Androgen-induced exosomal miR-379-5p release determines granulosa cell fate: The cellular mechanism of anovulatory polycystic ovaries

doi:10.21203/rs.3.rs-2599745/v1

Download PDF

Research Article

Androgen-induced exosomal miR-379-5p release determines granulosa cell fate: The cellular mechanism of anovulatory polycystic ovaries

https://doi.org/10.21203/rs.3.rs-2599745/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Polycystic ovarian syndrome (PCOS) is a complex multi-factorial syndrome associated with androgen excess and anovulatory infertility. In the current study, we investigated the role of dihydrotestosterone-induced exosomal miR-379-5p release in determining the destiny of the developing follicles. Our hypothesis was that androgen regulates granulosa cell miR-379-5p content by facilitating its exosomal release in a follicular-stage dependent manner, a process which determines granulosa cell fate. Compared to human non-PCOS subjects, individuals with PCOS exhibit higher follicular fluid free testosterone levels, lower exosomal miR-379-5p content and granulosa cell proliferation. Androgenized rats exhibited lower granulosa cell miR-379-5p but higher phosphoinositide-dependent kinase-1 (PDK1; a miR-379-5p target) content and proliferation. Androgen reduced granulosa cell miR-379-5p content by increasing its exosomal release in preantral follicles, but not in antral follicles in vitro. Studies with an exosomal release inhibitor confirmed that androgen-induced exosomal miR-379-5p release decreased granulosa cell miR-379-5p content and proliferation. Ovarian overexpression of miR-379-5p suppressed granulosa cell proliferation, and basal and androgen-induced preantral follicle growth in vivo. These findings suggest that increased exosomal miR-379-5p release in granulosa cells is a proliferative response to androgenic stimulation specific for the preantral stage of follicle development and that dysregulation of this response at the antral stage is associated with follicular growth arrest, as observed in human PCOS.

Molecular Biology

Immunology

Sexual & Reproductive Medicine

miR-379-5p

polycystic ovarian syndrome

exosomes

granulosa cells

PDK1

Using human derived samples and rat PCOS models, as well as granulosa cell and follicle cultures, we tested the hypothesis that androgen regulates granulosa cell miR-379-5p content by facilitating its exosomal release in a follicular-stage dependent manner, a process which determines granulosa cell fate and follicle destiny. Our studies demonstrate for the first time that granulosa cell exosomal miR-379-5p release in response to androgenic stimulation is specific to the preantral stage of follicle development but absent during the antral follicle growth. Our studies shed light on the cellular basis for the androgen-induced regulation of ovarian follicular growth in follicular stage-dependent manner and how its dysregulation could result in antral follicular growth arrest in PCOS.

Polycystic ovarian syndrome (PCOS) is a multi-factorial syndrome with complex pathology. PCOS is associated with androgen excess, increased ovarian preantral follicular growth, antral follicle growth arrest and chronic anovulation (1). Whereas a balanced level of androgen is important in normal ovarian follicular development (2–4), PCOS subjects exhibit androgen excess and suffer from anovulatory infertility (1). Studies with cell-specific androgen receptor knockout mice, indicate that granulosa cells are the main target cells for androgen and that reduced preantral follicle development in these mice demonstrates that androgen signalling is mainly involved in preantral follicle development (5, 6); potentially through proteasomal degradation and reduced androgen receptors in antral follicle granulosa cells (7–9). To study the pathological basis of PCOS, we and others have established a chronically androgenized rodent model with a relatively similar reproductive and metabolic phenotypes of human PCOS (9–17).

MicroRNAs (miRNAs) are small non-coding RNAs which play important roles in the regulation of various physiological and pathological processes by post-transcriptionally down-regulating target gene expression (18). Abnormal miRNA expression has been linked to diseases, including PCOS (18–26). We and others have demonstrated that androgen regulates cell fate (proliferation versus apoptosis) via up- or down-regulation of miRNAs (18, 26–30). However, most miRNA functional studies have been conducted in cancer cells (31–34) and less is known about the roles of androgen-regulated miRNAs (e.g. miR-379-5p (18)) in reproductive endocrinopathies.

We have previously assessed the changes in the ovarian miRNA profile associated with PCOS in a androgenized rat PCOS model (18). Our results indicate that 89 miRNAs, including miR-379-5p, are differentially expressed between dihydrotestosterone (DHT)-treated and control rats. MiR-379-5p is a member of the miRNA cluster DLK1-DIO3 (35). Up-regulation of miR-379-5p in granulosa cells from subjects with premature ovarian insufficiency is associated with suppressed cell proliferation (36), suggesting that miR-379-5p is involved in ovarian follicular development. However, further studies are needed to determine its function and regulation during ovarian follicular growth and development.

Extracellular vesicles, including small (exosomes) and large (microvesicles) vesicles, are involved in cell-cell communication (37–39) by selectively packaging and transferring bioactive materials (e.g., miRNAs (40, 41)). Exosomes are vesicles of approximately 30–150 nm in size and are formed within endosomes by membrane invagination. Microvesicles range from 0.1 to 1.0 µm and are produced via membrane blebbing in response to cell stress (42). It has been demonstrated that androgen attenuates follicular atresia through miR-125b-mediated suppression of granulosa cell pro-apoptotic protein expression (30), suggesting that granulosa cell miRNAs may have a direct regulatory role in ovarian follicular growth (43). MiRNA profiling studies have identified several differentially expressed miRNAs in follicular fluid from PCOS women (25, 41, 44). MiRNAs are present in both extracellular vesicles and extracellular vesicle-free media (44) and these miRNAs are regulators of reproductive, endocrine, and metabolic processes (25, 44). Earlier hypotheses suggested that exosomes may function by expelling unusable cellular constituents from cells. However, the role of exosome secretion in regulating the cellular content of miRNAs and the function of exosome-secreting cells is largely unexplored.

In the current study, our specific objectives were to examine (1) the regulation of granulosa cell-extracellular vesicle release in response to androgen exposure; (2) if and how androgen regulates the cellular and extracellular content of miR-379-5p in granulosa cells; and (3) whether regulation of these processes is follicular stage-specific. Our overall hypothesis was that androgenic regulation of ovarian follicular development is follicular stage-specific, a phenomenon which is dependent on exosomal miR-379-5p release. In this study, we have demonstrated, for the first time, that androgen regulates ovarian exosome dynamics and their miRNA content in the control of follicle growth, and how this process may be compromised in PCOS. Our findings indicate that androgen regulates follicle growth in a stage-dependent manner via changes in the cellular and extracellular content of miR-379-5p, with phosphoinositide-dependent kinase-1 (PDK1) being its molecular target and responsible for the activation of the AKT pathway. Results from our animal studies were supported in part by our clinical observations that PCOS subjects at the preovulatory stage had lower exosomal miR-379-5p content and attenuated granulosa cell proliferation. These findings suggest that increased exosomal miR-379-5p release is a proliferation stimulus that is specific for preantral follicle granulosa cells, and this is subject to androgenic regulation.

A) Human studies

2.1 Human PCOS subjects had a lower exosomal miR-379-5p content in follicular fluids and granulosa cell proliferation from large dominant follicles. To investigate the possible role of mir-379-5p in PCOS pathogenesis, its exosomal and extracellular vesicle-depleted follicular fluid content as well as granulosa cell proliferation in PCOS and non-PCOS subjects were compared. Dominant follicles (≥ 20 mm) of PCOS subjects had significantly higher ovarian follicular fluid free testosterone levels compared with that of non-PCOS. PCOS subjects also exhibited a lower exosomal miR-379-5p content (relative to miR-92a-3p, the content of which remained constant through the current study; Supplementary Fig. 1A) and granulosa cell proliferation (Supplementary Fig. 1B,C). MiR-379-5p was detected in follicular fluid depleted of extracellular vesicle, but its levels were not different from those from non-PCOS subjects (Supplementary Fig. 1A), suggesting that androgen excess in PCOS subjects dysregulates the association between cellular and exosomal content of miR-379-5p and subsequently cell proliferation in large dominant follicles.

B) Rat studies

2.2 DHT reduced miR-379-5p and induced AKT pathway in rat granulosa cells. To investigate if and how androgen excess regulates miRNA expression in granulosa cells, several candidate miRNAs (miR-24, miR-9, miR-379-5p, and let-7d) were selected based on our previous miRNA profile analysis of the whole ovary of androgenized rat PCOS model (18). Expression of candidate miRNAs in granulosa cells isolated from immature rats and those treated with DHT for 24 h and 36 h in vitro were compared with controls. DHT significantly reduced the cellular content of miR-379-5p (Fig. 1A) but not that of miR-24, miR-9, let-7d (Supplementary Fig. 2). Using the bioinformatics website (TargetScan and RNA22 v2 microRNA target detection) and based on published findings (33), transforming growth factor beta receptor I (TGFBR1) and phosphoinositide-dependent kinase-1 (PDK1, responsible to activate AKT by phosphorylation of Thr 308) were identified as potential downstream targets of miR-379-5p. PDK1 protein content was significantly increased 24 h and 36 h post-DHT treatment and was associated with higher granulosa cell proliferation, as evidenced by increased MCM2 content (Fig. 1A). However, TGFBR1 protein content was not affected by DHT treatment (Supplementary Fig. 3). Therefore, we focused on PDK1 as a potential target gene regulated by miR-379-5p in subsequent studies.

2.3 Androgen-induced exosomal miR-379-5p release reduces its granulosa cell content, resulting in higher PDK1-mediated cell proliferation in rat preantral but not in antral follicles. DHT significantly reduced granulosa cell miR-379-5p content 24 h post-treatment at the preantral but not in at the antral follicle stage (Fig. 1A). The reduction of granulosa cell miR-379-5p content was accompanied by increases in PDK1 and P-PDK1 content at 24 h and 36 h in preantral follicles, but not in antral follicle in vitro (Fig. 1A). Moreover, DHT treatment significantly increased P-AKT, P-AKT/AKT ratio and proliferation (MCM2, Fig. 1A) in preantral but not antral follicle granulosa cells.

To investigate if changes in granulosa cell miR-379-5p content is regulated by changing its cellular synthesis or through its packaging and release from cell, and whether these responses are follicular stage-specific, we assessed cellular content of pri-miR-379 (precursor of miR-379-5p), the number of microvesicles, exosomes and their miR-379-5p content in the conditioned medium from preantral and antral follicle granulosa cells at 24 h and 36 h post-DHT treatment in vitro. DHT significantly increased granulosa cell pri-miR-379 content (Supplementary Fig. 4), indicating that miR-379-5p is an androgen-responsive miRNA. Antral follicle granulosa cells also had higher cellular pri-miR-379 content than that of preantral follicle stage. However, there were no significant interactions between follicle stage and DHT treatment (stage x DHT) nor between follicle stage, DHT treatment and culture duration (stage x DHT treatment x culture duration). DHT treatment had no influence on the size of granulosa cell-derived microvesicles and exosomes irrespective of follicle stage or culture duration (Supplementary Fig. 5). DHT treatment for 36 h reduced microvesicle release from both preantral and antral follicular granulosa cells (Fig. 1B), and reduced exosome release in antral but not preantral follicle granulosa cells (Fig. 1B). The ratio of microvesicle to exosome released was reduced 36 h post-DHT treatment in preantral follicle granulosa cells (Fig. 1B). miR-379-5p content in exosomes but not in microvesicles was increased 36 h following exposure of preantral follicle granulosa cells with androgen in vitro (Fig. 1C). DHT had no significant influence on the microvesicle and exosomal content of miR-379-5p from antral follicle granulosa cells (Fig. 1C). miR-379-5p was not detectable in extracellular vesicle-depleted conditioned media of preantral and antral follicular granulosa cells (data not presented). These results suggest that preantral follicle granulosa cells selectively package and release mir-379-5p through exosomes in response to androgen, rather than increasing the number of exosome release.

To determine if androgen regulates exosomal miRNA release and if this response is miR-379-5p-specific, exosomal miR-24, miR-9, and let-7d contents were also assessed in preantral follicle granulosa cell cultures for 24 h and 36 h in the absence or presence of DHT. Our results indicate that DHT treatment did not affect their exosomal contents, suggesting that the androgenic regulation of exosomal miRNA contents in preantral granulosa cells is miR-379-5p-specific (Supplementary Fig. 2).

2.4 Androgenized rats had lower granulosa cell miR-379-5p content and higher cellular PDK1 content and proliferation. To investigate if androgen excess is associated with down-regulation of granulosa cell miR-379-5p content in vivo, its expression was evaluated in granulosa cells isolated from an androgenized PCOS rat model. One month of DHT treatment in vivo significantly reduced granulosa cell miR-379-5p content (P < 0.05; Fig. 2). This response was associated with increased PDK1 and P-PDK1 contents (Fig. 2). Although total AKT content was not altered by androgen treatment in vivo, DHT-treated rats exhibited higher P-AKT (Thr308) levels and a higher P-AKT/AKT ratio (Fig. 2). Furthermore, activation of the AKT pathway was accompanied by increased granulosa cell proliferation, as indicated by an increased cellular MCM2 content in granulosa cells from DHT-treated rats (Fig. 2).

2.5 miR-379-5p regulates rat granulosa cell proliferation through PDK1 suppression. To determine whether miR-379-5p directly regulates PDK1 expression, we first examined its binding to the 3ʹ UTR section of PDK1 mRNA, using a dual-reporter luciferase assay with a reporter vector containing a complementary miR-379-5p sequence in its 3ʹ-UTR (Fig. 3A). The binding of miR-379-5p mimic to the PDK1 3′-UTR, but not to its mutant (binding site deleted; Fig. 3A), significantly reduced luciferase activity in preantral follicle granulosa cells transfected with the reporter vector, confirming binding of the miRNA to the PDK1 mRNA (Fig. 3A). Further studies with miR-379-5p mimic and inhibitor demonstrate that miR-379-5p mimic significantly suppressed granulosa cell PDK1 protein content and proliferation (Fig. 3B,C), two down-stream responses readily attenuated by its inhibitor (Fig. 3B,C), suggesting that miR-379-5p reduces granulosa cell proliferation through PDK1 suppression.

2.6 miR-379-5p exosomal release is a cellular mechanism through which androgen up-regulates granulosa cell PDK1 content and proliferation in rat preantral follicles. The role of exosomal miR-379-5p release on its cellular content and those of PDK1 was studied in vitro, using the exosome release inhibitor GW4869 (chemical inhibitors of N-sphingomyelinase, nSMase) (45, 46). To determine the optimal concentration of GW4869 for inhibition of exosome release, preantral follicle granulosa cells were pre-treated with different concentration of GW4869 (0, 10 and 20 nM) for 24 h and then cultured in the absence and presence of DHT (1µM; 36 h). Concentration-response studies indicate GW4869 treatment at 20 nM significantly suppressed basal and DHT (1µM; 36h)-induced exosome release from preantral follicle granulosa cells, as evidenced by increased granulosa cell CD63 content (exosome marker; Fig. 4A), and its reduction in culture conditioned medium (Supplementary Fig. 6). Inhibition of exosome release increased granulosa cell miR-379-5p content in the presence of DHT (Fig. 4A). To further investigate if exosomal miR-379-5p release regulates granulosa cell proliferation, preantral follicle granulosa cells were pre-treated with GW4869 (20 nM; 24 h), and then treated with or without DHT (1µM; 36 h). Inhibition of exosome release suppressed DHT-induced granulosa cell proliferation (Fig. 4B), supporting our hypothesis that exosomal miR-379-5p release is a regulatory mechanism by which androgen increases granulosa cell proliferation in preantral follicles.

2.7 The induction of exosome release reduces granulosa cell miR-379-5p content in the rat antral follicle stage. Cortactin is involved in exosome docking and release and its overexpression induces exosome release without affecting its content (47). In contrast to the more responsive granulosa cells from preantral follicles, DHT failed to influence granulosa cell cortactin content (Fig. 4C) and proliferation in antral follicles in vitro, suggesting that the failure of androgen to induce granulosa cell proliferation in antral follicles could be due to dysregulated or non-responsive granulosa cell cortactin-mediated exosome release. To test this possibility, cortactin was overexpressed in granulosa cells from antral follicles to determine if cortactin-induced exosome release increases exosomal miR-379-5p release and reduces its cellular content. The overexpression of cortactin increased exosome release by antral follicular granulosa cell 24 h and 36 h post-DHT treatment in vitro (Cttn + DHT; Fig. 4D). Exosome release induction in Cttn + DHT increased exosomal miR-379-5p release and reduced its cellular content at 24 h (Fig. 4D), suggesting that the failure of androgen to induce granulosa cell proliferation in antral follicles could be due to lack of cortactin activation and miR-379-5p exosomal release.

2.8 Inhibition of exosome release suppresses DHT-induced rat preantral follicular growth. To determine if androgen-induced preantral follicular growth is mediated through exosomal miR-379-5p release, preantral follicles pre-treated with and without GW4869 (20 nM; 24 h) were cultured in the absence and presence of DHT (1 µM). Follicular growth in the four experimental groups: control, GW4869, DHT and DHT + GW4869 was assessed on days 2 and 4 post-DHT treatment. Our results indicate that, although there was no difference in follicular volume between control and GW4869 on days 2 and 4, inhibition of exosomal release suppressed the DHT-induced follicular growth (DHT + GW4869) compared to control, GW4869 or DHT treatment, alone (Fig. 5A).

2.9 MiR-379-5p suppresses the follicular growth of rat preantral follicles. To determine the role of miR-379-5p in follicular growth, preantral follicles were transfected with miR-379-5p mimic and cultured with or without DHT (1 µM), and follicular growth in vitro was assessed on days 2 and 4 post-treatment. Alexa-647-labelled miR-379-5p mimic was used to confirm successful transfection of granulosa cells in preantral follicles. Image analysis of the transfected preantral follicles indicates that a subset of granulosa cells was successfully transfected with Alexa-647-labelled miR-379-5p mimic (Fig. 5B), which significantly suppressed the follicle growth compared to that of control and DHT groups (Fig. 5C).

2.10 Reconstitution of DHT-treated rat granulosa cells with miR-379-5p-enriched exosomes attenuates the DHT-induced proliferative response in vitro. As we showed earlier, DHT-induced exosomal miR-379-5p release reduces its granulosa cell content and increases PDK1 content and proliferation in rat preantral follicle granulosa cells in vitro. In addition to the increased exosomal miR-379-5p release in response to androgen, the decreased granulosa cell miR-379-5p content could be due to decreased exosome uptake. To address this possibility, we investigated whether DHT alters granulosa cell exosome uptake by assessing if reconstitution of DHT-treated granulosa cells (miR-379-5p down-regulated) with miR-379-5p-enriched exosomes (DHT-exo) could attenuate the DHT-induced proliferative response in vitro (Supplementary Fig. 7A). Proliferation assays indicate that androgen increased cell proliferation in granulosa cells treated with exogenous control exosomes (CTR-exo). However, this response was attenuated by reconstitution of miR-379-5p by utilizing exosome-enriched in miR-379-5p isolated from DHT-treated donor granulosa cells (DHT-exo; Supplementary Fig. 7B,C). Exosome uptake was assessed by flow cytometry. Our results indicate that, while exosomes were effectively taken up by recipient granulosa cells, this response was not affected by DHT treatment of either donor or recipient granulosa cells (Supplementary Fig. 7C). This suggests that, despite the stimulatory role of androgen on granulosa cell exosomal miR-379-5p secretion, it plays minimal or no role in the regulation of exosome uptake in recipient cells.

2.11 MiR-379-5p overexpression suppresses the basal and androgen-induced rat preantral follicle growth by reduced granulosa cell proliferation. To further examine the role of miR-379-5p in follicular growth in vivo and whether miR-379-5p overexpression could suppress DHT-induced preantral follicle development, lentiviral miR-379-5p mimic was introduced under the ovarian bursa of control and androgenized rats (Fig. 6A). Ovarian growth (weight and length), follicular stage, number of follicles and their cell proliferation (Ki67) at each stage were compared between sham-control and DHT-treated rats. Our results indicate that ovarian miR-379-5p overexpression suppressed ovarian growth (length and weight; Fig. 6B,C) in control rats. In addition, ovarian miR-379-5p overexpression reduced the number of preantral follicle (Fig. 6B,C) and their granulosa cell proliferation (Fig. 6D,E) and total number of follicles (Fig. 6B,C) in both control and androgenized rats. These results provide further support that miR-379-5p is mainly involved in the growth and development of preantral follicles.

Androgen promotes both follicular growth and atresia in a follicular stage-dependent manner (6, 9, 48, 49) possibly through changes in the cellular content of miRNAs (18). Emerging evidence suggests that exosomes contribute to many aspects of reproductive physiology and disorders through intercellular communication (42, 46). However, the role of exosome secretion in determining miRNA content, and their down-stream consequences, in exosome-secreting cells remains relatively unexplored. In the current study, we have demonstrated if and how androgen-induced exosomal miR-379-5p release in granulosa cells determines the destiny of a growing follicle. Our results indicate that androgen regulates follicular growth in a stage-dependent manner, and this is via differential changes in cellular and extracellular content of miR-379-5p. Mechanistic studies indicate that PDK1, responsible for phosphorylation and activation of AKT, is a downstream target of miR-379-5p; and that androgenic stimulation of preantral follicles increases exosomal miR-379-5p release and reduces its granulosa cell content, resulting in enhanced PDK1-mediated granulosa cell proliferation. These responses appear to be specific to preantral follicles, since DHT failed to stimulate proliferation of granulosa cells from antral follicles, nor did it increase exosomal miR-379-5p release or decrease granulosa cell content. These findings may offer important mechanistic insight into developmental differences in the follicle growth dynamics associated with PCOS, a condition often associated with androgen excess.

Exosomes play important roles in intercellular communication by serving as vehicles for transferring various cellular constituents, such as proteins, lipids and nucleic acids, between cells. However, very little is known about the role of exosome release in determining the cellular miRNA content from exosome-releasing cells. It has been suggested that exosome release plays a crucial role in maintaining cellular homeostasis in exosome-releasing cells and inhibition of this process induces reactive oxygen species-dependent DNA damage and apoptosis in normal human cells (50). In the present studies, we have demonstrated that androgen upregulates exosomal miR-379-5p release and proliferation of granulosa cells from preantral, but not antral follicles. The androgen-induced miR-379-5p release from preantral granulosa cells appears to be specific to mir-379-5p and is mediated by exosomes but not by microvesicles or simple diffusion (evident by its absence in extracellular vesicle-depleted conditioned medium). This may represent an androgen-induced proliferative mechanism to reduce its cellular content in preantral follicle granulosa cells. Our findings support the concept that miRNAs are selectively packaged into exosomes (51–53). Based on an miRNA’s motif sequence, RNA-binding proteins can bind to the specific miRNA molecules, and assist the miRNA sorting process into exosomes. Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is one such RNA-binding protein capable of targeting specific miRNAs to control their sorting and loading into exosomes (54). Recently, it has been shown that hnRNPA2B1 protein recognizes and specifically packages miRNA-17 and − 93 into exosomes through binding into their AGG/UAG motifs (55, 56). These binding motifs could provide a potential mechanism whereby hnRNPA2B1 exerts regulatory control over miRNA sorting. Interestingly, we also identified that mir-379-5p has a similar motif sequence and hence hnRNPA2B1 may be involved in selective packaging of mir-379-5p into exosomes in response to androgen. However, further investigations are required to confirm if this is indeed the case. The notion of exosomal packaging of mir-379-5p is further supported by the current observation that, during preantral follicle development, inhibition of exosome release with GW4869 increased granulosa cell miR-379-5p levels, and reduced proliferation, in response to androgen. Collectively, these results suggest that it is not the release of exosomes, but increased the exosomal content of miR-379-5p released from granulosa cells in response to androgenic stimulation is a proliferative mechanism specific for the preantral stage of follicle development.

The androgen receptor (AR) plays important regulatory roles in ovarian follicular development and AR cellular content determines the capability of the target cell to respond to androgen stimulation. Ring finger protein 6 (RNF6), a member of E3 ligase family, induces AR ubiquitination (9). In previous studies, we have observed that androgen induced ovarian follicular growth is dependent on RNF6-mediated, lysine site-specific, AR polyubiquitination, thus determining AR transcriptional activity, stability and abundance. Also, the androgenized rat PCOS model exhibits increased RNF6-mediated AR ubiquitination (K48), enhanced AR degradation, and decreased granulosa cell proliferation in the antral but not preantral follicle stage (9, 48). In this study, and in contrast to the more responsive preantral follicle granulosa cells, granulosa cells in antral follicles failed to respond to DHT with increased cortactin expression and proliferation in vitro. On the other hand, cortactin overexpression in the antral follicle stage, increases its exosomal miR-379-5p release and reduces granulosa cell content in response to androgen, suggesting that the failure of androgen-induced granulosa cell proliferation in antral follicle could be due to AR degradation, lack of cortactin activation and mir-379-5p exosomal release. In preantral follicle stage, whereas DHT significantly increased cortactin protein content, it did not change that of exosome release. Therefore, exosome release may possibly be regulated differentially in preantral and antral follicle stages. Further experiments are required to test this hypothesis.

The content of cellular constituents, such as miRNAs and proteins, is determined by a balance between exosome release and uptake. Preferential uptake of extracellular vesicles derived from follicular fluid of small antral follicles, compared to those isolated from large follicles, stimulated granulosa cell proliferation through Src, PI3K/AKT, and MAPK signaling pathways (57). The proliferative response could be explained by both the differential cellular contents and by the preferential uptake of extracellular vesicles (57). In the current study, androgen treatment of donor and recipient cells did not alter exosome uptake. However, exosomal uptake was not assessed between different follicle stages, an aspect being considered for future studies. Although exosome uptake did not differ in response to androgen, a higher exosomal miR-379-5p content of DHT-exo, suppressed androgen-induced proliferation by increasing its cellular content in preantral follicle granulosa cells. In breast cancer cells, miR-379 has been reported to be a tumor suppressor, and its expression is significantly lower in cancer compared with healthy breast tissues (58). Recently, mesenchymal stem cells stably transduced to express miR-379 and exosome-enriched with miR-379-5p have been developed for breast cancer therapy. Systemic administration of exosome enriched with miR-379 has been shown to have therapeutic effect, with a significant reduction in tumor activity in animals (58). These results support the regulatory role of exosomes in the determination of cellular miR-379-5p content and proliferation.

Recently, we have demonstrated that one-month of DHT treatment reduces ovarian weight and length, but without affecting pre-antral follicles atresia and systemic changes, including body weight and the insulin sensitivity index (14). Although all stage of follicles and corpus luteum can be found in control ovaries, DHT ovaries mainly contain pre- and early antral follicles. In the current study, overexpression of miR-379-5p in ovaries using lentiviral injection allowed us to further evaluate its function in follicular development. MiR-379-5p injection suppressed preantral follicle granulosa cell proliferation and development without affecting other stages of follicular development in both control and androgenized model, suggesting that miR-379-5p is mainly involved in the development of preantral follicles.

MiR-379-5p content in follicular fluid from PCOS subjects was significantly reduced in exosomes with no changes in extracellular vesicle-depleted follicular fluid, whereas androgen-induced miR-379-5p changes in rat granulosa cells was exosome-specific and was not detectable in extracellular vesicle-depleted condition medium. This reason(s) for this apparent discrepancy is not known, but it may be related to species differences (rat vs. human) and their response to androgen excess with respect to regulation of cellular and extracellular miRNA content. Additionally, it is also possible that the reduced exosomal mir-379-5p content in follicular fluid may be associated with the higher granulosa cell content and reduced cellular PDK1 and proliferation in PCOS subjects. Whether this indeed is the case remains to be determined.

Whereas, androgen did not influence exosomal miR-379-5p content in antral follicles stage in rat, human PCOS significantly had lower exosomal miR-379-5p content. This is important to consider that human granulosa cells and follicular fluids were collected at the preovulatory follicle stage; however those of rat collected from antral follicles. Therefore, the differences of exosomal miR-379-5p content could have be resulted from differences in follicular stage between the rat and the human models.

In conclusion, our findings suggest that increased exosomal miR-379-5p release from granulosa cells in response to androgen action is a proliferative mechanism specific to preantral follicle development. To facilitate future investigation into the role and regulation of miR-379-5p in the androgenic control of ovarian follicular development, the following hypothetical model is proposed (Supplementary Fig. 8). In preantral follicles, androgen excess reduces granulosa cell miR-379-5p content by increased exosomal release. Reduced cellular miR-379-5p up-regulates PDK1, which phosphorylates and activates AKT, and induces cell proliferation. In contrast, these cellular mechanisms were not evident at the antral follicle stage, resulting in attenuation of the proliferative response of granulosa cells to androgenic stimulation and of antral follicle growth. Although it is well established that exosomes facilitate miRNA-mediated cell-cell communication, whether and how target cell uptake of exosomes enriched in miR-379-5p within the ovarian microenvironment regulates follicular growth in normal ovarian physiology, and in the etiology of PCOS, is unknown and requires further investigation.

Additional information is provided in SI Appendix.

4.1 Animals and DHT implant in vivo

Female Sprague Dawley rats (Charles River, Montreal, Canada) were maintained on 12 h cycle (light and dark) and given food and water ad libitum. Immature female rats at 21 days of age were implanted subcutaneously with silicone capsules without (control, sham control) or with DHT (DHT, Steraloids Inc., Newport, USA), as previously described (12, 15) to continuously release 83 µg DHT/day for 28 days. Sham control animals received identical pellets lacking the steroid. Additional information on sample collection is provided in the SI Appendix.

4.2 Primary culture of rat follicles, granulosa cells, miRNA transfection and exosome release inhibition

Granulosa cells from preantral follicles (DES-primed 21-day old rats; 1 mg/d, s.c. for 3 consecutive days) and antral follicles (eCG–injected 22-day old rats; 10 IU i.p., animals sacrificed 2 days post-injection) were isolated by follicular puncture as described previously (9, 15). Granulosa cells were plated (1 × 10⁶ per well in a 6 well plate) overnight and treated with or without DHT (1 µM; 24 h and 36 h; (n = 3 replicates, each from 2 rats)). To manipulate miR-379-5p expression, granulosa cells were transfected with miR-379-5p mimic (50 and 100 nM) or inhibitor (100 nM; mirVana Mimics & Inhibitors) or scrambled sequence using Lipofectamine 2000 for 24 h before DHT treatment in vitro. To inhibit exosome release, granulosa cells were treated with GW4869 (0, 10 and 20 mM) for 24 h before DHT treatment. Additional information is provided in the SI Appendix.

4.3 Human samples:

Human ovarian follicular fluids (FF) were collected from pre-ovulatory follicle of lean PCOS (based on Rotterdam criteria; BMI < 30; Supplementary table 2) and non-PCOS subjects (premenopausal, 18–40 years) with normal thyroid function and prolactin levels, undergoing assisted reproduction treatment at the CReATe Fertility Centre, Toronto, Canada. Exclusion criteria include: (1) known causes of oligomenorrhea other than PCOS; and (2) use of hormone treatment, birth control pill, insulin sensitizers, lipid lowering agents, or medications known to influence insulin sensitivity or serum androgens, within 3 months of the study onset. Moreover, follicular fluids were collected individually from pre-ovulatory follicle of right or left ovary without buffer and those contaminated with visible blood were excluded (Supplementary Fig. 9). All FF (PCOS, n = 13 & non-PCOS, n = 25) and granulosa cell (Ki67: pre-ovulatory follicle; PCOS, n = 12 & non-PCOS, n = 11) specimens were obtained from the CReATe Biobank, CReATe Fertility Centre (59).

4.4 Statistics

T-tests, and one-, two-, or three-way analyses of variance (ANOVA) were used to assess the effects of, and interactions between variables, followed by multiple comparison by the Tukey post hoc test, using Prism v.7 (GraphPad, San Diego, CA) and Sigma plot v.12 (Systat Software, San Jose, CA). P < 0.05 was considered statistically significant.

4.5 Study approval

All FF and granulosa cell specimens were obtained from the CReATe Biobank, CReATe Fertility Centre. The CReATe Biobank (banking protocols approved by Veritas IRB), collects biological materials from consenting patients, according to the best practice-based standards of biobanking (59). All samples from the Biobank were approved for use in this study by the University of Toronto REB (Protocol#29236) and The Ottawa Hospital REB (Protocol #20170453-01H)]. All animal procedures were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals, Canadian Council on Animal Care, and were approved by the University of Ottawa Animal Care Committee.

Conflict of interest: The authors declare no conflict of interest.

Acknowledgments:

This work was supported by grant MOP-119381 from the Canadian Institutes of Health Research (to BKT); postdoctoral fellowships from the Lalor Foundation, Mathematics of Information Technology and Complex Systems (Mitacs; to RS) and CReATe Fertility Centre's funding contribution to MITACS fellowship (to RS). Human biological materials and associated de-identified data were collected by CReATe Biobank personnel. The CReATe Biobank is certified by the CTRNet Biobank Program. The collection and biobanking of biological materials for research was approved by an independent, accredited research ethics board.

Author contributions:

RS and BKT designed the experiments; RS, AA, BP, YX, YU, AG and JLV performed the experiments; BW, SJ and CLL provided human samples; KX prepared adenovirus; KDB, DB, BV, JL contributed analytic tools; RS analyzed experimental data; RS prepared the manuscript with input from MA-W, FE, BKT, BV, DB, YO, KDB, JL, and CLL; All authors reviewed and revised manuscript; BKT, YO and CLL provided financial support for the project.

Dunaif A (1997) Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocrine reviews 18(6):774–800.
Astapova O, Minor BMN, & Hammes SR (2019) Physiological and Pathological Androgen Actions in the Ovary. Endocrinology 160(5):1166–1174.
Sellix MT & Sen A (2017) Finding the Right Balance: Androgens at the Tipping Point of Fertility and Metabolism in Women. Endocrinology 158(3):467–469.
Prizant H, Gleicher N, & Sen A (2014) Androgen actions in the ovary: balance is key. The Journal of endocrinology 222(3):R141-151.
Walters KA, et al. (2012) Targeted loss of androgen receptor signaling in murine granulosa cells of preantral and antral follicles causes female subfertility. Biology of reproduction 87(6):151.
Sen A & Hammes SR (2010) Granulosa cell-specific androgen receptors are critical regulators of ovarian development and function. Molecular endocrinology 24(7):1393–1403.
Hillier SG, Tetsuka M, & Fraser HM (1997) Location and developmental regulation of androgen receptor in primate ovary. Human reproduction 12(1):107–111.
Tetsuka M, et al. (1995) Developmental regulation of androgen receptor in rat ovary. The Journal of endocrinology 145(3):535–543.
Lim JJ, Han CY, Lee DR, & Tsang BK (2017) Ring Finger Protein 6 Mediates Androgen-Induced Granulosa Cell Proliferation and Follicle Growth via Modulation of Androgen Receptor Signaling. Endocrinology 158(4):993–1004.
Kim JY, et al. (2013) Chemerin suppresses ovarian follicular development and its potential involvement in follicular arrest in rats treated chronically with dihydrotestosterone. Endocrinology 154(8):2912–2923.
Lim JJ, Lima PDA, Salehi R, Lee DR, & Tsang BK (2017) Regulation of androgen receptor signaling by ubiquitination during folliculogenesis and its possible dysregulation in polycystic ovarian syndrome. Scientific reports 7(1):10272.
Lima PDA, et al. (2018) Polycystic ovary syndrome: possible involvement of androgen-induced, chemerin-mediated ovarian recruitment of monocytes/macrophages. Biology of reproduction 99(4):838–852.
Manneras L, et al. (2007) A new rat model exhibiting both ovarian and metabolic characteristics of polycystic ovary syndrome. Endocrinology 148(8):3781–3791.
Salehi R, et al. (2020) Ovarian mitochondrial dynamics and cell fate regulation in an androgen-induced rat model of polycystic ovarian syndrome. Scientific reports 10(1):1021.
Wang Q, et al. (2012) Chemerin, a novel regulator of follicular steroidogenesis and its potential involvement in polycystic ovarian syndrome. Endocrinology 153(11):5600–5611.
Brown RE, Wilkinson DA, Imran SA, Caraty A, & Wilkinson M (2012) Hypothalamic kiss1 mRNA and kisspeptin immunoreactivity are reduced in a rat model of polycystic ovary syndrome (PCOS). Brain research 1467:1–9.
Caldwell ASL, et al. (2017) Neuroendocrine androgen action is a key extraovarian mediator in the development of polycystic ovary syndrome. Proceedings of the National Academy of Sciences of the United States of America 114(16):E3334-E3343.
Hossain MM, et al. (2013) Altered expression of miRNAs in a dihydrotestosterone-induced rat PCOS model. Journal of ovarian research 6(1):36.
Chen B, Xu P, Wang J, & Zhang C (2019) The role of MiRNA in polycystic ovary syndrome (PCOS). Gene 706:91–96.
Tu J, Cheung AH, Chan CL, & Chan WY (2019) The Role of microRNAs in Ovarian Granulosa Cells in Health and Disease. Frontiers in endocrinology 10:174.
Zhang J, Xu Y, Liu H, & Pan Z (2019) MicroRNAs in ovarian follicular atresia and granulosa cell apoptosis. Reproductive biology and endocrinology: RB&E 17(1):9.
Fitzgerald JB, George J, & Christenson LK (2016) Non-coding RNA in Ovarian Development and Disease. Advances in experimental medicine and biology 886:79–93.
Santonocito M, et al. (2014) Molecular characterization of exosomes and their microRNA cargo in human follicular fluid: bioinformatic analysis reveals that exosomal microRNAs control pathways involved in follicular maturation. Fertility and sterility 102(6):1751–1761 e1751.
Roth LW, et al. (2014) Altered microRNA and gene expression in the follicular fluid of women with polycystic ovary syndrome. Journal of assisted reproduction and genetics 31(3):355–362.
Sorensen AE, Wissing ML, Englund AL, & Dalgaard LT (2016) MicroRNA Species in Follicular Fluid Associating With Polycystic Ovary Syndrome and Related Intermediary Phenotypes. The Journal of clinical endocrinology and metabolism 101(4):1579–1589.
Xue Y, et al. (2018) Identification of microRNAs and genes associated with hyperandrogenism in the follicular fluid of women with polycystic ovary syndrome. Journal of cellular biochemistry 119(5):3913–3921.
Murri M, Insenser M, Fernandez-Duran E, San-Millan JL, & Escobar-Morreale HF (2013) Effects of polycystic ovary syndrome (PCOS), sex hormones, and obesity on circulating miRNA-21, miRNA-27b, miRNA-103, and miRNA-155 expression. The Journal of clinical endocrinology and metabolism 98(11):E1835-1844.
Chen YH, et al. (2013) miRNA-93 inhibits GLUT4 and is overexpressed in adipose tissue of polycystic ovary syndrome patients and women with insulin resistance. Diabetes 62(7):2278–2286.
Huang X, et al. (2016) Identification of altered microRNAs and mRNAs in the cumulus cells of PCOS patients: miRNA-509-3p promotes oestradiol secretion by targeting MAP3K8. Reproduction 151(6):643–655.
Sen A, et al. (2014) Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression. Proceedings of the National Academy of Sciences of the United States of America 111(8):3008–3013.
Gururajan M, et al. (2014) miR-154* and miR-379 in the DLK1-DIO3 microRNA mega-cluster regulate epithelial to mesenchymal transition and bone metastasis of prostate cancer. Clinical cancer research: an official journal of the American Association for Cancer Research 20(24):6559–6569.
Jee YH, et al. (2018) mir-374-5p, mir-379-5p, and mir-503-5p Regulate Proliferation and Hypertrophic Differentiation of Growth Plate Chondrocytes in Male Rats. Endocrinology 159(3):1469–1478.
Li Z, Shen J, Chan MT, & Wu WK (2017) MicroRNA-379 suppresses osteosarcoma progression by targeting PDK1. Journal of cellular and molecular medicine 21(2):315–323.
Zhou F, Nie L, Feng D, Guo S, & Luo R (2017) MicroRNA-379 acts as a tumor suppressor in non-small cell lung cancer by targeting the IGF1R-mediated AKT and ERK pathways. Oncology reports 38(3):1857–1866.
Han Z, et al. (2012) Spatiotemporal expression pattern of Mirg, an imprinted non-coding gene, during mouse embryogenesis. Journal of molecular histology 43(1):1–8.
Dang Y, et al. (2018) MicroRNA-379-5p is associate with biochemical premature ovarian insufficiency through PARP1 and XRCC6. Cell death & disease 9(2):106.
Burns GW, Brooks KE, & Spencer TE (2016) Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep. Biology of reproduction 94(3):56.
O'Neil EV, Burns GW, Ferreira CR, & Spencer TE (2020) Characterization and regulation of extracellular vesicles in the lumen of the ovine uterusdagger. Biology of reproduction 102(5):1020–1032.
O'Neil EV, Burns GW, & Spencer TE (2020) Extracellular vesicles: Novel regulators of conceptus-uterine interactions? Theriogenology 150:106–112.
Navakanitworakul R, et al. (2016) Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles. Scientific reports 6:25486.
Hu J, et al. (2020) The expression of small RNAs in exosomes of follicular fluid altered in human polycystic ovarian syndrome. PeerJ 8:e8640.
Burger D, et al. (2013) Microparticles: biomarkers and beyond. Clinical science 124(7):423–441.
Imbar T & Eisenberg I (2014) Regulatory role of microRNAs in ovarian function. Fertility and sterility 101(6):1524–1530.
Sang Q, et al. (2013) Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo. The Journal of clinical endocrinology and metabolism 98(7):3068–3079.
Trajkovic K, et al. (2008) Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319(5867):1244–1247.
Vinas JL, et al. (2016) Transfer of microRNA-486-5p from human endothelial colony forming cell-derived exosomes reduces ischemic kidney injury. Kidney international 90(6):1238–1250.
Sinha S, et al. (2016) Cortactin promotes exosome secretion by controlling branched actin dynamics. The Journal of cell biology 214(2):197–213.
Lim JJ, Lima PDA, Salehi R, Lee DR, & Tsang BK (2017) Regulation of androgen receptor signaling by ubiquitination during folliculogenesis and its possible dysregulation in polycystic ovarian syndrome. Scientific reports 7(1):10272.
Harlow CR, Shaw HJ, Hillier SG, & Hodges JK (1988) Factors influencing follicle-stimulating hormone-responsive steroidogenesis in marmoset granulosa cells: effects of androgens and the stage of follicular maturity. Endocrinology 122(6):2780–2787.
Takahashi A, et al. (2017) Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nature communications 8:15287.
Lee H, Groot M, Pinilla-Vera M, Fredenburgh LE, & Jin Y (2019) Identification of miRNA-rich vesicles in bronchoalveolar lavage fluid: Insights into the function and heterogeneity of extracellular vesicles. Journal of controlled release: official journal of the Controlled Release Society 294:43–52.
Nolte-'t Hoen EN, et al. (2012) Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions. Nucleic acids research 40(18):9272–9285.
Skog J, et al. (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nature cell biology 10(12):1470–1476.
Villarroya-Beltri C, et al. (2013) Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nature communications 4:2980.
Lee H, et al. (2019) Caveolin-1 selectively regulates microRNA sorting into microvesicles after noxious stimuli. The Journal of experimental medicine 216(9):2202–2220.
Wu B, et al. (2018) Molecular basis for the specific and multivariant recognitions of RNA substrates by human hnRNP A2/B1. Nature communications 9(1):420.
Hung WT, et al. (2017) Stage-specific follicular extracellular vesicle uptake and regulation of bovine granulosa cell proliferation. Biology of reproduction 97(4):644–655.
O'Brien KP, et al. (2018) Employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (EV)-encapsulated microRNA-379. Oncogene 37(16):2137–2149.
Campbell LD, et al. (2012) Development of the ISBER Best Practices for Repositories: Collection, Storage, Retrieval and Distribution of Biological Materials for Research. Biopreservation and biobanking 10(2):232–233.

SupplementaryMaterialandMethodsinformationRevised2022New.2.pdf
Supplementary materials and methods
SIPNASBKT2022clean.New.2.pdf
Supplementary figures and tables

Download PDF

Version 1

posted

You are reading this latest preprint version

Androgen-induced exosomal miR-379-5p release determines granulosa cell fate: The cellular mechanism of anovulatory polycystic ovaries

Status:

Version 1

Abstract

Figures

Significance

1. Introduction

2. Results

3. Discussion

4. Materials And Methods

4.1 Animals and DHT implant in vivo

4.2 Primary culture of rat follicles, granulosa cells, miRNA transfection and exosome release inhibition

4.3 Human samples:

4.4 Statistics

4.5 Study approval

Declarations

References

Supplementary Files

Status:

Version 1