Acupuncture regulates the apoptosis of ovarian granulosa cells in polycystic ovarian syndrome-related abnormal follicular development through LncMEG3-mediated inhibition of miR-21-3p.

BACKGROUND: The main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulatory dysfunction, which are caused by excessive apoptosis of ovarian granulosa cells. Acupuncture has been shown to improve follicular development abnormalities in patients with PCOS, but its mechanism is unknown. This study hypothesized that the mechanism of acupuncture on follicular development abnormalities in PCOS patients is the inhibition of granulosa cell apoptosis through LncMEG3-mediated regulation of miR-21-3p. METHODS: A PCOS-like rat model was established using subcutaneous injection of dehydroepiandrosterone (DHEA). Acupuncture was performed on rats for 15 d (CV-4, RN-3, CV-6, SP-6 and EX-CA 1). Ovarian morphology was observed by HE staining, and sex hormone and AMH levels were detected by ELISA. Primary granulosa cells were isolated from each group of rats to assess the association of acupuncture treatment, LncMEG3, miR-21-3p, and granulosa cell apoptosis in rats with PCOS. RESULTS: LncMEG3 and miR-21-3p were highly expressed in the ovarian granulosa cells of rats with PCOS, and LncMEG3-mediated regulation of miR-21-3p was involved in the development of PCOS in rats. Silencing of MEG3 attenuated sex hormone dysregulation and ovarian histopathological changes in PCOS rats and promoted follicle cell development and maturation. In addition, silencing MEG3 increased the viability and number of granulosa cells. In addition, silencing MEG3 further inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats. Acupuncture improved polycystic ovarian morphology and sex hormone levels in PCOS rats. Acupuncture intervention increased the viability and number of granulosa cells. Acupuncture intervention inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats by targeting miR-21-3p via LncMEG3. CONCLUSION: These results suggest that acupuncture can downregulate LncMEG3, thereby targeting and regulating miR-21-3p to suppress early and late granulosa cell apoptosis and normalize their proliferation. These factors ultimately compensate for abnormal follicular development. These findings shed light on the clinical potential of acupuncture as a safe treatment for follicular developmental abnormalities in PCOS.

TGFBR2 is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells.

The ubiquitin-specific peptidase 9 X-linked (USP9X) is one of the highly conserved members belonging to the ubiquitin-specific proteases (USPs) family, which has been reported to control substrates-mediated biological functions through deubiquitinating and stabilizing substrates. Here, we have found that TGFBR2, the type II receptor of the transforming growth factor beta (TGF-ß) signaling pathway, is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells (GCs). Mechanically, USP9X positively influences the expression of TGFBR2 at different levels through two independent ways: (i) directly targets and deubiquitinates TGFBR2, which maintains the protein stability of TGFBR2 through avoiding degradation mediated by ubiquitin-proteasome system; (ii) indirectly maintains TGFBR2 messenger RNA (mRNA) expression via SMAD4/miR-143 axis. Specifically, SMAD4, another substrate of USP9X, acts as a transcription factor and suppresses miR-143 which inhibits the mRNA level of TGFBR2 by directly binding to its 3'-untranslated region. Functionally, the maintenance of TGFBR2 by USP9X activates the TGF-ß signaling pathway, which further represses GC apoptosis. Our study highlights a functional micro-regulatory network composed of deubiquitinase (USP9X), small noncoding RNA (miR-143) and the TGF-ß signaling pathway, which plays a crucial role in the regulation of GC apoptosis and female fertility.

SMARCA2 is regulated by NORFA-miR-29c, a novel pathway that controls granulosa cell apoptosis and is related to female fertility.

SMARCA2, an evolutionarily conserved catalytic ATPase subunit of SWI/SNF complexes, has been implicated in development and diseases; however, its role in mammalian ovarian function and female fertility is unknown. Here, we identified and characterized the 3'-UTR of the porcine SMARCA2 gene and identified a novel adenylate number variation. Notably, this mutation was significantly associated with sow litter size traits and SMARCA2 levels, due to its influence on the stability of SMARCA2 mRNA in ovarian granulosa cells (GCs). Immunohistochemistry and functional analysis showed that SMARCA2 is involved in the regulation of follicular atresia by inhibiting GC apoptosis. In addition, miR-29c, a pro-apoptotic factor, was identified as a functional miRNA that targets SMARCA2 in GCs and mediates regulation of SMARCA2 expression via the NORFA-SMAD4 axis. Although a potential miR-29c-responsive element was identified within NORFA, negative regulation of miR-29c expression by NORFA was not due to activity as a competing endogenous RNA. In conclusion, our findings demonstrate that SMARCA2 is a candidate gene for sow litter size traits, because it regulates follicular atresia and GC apoptosis. Additionally, we have defined a novel candidate pathway for sow fertility, the NORFA-TGFBR2-SMAD4-miR-29c-SMARCA2 pathway.This article has an associated First Person interview with the first author of the paper.

Decreased fatty acids induced granulosa cell apoptosis in patients with diminished ovarian reserve.

PURPOSE: To investigate whether fatty acid changes in granulosa cells (GCs) underly the pathogenic mechanisms of diminished ovarian reserve (DOR). METHODS: GCs were obtained from patients with DOR (n = 70) and normal ovarian reserve (NOR, n = 70). Analysis of fatty acids changes in GCs was then analyzed. RESULTS: Patients with DOR had significantly lower levels of antral follicle count and anti-Mullerian hormone and higher levels of follicle-stimulating hormone compared with NOR patients (P < 0.001). The good-quality embryo rate was notably decreased in DOR patients (51.99 vs 39.52%, P < 0.05). A total of 15 significantly decreased fatty acids in GCs from patients with DOR. The ATP levels were markedly lower in DOR patients than in NOR patients (39.07 ± 12.89 vs 23.21 ± 13.69%, P < 0.05). Mitochondrial membrane potential decreased in DOR patients (P < 0.01). In GCs from DOR patients, the ß-oxidation genes (HADHA and ACSL) and DNA repair genes (PRKDC and RAD50) were significantly downregulated (P < 0.05). The γH2AX foci/nucleus ratio in DOR patients markedly increased relative to that of NOR patients (0.31 ± 0.03 vs 0.87 ± 0.07, P < 0.001). Meanwhile, the apoptosis rate of GCs was significantly higher in DOR patients (6.43 ± 2.11 vs 48.06 ± 6.72%, P < 0.01). CONCLUSION: GC apoptosis resulting from the decrease of fatty acids, and associated with reduced ATP production and DNA damage, may contribute to the pathogenic mechanisms responsible for DOR.

circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging.

Ovarian granulosa cell (GC) apoptosis is the major cause of follicular atresia. Regulation of non-coding RNAs (ncRNAs) was proved to be involved in regulatory mechanisms of GC apoptosis. circRNAs have been recognized to play important roles in cellular activity. However, the regulatory network of circRNAs in follicular atresia has not been fully validated. In this study, we report a new circRNA, circSLC41A1, which has higher expression in healthy follicles compared to atretic follicles, and confirm its circular structure using RNase R treatment. The resistant function of circSLC41A1 during GC apoptosis was detected by si-RNA transfection and the competitive binding of miR-9820-5p by circSLC41A1 and SRSF1 was detected with a dual-luciferase reporter assay and co-transfection of their inhibitors or siRNA. Additionally, we predicted the protein-coding potential of circSLC41A1 and analyzed the structure of circSLC41A1-134aa. Our study revealed that circSLC41A1 enhanced SRSF1 expression through competitive binding of miR-9820-5p and demonstrated a circSLC41A1-miR-9820-5p-SRSF1 regulatory axis in follicular GC apoptosis. The study adds to knowledge of the post-transcriptional regulation of follicular atresia and provides insight into the protein-coding function of circRNA.

Does LH supplementation in poor responders affect granulosa cells apoptosis rate in ART? A prospective randomised controlled trial.

The aim was to compare granulosa cell's (GCs) apoptosis rate with (group A) or without (group B) luteinising hormone (LH) supplementation in poor ovarian responders (PORs) during controlled ovarian stimulation (COS). After oocyte retrieval, the follicular fluid was analysed by cytoflowmetry. Primary outcomes were GCs apoptosis rate in terms of viability, early apoptosis, late apoptosis and necrosis. Secondary outcome was clinical pregnancy rate. The viability was 96.7{IQR: 8} and 83.5{IQR: 20} for groups A and B, respectively (p < .001). Late apoptosis rates were significantly lower in group A (median 1.5, {IQR: 3.1}) than group B (median 9.5, {IQR: 20.6}) (p < .001). Median early apoptosis rates were 1.4 {IQR: 2.9} and 5.2 {IQR: 6.5} for group A and B respectively (p = .04). No significant difference was observed in the clinical pregnancy rate. Although LH seems necessary in PORs to decrease late granulosa apoptosis rates, this does not improve clinical pregnancy rates.IMPACT STATEMENTWhat is already known on this subject? LH supplementation during COS has long been an issue in PORs to overcome the rFSH responsiveness due to the LH polymorphism. LH receptors have also been on GCs and their expression increases in preovulatory follicles. GCs apoptosis rates may show the oocyte quality and reproductive potential of oocyte retrieved and the requirement for LH supplementation.What do the results of this study add? The present study shows that LH supplementation during COS for PORs promotes the GC viability and reduces early/late apoptosis rates. Similarly, the number of MII oocytes was significantly higher in the LH regimen group. However, there was no significant difference in terms of clinical pregnancy rates.What are the implications of these findings for clinical practice and/or further research? The oocyte quality parameters such as higher GC viability and lower GC early/late apoptosis rates verify the LH supplementation in PORs during COS. However, the limited size of this study requires further multi-centre research in a larger cohort of patients. Results obtained with a sensitive and validated method will help clinicians to make better decisions in patient care.

circRNA-Mediated Inhibin-Activin Balance Regulation in Ovarian Granulosa Cell Apoptosis and Follicular Atresia.

Ovarian granulosa cells (GC) play an essential role in the development and atresia of follicles. Emerging studies suggest that non-coding RNAs are involved in the regulation of GC apoptosis. Here, we aimed to analyze the function of ssc-circINHA-001, coded by the first exon of the inhibin subunit α gene (INHA), in resisting GC apoptosis and follicular atresia by enhancing the expression of the inhibin subunit ß A (INHBA) through a cluster of miRNAs. A higher expression of ssc-circINHA-001 in healthy follicles compared to early atretic follicles was detected by qRT-PCR. Its circular structure was confirmed by RNase R treatment and reversed PCR. The function of ssc-circINHA-001 in GC resistance to apoptosis was detected by in vitro transfection of its si-RNA. Furthermore, the dual-luciferase reporter assay suggested that ssc-circINHA-001 adsorbed three miRNAs, termed miR-214-5p, miR-7144-3p, and miR-9830-5p, which share the common target INHBA. A low expression of ssc-circINHA-001 increased the levels of the free miRNAs, inhibited INHBA expression, and thus raised GCs apoptosis through a shift from the secretion of activin to that of inhibin. Our study demonstrated the existence of a circRNA-microRNAs-INHBA regulatory axis in follicular GC apoptosis and provides insight into the relationship between circRNA function and its coding gene in inhibin/activin balance and ovarian physiological functions.

Correlation between leptin and IFN-γ involved in granulosa cell apoptosis in PCOS.

OBJECTIVES: Our study aimed to explore the relationship between leptin and IFN-γ in PCOS patients, and confirmed the effect of leptin-induced IFN-γ on granulosa cells furtherly. METHODS: 29 patients with PCOS and 36 healthy controls were enrolled. Leptin level and the proportion of Th1 cells were detected and association between them were analyzed. Meanwhile, peripheral blood mononuclear cells (PBMCs) isolated from PCOS patients were treated with leptin and then the proportion of Th1 was analyzed. Besides that, the apoptotic level of KGN cells was monitored after IFN-γ treatment. RESULTS: In the circulation of PCOS patients, leptin level dramatically increased compared with controls. And, this was associated with upregulated Th1 cells proportion and IFN-γ level. In vitro, Th1 cells proportion increased after leptin treated PBMCs from PCOS patients. Furthermore, for KGN cells, the percentage of live cells decreased and later apoptosis cells increased after IFN-γ treatment. CONCLUSIONS: Our results indicated that leptin takes part in process of PCOS via inducing expression of IFN-γ. Our findings highlight the importance of the connection between leptin and inflammation in PCOS and provide new insights therapeutic strategy for this disease.

Characteristics of Circular RNA Expression Profiles of Porcine Granulosa Cells in Healthy and Atretic Antral Follicles.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions-circ_CBFA2T2 and circ_KIF16B-were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes-the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

MicroRNAs in ovarian follicular atresia and granulosa cell apoptosis.

MicroRNAs (miRNAs) are short, noncoding RNAs that posttranscriptionally regulate gene expression. In the past decade, studies on miRNAs in ovaries have revealed the key roles of miRNAs in ovarian development and function. In this review, we first introduce the development of follicular atresia research and then summarize genome-wide studies on the ovarian miRNA profiles of different mammalian species. Differentially expressed miRNA profiles during atresia and other biological processes are herein compared. In addition, current knowledge on confirmed functional miRNAs during the follicular atresia process, which is mostly indicated by granulosa cell (GC) apoptosis, is presented. The main miRNA families and clusters, including the let-7 family, miR-23-27-24 cluster, miR-183-96-182 cluster and miR-17-92 cluster, and related pathways that are involved in follicular atresia are thoroughly summarized. A deep understanding of the roles of miRNA networks will not only help elucidate the mechanisms of GC apoptosis, follicular development, atresia and their disorders but also offer new diagnostic and treatment strategies for infertility and other ovarian dysfunctions.

Smad4 Feedback Enhances BMPR1B Transcription in Ovine Granulosa Cells.

BMPR1B is a type 1B receptor of the canonical bone morphogenetic protein (BMP)/Sma- and mad-related protein (Smad) signaling pathway and is well known as the first major gene associated with sheep prolificacy. However, little is known about the transcriptional regulation of the ovine BMPR1B gene. In this study, we identified the ovine BMPR1B gene promoter and demonstrated that its transcription was regulated by Smad4. In sheep ovarian follicles, three transcriptional variants of BMPR1B gene with distinct transcription start sites were identified using 5' RACE assay while variants II and III were more strongly expressed. Luciferase assay showed that the region -405 to -200 nt is the PII promoter region of variant II. Interestingly, two putative Smad4-binding elements (SBEs) were detected in this region. Luciferase and ChIP assay revealed that Smad4 enhances PII promoter activity of the ovine BMPR1B gene by directly interacting with SBE1 motif. Furthermore, in the ovine granulosa cells, Smad4 regulated BMPRIB expression, and BMPRIB-mediated granulosa cells apoptosis. Overall, our findings not only characterized the 5' regulatory region of the ovine BMPR1B gene, but also uncovered a feedback regulatory mechanism of the canonical BMP/Smad signaling pathway and provided an insight into the transcriptional regulation of BMPR1B gene and sheep prolificacy.

miR-181b-induced SMAD7 downregulation controls granulosa cell apoptosis through TGF-ß signaling by interacting with the TGFBR1 promoter.

SMAD7 disrupts the TGF-ß signaling pathway by influencing TGFBR1 stability and by blocking the binding of TGFBR1 to SMAD2/3. In this study, we showed that SMAD7 attenuated the TGF-ß signaling pathway in ovarian granulosa cells (GCs) by regulating TGFBR1 transcriptional activity. To function as a transcription factor, SMAD7 downregulated the mRNA levels of TGFBR1 via direct binding to the SMAD-binding elements (SBEs) within the promoter region of pig TGFBR1. We also showed that SMAD7 enhanced porcine GC apoptosis by interrupting TGFBR1 and the TGF-ß signaling pathway. Interestingly, miR-181b, a microRNA that is downregulated during porcine follicular atresia, was identified to be directly targeting SMAD7 at its 3'-UTR. By inhibiting SMAD7, miR-181b could inhibit GC apoptosis by activating the TGF-ß signaling pathway. Our findings provide new insights into the mechanisms underlying the regulation of the TGF-ß signaling pathway by SMAD7 and miR-181b.

Alleviation of endoplasmic reticulum stress protects against cisplatin-induced ovarian damage.

BACKGROUND: Cisplatin (CDDP), a widely used chemotherapeutic agent, can induce excessive granulosa cell apoptosis, follicle loss and even premature ovarian insufficiency (POI). However, the mechanism remains elusive, although some studies have indicated the involvement of endoplasmic reticulum stress (ERS). The aim of our study was to investigate the possible mechanism ERS in CDDP-induced granulosa cell apoptosis and follicle loss. METHODS: A POI mouse model was generated by CDDP. The ovaries samples were collected and processed for isobaric tags for relative and absolute quantification analysis (iTRAQ) to screen out our interested proteins of HSPA5 and HSP90AB1, and the decline in their expression were verified by a real-time quantitative PCR and a western blotting assay. In vitro, human granulosa cells, KGN and COV434 cells were transfected with siRNA targeting HSPA5 and HSP90AB1 and then treated with CDDP, or treated with CDDP with/without CDDP+ 4-phenylbutyric acid (4-PBA) and 3-methyladenine (3-MA). The levels of ERS, autophagy and apoptosis were evaluated by western blotting, DALGreen staining and flow cytometry. In vivo, ovaries from mice that received intraperitoneal injections of saline, CDDP, CDDP+ 4-PBA and CDDP+ 3-MA were assayed by immunofluorescence, hematoxylin and eosin (H&E) staining for follicle counting, and terminal-deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining for cell apoptosis assay. The plasma hormone levels were measured by an enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: We have clarified the relationships between ERS, autophagy, and apoptosis in CDDP-induced granulosa cell apoptosis, both in vitro and in vivo. Alleviating ERS by inhibiting HSPA5 and HSP90AB1 attenuated CDDP-induced autophagy and apoptosis. 4-PBA treatment significantly attenuated CDDP-induced cell autophagy and apoptosis in cultured KGN and COV434 cells. However, inhibiting cell autophagy with 3-MA negligibly restored the CDDP-induced changes in ERS and apoptosis. In vivo experiments also demonstrated that treatment with 4-PBA, but not 3-MA, prevented CDDP-induced ovarian damage and hormone dysregulation. CONCLUSIONS: CDDP-induced ERS could promote autophagy and apoptosis in granulosa cells, causing excessive follicle loss and endocrine disorders. Alleviation of ERS with 4-PBA, but not of autophagy with 3-MA, protect against CDDP-induced granulosa cell apoptosis and ovarian damage. Thus, 4-PBA can be used to protect the ovary during chemotherapy in women.

Morphological study of apoptosis in granulosa cells and ovulation in a model of atresia in rat preovulatory follicles.

SummaryPrevious studies have established a model of atresia in preovulatory follicles after stimulation of immature rats with equine chorionic gonadotropin (eCG). This gonadotropin recruits a follicular pool and the deprivation of preovulatory luteinizing hormone (LH) surge induces the atresia in preovulatory follicles. The present study investigated the occurrence of ovulation and provided some morphological features of granulosa cell (GC) apoptosis of atretic follicles at 0, 48, 72 and 120 h after eCG stimulation. Histological sections of ovaries from untreated animals (0 h) showed primordial, primary, secondary and early antral follicles. After 48 h ovaries showed large antral follicles. Preovulatory follicles were observed at 72 h, and two out of five rats displayed cumulus-oocyte complexes (COCs) in the oviducts. All animals exhibited corpora lutea after 120 h. We observed increased estradiol (E2) levels 48 h after eCG treatment that might trigger an endogenous preovulatory gonadotropin surge. Higher progesterone (P4) level, which is the hallmark of a functional corpus luteum, was observed at 120 h. Atresia in secondary and antral follicles was observed by pyknotic granulosa cell nuclei in histology and positive immunolabelling for cleaved caspase 3. We also observed macrophages in secondary and antral follicles in atresia. Transmission electron microscopy revealed GCs with compacted chromatin against the nuclear envelope, nuclear fragmentation, cell shrinkage and fragmentation. No preovulatory follicles showed apoptosis of GCs. In conclusion, our results suggested the occurrence of an endogenous gonadotropin surge, promoting ovulation and preventing atresia of preovulatory follicles.

MicroRNA-26b functions as a proapoptotic factor in porcine follicular Granulosa cells by targeting Sma-and Mad-related protein 4.

Sma- and Mad-related protein 4 (SMAD4) is the central mediator of the transforming growth factor beta signaling pathway and is closely related to mammalian reproductive ability and the development of ovarian follicles. However, little is currently known about the role of SMAD4 in mammalian follicular granulosa cell (GC) apoptosis or its regulation by miRNAs. Here, we found that the porcine SMAD4 protein was expressed at high levels in GCs and oocytes from primary, preantral, and antral follicles, and only slightly expressed in theca cells; its expression level was down-regulated in apoptotic ovarian GCs, suggesting that SMAD4 may be involved in ovary development and selection. Overexpression and knockdown of SMAD4 increased the proliferation and apoptosis of cultured porcine GCs, respectively. In addition, the use of miRNA mimics and luciferase reporter assays revealed that miRNA-26b (miR-26b) functions as a proapoptotic factor in porcine follicular GCs by targeting the 3'-untranslated region of the SMAD4 gene. Overexpression of miR-26b in follicular GCs suppressed SMAD4 mRNA and protein levels, resulting in down-regulation of the antiapoptotic BCL-2 gene and the promotion of GC apoptosis. Furthermore, transforming growth factor beta 1 (TGF-beta1) down-regulates miR-26b expression in porcine GCs. Taken together, these data suggest that SMAD4 plays a critical role in porcine follicular GC apoptosis and follicular atresia and that miR-26b may have a proapoptotic role in GCs by regulating the expression of SMAD4 in the transforming growth factor beta signaling pathway.

miR-423 sponged by lncRNA NORHA inhibits granulosa cell apoptosis.

BACKGROUND: Atresia and degeneration, a follicular developmental fate that reduces female fertility and is triggered by granulosa cell (GC) apoptosis, have been induced by dozens of miRNAs. Here, we report a miRNA, miR-423, that inhibits the initiation of follicular atresia (FA), and early apoptosis of GCs. RESULTS: We showed that miR-423 was down-regulated during sow FA, and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo. The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis, especially early apoptosis in GCs. Mechanically speaking, the miR-423 targets and interacts with the 3'-UTR of the porcine SMAD7 gene, which encodes an apoptosis-inducing factor in GCs, and represses its expression and pro-apoptotic function. Interestingly, FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423. Additionally, we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths (NSB) trait of sows. CONCLUSION: These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis, suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.

Downregulation of miR-192 Alleviates Oxidative Stress-Induced Porcine Granulosa Cell Injury by Directly Targeting Acvr2a.

Follicular atresia is primarily caused by breakdown to granulosa cells (GCs) due to oxidative stress (OS). MicroRNAs (miRNAs) elicit a defense response against environmental stresses, such as OS, by acting as gene-expression regulators. However, the association between miRNA expression and OS in porcine GCs (PGCs) is unclear. Here, we examined the impact of H2O2-mediated OS in PGCs through miRNA-Seq. We identified 22 (14 upregulated and 8 downregulated) and 33 (19 upregulated and 14 downregulated) differentially expressed miRNAs (DEmiRNAs) at 100 µM and 300 µM H2O2, respectively, compared with the control group. Among the DEmiRNAs, mi-192 was most induced by H2O2-mediated OS, and the downregulation of miR-192 alleviated PGC oxidative injury. The dual-luciferase reporter assay results revealed that miR-192 directly targeted Acvr2a. The Acvr2a level was found to be remarkably decreased after OS. Furthermore, grape seed procyanidin B2 (GSPB2) treatment significantly reduced the H2O2-induced upregulation of miR-192, and decreased PGC apoptosis and oxidative damage. Meanwhile, GSPB2 prevented an H2O2-induced increase in caspase-3 activity, which was enhanced by the application of the miR-192 inhibitor. These results indicate that GSPB2 protects against PGC oxidative injury via the downregulation of miR-192, the upregulation of Acvr2a expression, and the suppression of the caspase-3 apoptotic signaling pathway.

NORHA, a novel follicular atresia-related lncRNA, promotes porcine granulosa cell apoptosis via the miR-183-96-182 cluster and FoxO1 axis.

BACKGROUND: Follicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility, which are regulated by many factors such as microRNAs (miRNAs), which constitute a class of noncoding RNAs (ncRNAs). However, little is known about long noncoding RNAs (lncRNAs), which constitute another ncRNA family that regulate follicular atresia. RESULTS: A total of 77 differentially expressed lncRNAs, including 67 upregulated and 10 downregulated lncRNAs, were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing. We characterized a noncoding RNA that was highly expressed in atretic follicles (NORHA). As an intergenic lncRNA, NORHA was one of the upregulated lncRNAs identified in the atretic follicles. To determine NORHA function, RT-PCR, flow cytometry and western blotting were performed, and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress. To determine the mechanism of action, bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation assay were performed, and the results showed that NORHA acted as a 'sponge', that directly bound to the miR-183-96-182 cluster, and thus prevented its targeted inhibition of FoxO1, a major sensor and effector of oxidative stress. CONCLUSIONS: We provide a comprehensive perspective of lncRNA regulation of follicular atresia, and demonstrate that NORHA, a novel lncRNA related to follicular atresia, induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.

SMAD4-induced knockdown of the antisense long noncoding RNA BRE-AS contributes to granulosa cell apoptosis.

Antisense long noncoding RNAs (AS-lncRNAs), a sub-class of lncRNAs, are transcribed in the opposite direction from their overlapping protein-coding genes and are implicated in various physiological and pathological processes. However, their role in female reproduction remains largely unknown. Here, we report that BRE-AS, an AS-lncRNA transcript from intron 10 of the protein-coding gene BRE, is involved in granulosa cell (GC) apoptosis. Based on our previous RNA sequencing data, we identified 28 AS-lncRNAs as important in the initiation of porcine follicular atresia, with BRE-AS showing the most significant upregulation in early atretic follicles. In this study, gain- and loss-of-function assays demonstrated that BRE-AS induces early apoptosis in GCs. Mechanistically, BRE-AS acts in cis to suppress the expression of BRE, an anti-apoptotic factor, via direct interaction with the pre-mRNA transcript of the latter, inducing increased GC apoptosis. Notably, we also found that BRE-AS was upregulated in SMAD4-silenced GCs. SMAD4 was identified as a transcriptional repressor of BRE-AS because it inhibits BRE-AS expression and BRE-AS-mediated GC apoptosis. In conclusion, we not only identified a novel AS-lncRNA related to the early apoptosis of GCs and initiation of follicular atresia but also described a novel regulatory pathway, SMAD4/BRE-AS/BRE, coordinating GC function and female fertility.

Upregulation of miR-146b promotes porcine ovarian granulosa cell apoptosis by attenuating CYP19A1.

MicroRNAs (miRNAs) are 21- to 24-nucleotide long small noncoding RNAs, which play an important role in follicular atresia and granulosa cell (GC) apoptosis in the mammalian ovary. Here, we report that miR-146b, a conserved and ovary-enriched miRNA, modulates estradiol (E2) secretion, GC apoptosis, and follicular atresia in pigs. Genome-wide analysis and quantitative real-time PCR revealed that miR-146b was significantly upregulated during follicular atresia, and fluorescence-activated cell sorting showed that miR-146b functioned as a proapoptotic factor to induce GC apoptosis. MicroRNA-mRNA network analysis and luciferase reporter assays showed that CYP19A1, the pivotal enzyme for E2 synthesis signaling, was directly targeted by miR-146b. Furthermore, miR-146b interacted with the 3'untranslated region of CYP19A1 to prevent translation, thereby regulating CYP19A1-mediated E2 secretion and GC apoptosis. However, miR-146b was not regulated by the transcription factor SMAD4 or oxidative stress, both of which are critical regulators of CYP19A1. We, thus, conclude that miR-146b is a novel epigenetic factor regulating GC functions, follicular development, and female reproduction.