TGF-β family signaling pathways, including TGF-β and BMP pathways, are widely involved in the regulation of health and diseases through downstream SMADs, which are also regulated by multiple validated mechanisms, such as genetic regulation, epigenetic regulation, and feedback regulation. However, it is still unclear whether R-SMADs or Co-SMAD can feedback regulate the TGF-β family signaling pathways in granulosa cells (GCs). In this study, we report a novel mechanism underlying the feedback regulation of TGF-β family signaling pathways, i.e., SMAD4, the only Co-SMAD, positive feedback activates the TGF-β family signaling pathways in GCs with a basal level of TGF-β ligands by interacting with the core promoters of its upstream receptors. Mechanistically, SMAD4 acts as a transcription factor, and feedback activates the transcription of its upstream receptors, including ACVR1B, BMPR2, and TGFBR2, of the canonical TGF-β signaling pathways by interacting with three coactivators (c-JUN, CREB1, and SP1), respectively. Notably, three different interaction modes between SMAD4 and coactivators were identified in SMAD4-mediated feedback regulation of upstream receptors through reciprocal ChIP assays. Our findings in the present study indicate for the first time that SMAD4 feedback activates the canonical TGF-β family signaling pathways in GCs, which improves and expands the regulatory mechanism, especially the feedback regulation modes of TGF-β family signaling pathways in ovarian GCs.

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.

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.

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.

Transforming growth factor-β receptor II (TGFBR2), the type II receptor of the TGF-β/SMA- and MAD-related protein (SMAD) signaling pathway, plays a crucial role in TGF-β signal transduction and is regulated by multiple factors. Nevertheless, the modulation of the non-coding RNA involved in the process of TGFBR2 expression in ovaries is not well studied. In our study, we isolated and characterized the 3′-untranslated region (UTR) of the porcine TGFBR2 gene and microRNA-1306 (miR-1306) was identified as the functional miRNA that targets TGFBR2 in porcine granulosa cells (GCs). Functional analysis showed that miR-1306 promotes apoptosis of GCs as well as attenuating the TGF-β/SMAD signaling pathway targeting and impairing TGFBR2 in GCs. Moreover, we identified the miR-1306 core promoter and found three potential SMAD4-binding elements (SBEs). Luciferase and chromatin immunoprecipitation (ChIP) assays revealed that the transcription factor SMAD4 directly binds to the miR-1306 core promoter and inhibits its transcriptional activity. Furthermore, the TGF-β/SMAD signaling pathway is modulated by SMAD4 positive feedback via inhibition of miR-1306 expression in GCs. Collectively, our findings provide evidence of an epigenetic mechanism that modulates as well as mediates the feedback regulation of the classical TGF-β/SMAD signaling pathway in GCs from porcine ovaries.

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.