study, androgens up-regulated miR-125b expression in granulosa cells, which in turn suppressed apoptotic pathways, thereby increasing granulosa cell survival (Shi et al. 2007; Sen et al. 2014). Additionally, using granulosa cells from androgen receptor knockout (ARKO) mice, miR-125b expression was decreased and this resulted in increased apoptotic protein levels. Because anomalies of androgen signaling are a hallmark of PCOS, these studies indicate a possible role for miR-125b in this disease (see discussion below). miRNA IN THE CORPUS LUTEUM

After ovulation, the mural granulosa cells differentiate to form the corpora luteum (CL). The CL is an endocrine tissue that undergoes rapid transformation following ovulation. This endocrine gland remains as hormonal support 10

for early pregnancy then regresses either near the time of birth or, in the absence of pregnancy, immediately before the initiation of the next menstrual/estrous cycle. Currently, one study has definitively addressed the question of miRNA involvement in luteal function, although several additional studies have identified a number of notable miRNA including let-7b, miR-17-5p, miR-122, miR-125, and miR-378 within luteal tissues at different physiological states (i.e., development, pregnancy, and CL regression). Studies by Otsuka et al. 2008 have implicated miRNA regulatory mechanisms in the function of the CL. Since total loss of dicer is embryonic lethal, these researchers created a dicer hypomorphic mouse (dicer hypo), that expresses low levels of dicer and survive into adulthood. Female dicer hypo mice are infertile owing to CL insufficiency, a condition known to cause recurrent miscarriage in women (Otsuka et al. 2008). The investigators propose that the lack of luteal tissue vascularization is caused by the loss of several miRNAs (such as let-7b and miR-175p), which may regulate the antiangiogenic factor, tissue inhibitor of metalloproteinase-1 (timp1). Replacement of let-7b and miR-17-5p by direct injection into the bursa of pregnant dicer hypo mice can partially rescue the phenotype with improved vasculature and increased progesterone levels. However, this single dose treatment was not able to maintain pregnancy, suggesting that other factors are likely also involved or that long-term/repeated inhibition would be needed to induce a sustained longterm effect (Otsuka et al. 2008). Ma et al. (2011) used a bovine model to compare miRNA expression patterns of fully functional CL producing high levels of progesterone versus CL undergoing regression (Ma et al. 2011). Not surprisingly, marked differences in miRNA expression were observed. miRNA378 showed the greatest amount of up-regulation in nonregressing CL and was examined further in an effort to identify a possible functional role for this miRNA in CL maintenance. miRNA-378 had previously been reported to be involved in apoptosis by causing a downregulation of interferon gamma receptor 1





(IFNGR1) gene expression. Quantitative RTPCR studies confirmed high levels of miR-378 and IFNGR1 mRNA in the CL at middle and late stages of CL maintenance, but much lower levels of miR-378 in the regressing CL. Although there was no change in the levels of IFNGR1 mRNA during luteal regression, there was a significant increase in protein expression supporting the likelihood of posttranscriptional regulation of IFNGR1 (Ma et al. 2011). Interestingly, miR-378 has also been reported to down-regulate CYP19 expression in porcine granulosa cells (Xu et al. 2011b). CYP19, also known as aromatase, is an estrogen synthesizing enzyme that is highly expressed immediately before ovulation and the initiation of CL formation. Interestingly, miR-378 and miR-378 are generated from a single hairpin pre-miRNA from within the first intron of the peroxisome proliferator-activated receptor-g coactivator 1b (PGC-1b) gene and are thus coexpressed (Carrer et al. 2012). Although we did not find any reports for activity of PGC-1b in ovarian cells, genetic variations of the closely related gene PGC-1a have been identified in women with PCOS (Wang et al. 2006; San-Millan and Escobar-Morreale 2010). Because of the multiple instances of miR-378 being implicated in ovarian function by in vitro experiments, in vivo studies of this particular miRNA are clearly needed to fully validate the biologic activity of this miRNA. miRNA IN THE OOCYTE

As the oocyte grows within the follicle, it accumulates maternal mRNA and proteins on which it relies until after fertilization and activation of the embryonic genome. Expression levels of genes involved in the miRNA biogenesis pathway were examined in primate oocytes and cleavage stage embryos within the PREGER gene expression database (Zheng et al. 2004; Mtango et al. 2009; www.preger.org). These studies indicated that mRNA levels of Drosha, DGCR8, and Dicer, essential components of the miRNA-processing pathway, increased during primate oocyte maturation. Interestingly, the majority of the miRNA biogenesis pathway expressed in oocytes and early embryos are insen-

sitive to a-amanitin, an RNA polymerase inhibitor. Thus, their expression in cleavage stage embryos is generated primarily from stored maternally derived mRNA (Mtango et al. 2009). Unique species differences with respect to miRNA biogenesis in the oocyte have been uncovered that dramatically impact our understanding of oocyte biology. These differences and their relevance to understanding the human oocyte are discussed below. The majority of oocyte miRNA studies have been conducted on mouse oocytes that, in addition to expressing full-length dicer, also produce an oocyte-specific form of dicer that is not expressed in other species (Flemr et al. 2013). The dicer gene of mice and rats includes an MTC retrotransposon promoter within intron 6 that generates an amino-terminally truncated isoform of dicer (Dicer o) that is particularly efficient at generating endogenous small interfering RNA (siRNA) and is essential for mouse oocyte function (Flemr et al. 2013). Therefore, it is predicted that the majority of small RNA species produced in the mouse oocyte are siRNAs rather than miRNAs. Double stranded siRNAs are generated through the Dicer/AGO2 pathway but, unlike miRNAs, siRNAs do not rely on DGCR8. Knockout of dicer o causes female infertility and abnormal metaphase spindles during maturation of mouse oocytes, similar to the original Dicer-zp3-cKO and AGO2zp3-cKO mice, thus confirming these earlier studies (Murchison et al. 2007; Kaneda et al. 2009; Flemr et al. 2013). DGCR8-zp3-cKO oocytes, on the other hand, fertilize and develop with no apparent abnormalities (Suh et al. 2010). These earlier studies have proven a requirement for dicer and ago2 but not dgcr8 pathways for mouse oocyte maturation. The precise cause of the metaphase failure during maturation is unknown, but is likely caused by the specific targeting of genes involved in the regulation of cytoskeleton and cell cycle progression by endogenous siRNAs (Tam et al. 2008; Watanabe et al. 2008; Flemr et al. 2013). As most oocyte-specific miRNA research has been conducted primarily in rodent species that express the truncated variant dicer o that is not present in other species, it becomes critical


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to examine the relative roles of miRNA and siRNA in additional species. Evidence in other species in which Dicer knockouts can be easily generated with or without the reintroduction of individual miRNAs have shown that, in species such as Zebrafish and Xenopus, miRNAs target maternal RNAs leading to select deadenylation and degradation of maternal transcripts. Thus, miRNAs are functional and essential in the oocytes of these species (Giraldez et al. 2006; Lund et al. 2009). Several interesting studies have been conducted in bovine oocytes that support an important role for miRNAs in that species also. One such study examined the role of miR-212 in the regulation of FIGLA. FIGLA is an oocyte-specific transcription factor that is essential for follicle development and for coordinated expression of the zona pellucida proteins, ZP1, ZP2, and ZP3 (Liang et al. 1997). Expression remains high throughout oocyte development then declines during oocyte maturation before reaching nondetectable levels in the embryo at the time of maternal-zygotic transition (Tripurani et al. 2013). To determine if miRNAs may regulate the expression of FIGLA, potential binding sites in the 30 UTR of bovine FIGLA mRNA were examined using MicroInspecto software based on an algorithm for detecting putative binding sites between mRNA and candidate miRNA sequences (Tripurani et al. 2013). This analysis predicted miR-212 as a regulator of FIGLA in bovine germ cells. miR-212 is produced in tandem with miR-132, and together they are upregulated in granulosa cells following the LH surge follicles (see section on miRNA in Antral Follicles) (Fiedler et al. 2008). Examination of the expression pattern of miR-212 in bovine tissues found it highly expressed in germinal vesicle oocytes with a tendency to increase in the cleavage stage embryo up to the eight-cell stage, the time at which the bovine embryo undergoes that transition from maternal to zygotic (embryonic) gene regulation. This pattern of expression matches other miRNAs predicted to control transcript turnover at the time of maternal-zygotic transition, including Zebrafish miR-430 (Giraldez et al. 2006), Xenopus miR427 (Lund et al. 2009), and mouse miR-290 12

(Tang et al. 2007). The expression of miR-212 in the oocyte and cleaving embryo is inversely correlated with FIGLA expression, suggesting a possible negative regulatory role of miR-212 (Tripurani et al. 2013). Using cell culture models transfected with FIGLA and/or miR-212, it was shown that miR-212 does indeed bind to the 30 UTR region of FIGLA mRNA. Microinjection of a miR-212-mimic into bovine embryos caused a reduction in FIGLA protein expression in eight-cell embryos, suggesting that miR-212 may play a role in the regulation of the transcription factor FIGLA (Tripurani et al. 2013). Similar studies have implicated miR-196a and miR181a in the regulation of oocyte-specific maternal effect genes, NOBOX and NPM2, respectively (Lingenfelter et al. 2011; Tripurani et al. 2011). To date, few studies have examined miRNA expression in human oocytes. In one such study, Kakourou et al. (2013) compared gene expression of human MII oocytes (immature oocytes collected at 40 h post-hCG then “matured” 4 h in vitro) to that of human blastocysts (surplus frozen/thawed blastocysts donated for research). Using a “genome survey microarray”, the mRNA levels of miRNA biosynthetic genes were compared between oocytes and blastocysts. A select list of candidate genes was created, including “housekeeping” genes and genes that have been characterized in human embryonic stem cells. A list of miRNAs was generated from the nonhuman primate PREGER gene expression database (Mtango et al. 2009). Similar to nonhuman primates, Dicer and Drosha gene products were detected in both human oocytes and blastocysts. The gene trinucleotide repeatcontaining gene 6B (TNRC6B), a component of the RISC complex, was particularly highly expressed in oocytes. Exportin 5 (XPO5), the protein that transports pre-miRNA from the nucleus to the cytoplasm, was expressed at significantly higher levels in the blastocyst. The increase in Drosha, Dicer, gem (nuclear organelle) associated protein 5 (GEMIN5), and TNRC6B in the oocyte is similar to that found between mouse oocytes and blastocysts (Zheng et al. 2004; Cui et al. 2007; Mtango et al. 2009). MOV10, which is also involved in the RISC complex of human cells (Meister et al. 2005), was




undetectable in the nonhuman primate but was highly expressed in the human blastocyst (Kakourou et al. 2013). PIWIL1, another member of the RISC complex, is involved in maintaining germline stem cells in drosophila, but was not detected in either nonhuman primate or human samples (Mtango et al. 2009; Kakourou et al. 2013). These studies show the complex interplay of proteins and miRNA expression, each depending on selective expression of the other. miRNA IN OVARIAN DISEASES


Ovarian Cancer

Several studies have implicated miRNAs in the development and pathogenesis of ovarian cancer (Di Leva and Croce 2013). Additionally, because the miRNA content of cancer cells is different from healthy cells, miRNA have been proposed as possible markers to detect and stage ovarian cancer (Banno et al. 2014). This field has been the subject of numerous recent reviews and, as such, will not be addressed herein (for further information, see Di Leva and Croce 2013; Banno et al. 2014; Titone et al. 2014). Early Reproductive Senescence in Females

Women with premature ovarian failure (POF) show evidence of early menopause, including amenorrhea, elevated levels of gonadotropins, and hypoestrogenism (Nelson et al. 2005). This disease afflicts 1% to 3% of women under the age of 40 (Pouresmaeili and Fazeli 2014). A number of genetic and immunologic disorders have been implicated in the etiology of POF (Laml et al. 2002), including such conditions such as autoimmune oophoritis (Bakalov et al. 2005; Jasti et al. 2012) and fragile X mental retardation syndrome (Corrigan et al. 2005). Whether miRNAs have a direct influence on POF is unknown, but a growing body of evidence suggests that miRNAs may be involved in fragile X syndrome disorders which are also known to cause POF (Laml et al. 2002; Jin et al. 2004; John et al. 2004; Li et al. 2008; AlvarezMora et al. 2013; Zongaro et al. 2013).

Circulating miRNAs were first identified in maternal plasma in 2008 (Chim et al. 2008) and have since been identified in most body fluids. As such, they are considered as possible biomarkers for the identification of specific cancers and other disease states (Yang et al. 2012). In an effort to better understand the causes of POF, serum levels of miRNAs from women with normal ovarian function were compared with women with POF (Yang et al. 2012). This microarray study detected 12 differentially expressed miRNAs, with 10 being greater (miR202, miR-146a, miR-125b-2, miR-139-3p, miR654-5p, miR-27a, miR-765, miR-23a, miR-3423p and miR-126) and two being significantly lower in POF patients (let-7c and miR-144). Quantitative RT-PCR validation studies using a larger cohort of 39 POF and 20 normal women confirmed increased expression of several selected miRNAs (including miR-23a, miR-27a, miR-126, and miR-146a). A more detailed analysis of miR-23a was undertaken because of its potential role in regulating expression of the Xlinked inhibitor of apoptosis (XIAP). XIAP has been implicated in the maintenance of ovarian function and is known to be regulated by miR23a in brain tissues (Siegel et al. 2011; Yang et al. 2012). XIAP also functions as an antiapoptotic agent in rat granulosa cells (Li et al. 1998). As predicted, transfection of miR-23a into human granulosa cells caused down-regulation of XIAP protein, whereas inhibition of miR-23a significantly increased XIAP mRNA and protein expression (Yang et al. 2012). Subsequent increases in cleaved caspase were also detected, suggesting that miR-23a can induce apoptosis and granulosa cell death (Yang et al. 2012). A direct in vivo cause and effect relationship in POF patients has not been shown, and it remains to be determined if the high levels of circulating miR-23a is a result or a cause of POF. Zhao and Rajkovic (2008) have proposed involvement of miRNAs in the ovarian syndrome associated with mutations of the fragile X mental retardation gene (FMR1). Mutation of this gene causes severe mental retardation and other developmental abnormalities. Women who carry a single copy of the mutated FMR1 gene (one X chromosome affected) do not have


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mental retardation, but can be afflicted with a related syndrome known as fragile X-associated primary ovarian insufficiency (FXPOI) which includes subfertility, irregular menstrual cycles, and/or early reproductive failure (menopause) (Corrigan et al. 2005). About 2% of women who experience ovarian insufficiency and 7% of women with a family history of ovarian insufficiency have mutations of FMR1 gene. FMR1 is an RNA-binding protein that interacts with components of the miRNA processing pathway, including Dicer, AGO1, and AGO2 (Jin et al. 2004; Li et al. 2008; Zhao and Rajkovic 2008). FMR1 mutations cause significant changes in gene expression, including miRNA levels (Alvarez-Mora et al. 2013; Zongaro et al. 2013). Taken together, the interplay between miRNAs and the FMR1 gene and protein are strong indications that miRNA may play a role in fragile-X-associated infertility (Zhao and Rajkovic 2008). This observation suggests that early ovarian failure can be caused, either directly or indirectly, by changes in miRNA pathways. Polycystic Ovarian Syndrome

PCOS in an endocrinopathy first identified in 1935 (Stein and Leventhal 1935), which afflicts 6% to 25% of women of reproductive age (Setji and Brown 2014). The specific criteria for diagnosis include hyperandrogenism, anovulation or oligo/amenorrhea, and subfertility (Setji and Brown 2014). Several studies have implicated miRNAs in the pathogenesis of PCOS. In one recent study, Roth et al. (2014) compared miRNAs isolated from follicular fluid of women with PCOS versus controls collected at the time of oocyte retrieval during ART procedures. Microarray analysis identified 235 miRNAs in human follicular fluid, of which 29 were differentially expressed between the two cohorts. Ten of the 29 miRNAs that were overexpressed in PCOS patients were selected for validation by qRT-PCR, and five (50%) of these were confirmed to be significantly increased in PCOS patients, including miRNA-32, miRNA34c, miRNA-135a, miRNA-18b, and miRNA-9. Pathway analysis suggested regulation of genes involved in insulin production and inflamma14

tion. Three genes that were identified as potential targets of these miRNAs have been found to be significantly reduced in patients with PCOS, namely interleukin 8, synaptogamin 1, and insulin receptor substrate 2 (Roth et al. 2014). Further studies are needed to determine whether miRNAs play a role in the etiology of PCOS or if they are downstream effects of other PCOS risk factors. Hossain et al. (2013) examined changes in miRNA expression in a rat model of dihydrotestosterone (DHT)-induced PCOS. This study found significant changes in miRNA content within follicular fluid (17 up- and 72 downregulated). Only three miRNAs (miR-9, miR32, and miR-489) were increased in both women with PCOS and the DHT-induced PCOS rats, emphasizing the importance of correlating in vivo animal models with clinical relevance in humans. miR-9 has been studied extensively for its role in regulating the expression of BRCA1 and other genes associated with ovarian cancer (Laios et al. 2008; Sun et al. 2013). Although, no direct link has been found in PCOS (Barry et al. 2014), the possible links between miR-9 and BRCA1 need further research. The evidence that miR-125 can impact folliculogenesis and regulate androgen action in the ovary suggests that it too may play a role in the PCOS phenotype in women (Sen and Hammes 2010; Sen et al. 2014). Androgens up-regulate miR-125b in granulosa cells which, in turn, suppresses apoptotic pathways thereby increasing granulosa cell survival (Shi et al. 2007; Sen et al. 2014). Granulosa cells from ARKO mice have high rates of follicular atresia, low miR-125b expression, and high levels of proapoptotic proteins. It would be interesting to know if miR-125b is up-regulated in granulosa cells of PCOS patients and whether modulation of its expression could provide a potential target for treatment. Assisted Reproductive Technologies

Visual assessment is currently the most common technique used for selecting an oocyte or embryo for transfer. New noninvasive techniques are needed that will enable the selection





of oocytes and embryos with the highest developmental potential. Recent breakthroughs in single-cell microarray technologies may provide a practical test for the ART clinic. miRNA and mRNA can be detected and quantified in the first polar body and these values are highly correlated with their sibling human oocytes (Reich et al. 2011). This procedure enables the testing of oocyte quality before fertilization and would be a useful option for patients with ethical objections to creating surplus embryos. After fertilization, a less invasive approach would be to test the embryo culture media instead of embryo biopsies for the presence of secreted miRNAs. Initial studies have shown that the types of miRNAs secreted into the culture media are differentially expressed depending on the method of fertilization (ICSI or IVF) and may correlate with pregnancy outcomes. These data suggest that miRNAs within the secretome of cleavage stage embryos may serve as potential biomarkers of embryo developmental potential (Rosenbluth et al. 2014). The detection of RNAs from polar body biopsy of oocytes and/or embryonic secretome during culture may be used in the future to select oocytes and/or embryos with the highest developmental potential. SUMMARY

Data from numerous studies suggest that miRNAs are expressed in different cell types throughout the ovarian cycle, but we have only just begun to understand the implications of these gene regulators in ovarian development and function. Increasing our understanding of the functions of miRNAs in the ovary will allow us to develop novel biomarkers to detect diseases of reproductive tissues and possibly to aid in the selection of oocytes and embryos for ART. With increased access to high throughput, nextgeneration RNA-sequencing capabilities, future studies will allow us to identify novel or low abundance miRNAs that are expressed in the ovary that may have been missed in initial microarray approaches. Furthermore, as miRNAs with validated targets and functions are better understood, we can begin to investigate their use as therapeutic or contraceptive targets.

ACKNOWLEDGMENTS

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MicroRNA in Ovarian Biology and Disease Lynda K. McGinnis, Lacey J. Luense and Lane K. Christenson Cold Spring Harb Perspect Med published online May 18, 2015 Subject Collection

Molecular Approaches to Reproductive and Newborn Medicine

MicroRNA in Ovarian Biology and Disease Lynda K. McGinnis, Lacey J. Luense and Lane K. Christenson The Human Placental Methylome Wendy P. Robinson and E. Magda Price The Function of TrophomiRs and Other MicroRNAs in the Human Placenta Yoel Sadovsky, Jean-Francois Mouillet, Yingshi Ouyang, et al. Human Endometrial Transcriptomics: Implications for Embryonic Implantation E. Gómez, M. Ruíz-Alonso, Jose Miravet, et al. Genetic Considerations in Recurrent Pregnancy Loss Kassie J. Hyde and Danny J. Schust Genomics of Preterm Birth Kayleigh A. Swaggart, Mihaela Pavlicev and Louis J. Muglia

Confrontation, Consolidation, and Recognition: The Oocyte's Perspective on the Incoming Sperm David Miller Molecular Regulation of Parturition: The Role of the Decidual Clock Errol R. Norwitz, Elizabeth A. Bonney, Victoria V. Snegovskikh, et al. The Amniotic Fluid Transcriptome as a Guide to Understanding Fetal Disease Lillian M. Zwemer and Diana W. Bianchi The Perinatal Microbiome and Pregnancy: Moving Beyond the Vaginal Microbiome Amanda L. Prince, Derrick M. Chu, Maxim D. Seferovic, et al. Placental Extracellular Vesicles and Feto-Maternal Communication M. Tong and L.W. Chamley

For additional articles in this collection, see http://perspectivesinmedicine.cshlp.org/cgi/collection/

Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved

MicroRNA biology and pain.

Computational Biology in microRNA.

Overview of microRNA biology.

MicroRNA Deregulation in Anaplastic Thyroid Cancer Biology.

Ovarian cancer, Part I: Biology.

Molecular biology of ovarian cancer.

Circulating microRNA expression profiles in ovarian cancer.

The MicroRNA Biology of the Mammalian Nucleus.

Myocardin in biology and disease.

MicroRNA-200c and microRNA-31 regulate proliferation, colony formation, migration and invasion in serous ovarian cancer.

Herpes virus microRNA expression and significance in serous ovarian cancer.

Smoking status impacts microRNA mediated prognosis and lung adenocarcinoma biology.

Application of microRNA in diagnosis and treatment of ovarian cancer.

Therapeutic evaluation of microRNA-15a and microRNA-16 in ovarian cancer.

MicroRNA and Cardiovascular Disease.

FGFR3 biology and skeletal disease.

Disease, dysfunction, and synthetic biology.

[Metabolic disease and molecular biology].

Molecular biology and coeliac disease.

MicroRNA-200c and microRNA-141 as potential diagnostic and prognostic biomarkers for ovarian cancer.

Noncoding RNAs in cardiovascular biology and disease.

A novel serum microRNA panel to discriminate benign from malignant ovarian disease.

MicroRNA 130b enhances drug resistance in human ovarian cancer cells.

MicroRNA maturation and human disease.