Single-cell transcriptomic atlas of goat ovarian aging.

BACKGROUND: The ovaries are one of the first organs that undergo degenerative changes earlier in the aging process, and ovarian aging is shown by a decrease in the number and quality of oocytes. However, little is known about the molecular mechanisms of female age-related fertility decline in different types of ovarian cells during aging, especially in goats. Therefore, the aim of this study was to reveal the mechanisms driving ovarian aging in goats at single-cell resolution. RESULTS: For the first time, we surveyed the single-cell transcriptomic landscape of over 27,000 ovarian cells from newborn, young and aging goats, and identified nine ovarian cell types with distinct gene-expression signatures. Functional enrichment analysis showed that ovarian cell types were involved in their own unique biological processes, such as Wnt beta-catenin signalling was enriched in germ cells, whereas ovarian steroidogenesis was enriched in granulosa cells (GCs). Further analysis showed that ovarian aging was linked to GCs-specific changes in the antioxidant system, oxidative phosphorylation, and apoptosis. Subsequently, we identified a series of dynamic genes, such as AMH, CRABP2, THBS1 and TIMP1, which determined the fate of GCs. Additionally, FOXO1, SOX4, and HIF1A were identified as significant regulons that instructed the differentiation of GCs in a distinct manner during ovarian aging. CONCLUSIONS: This study revealed a comprehensive aging-associated transcriptomic atlas characterizing the cell type-specific mechanisms during ovarian aging at the single-cell level and offers new diagnostic biomarkers and potential therapeutic targets for age-related goat ovarian diseases.

BMP6 regulates AMH expression via SMAD1/5/8 in goat ovarian granulosa cells.

Anti-Müllerian hormone (AMH) is produced by ovarian granulosa cells (GCs)and plays a major role in inhibiting the recruitment of primordial follicles and reducing the sensitivity of growing follicles to follicle-stimulating hormone (FSH). Bone morphogenetic protein 6 (BMP6) has similar spatiotemporal expression to AMH during follicular development, suggesting that BMP6 may regulate AMH expression. However, the specific mechanism by which BMP6 regulates AMH expression remains unclear. The objectives of this study were to examine the molecular pathway by which BMP6 regulates AMH expression. The results showed that BMP6 promoted the secretion and expression of AMH in goat ovarian GCs. Mechanistically, BMP6 upregulated the expression of sex-determining region Y-box 9 (SOX9) and GATA-binding factor 4 (GATA4), which was associated with the transcriptional initiation of AMH. AMH expression was significantly decreased by GATA4 knockdown. Moreover, BMP6 treatment promoted the phosphorylation of SMAD1/5/8, whereas inhibiting the SMAD1/5/8 signaling pathway significantly abolished BMP6-induced upregulation of AMH and GATA4 expression. Interestingly, the activation of SMAD1/5/8 alone did not affect the expression of AMH or GATA4. The results suggested that BMP6 upregulated GATA4 through the SMAD1/5/8 signaling pathway, which in turn promoted AMH expression.

BMP6 Promotes the Secretion of 17 Beta-Estradiol and Progesterone in Goat Ovarian Granulosa Cells.

The purpose of this study was to investigate the effects of BMP6 on the function of goat ovarian granulosa cells (GCs). The results showed that the exogenous addition of BMP6 did not affect the EdU-positive ratio of ovarian GCs and had no significant effect on the mRNA and protein expression levels of the proliferation-related gene PCNA (p > 0.05). Meanwhile, BMP6 had no significant effect on the cycle phase distribution of GCs but increased the mRNA expression of CDK4 (p < 0.05) and CCND1 (p < 0.01) and decreased the mRNA expression of CCNE1 (p < 0.01). Moreover, BMP6 had no significant effect on the apoptosis rate of GCs and did not affect the mRNA expression levels of apoptosis-related genes BAX, BCL2, and Caspase3 (p > 0.05). Importantly, BMP6 upregulated the secretion of 17 beta-estradiol (E2) and progesterone (P4) in ovarian GCs (p < 0.01). Further studies found that BMP6 inhibited the mRNA expression of 3ß-HSD and steroid synthesis acute regulator (StAR) but significantly promoted the mRNA expression of the E2 synthesis rate-limiting enzyme CYP19A1 and the P4 synthesis rate-limiting enzyme CYP11A1 (p < 0.01). Taken together, these results showed that the exogenous addition of BMP6 did not affect the proliferation, cell cycle, and apoptosis of goat ovarian GCs but promoted the secretion of E2 and progesterone P4 in ovarian GCs by upregulating the mRNA expressions of CYP19A1 and CYP11A1.

Liver Receptor homolog-1 Regulates Apoptosis of Bovine Ovarian Granulosa Cells by Progestogen Receptor Signaling Pathway.

The purpose of the present investigation was to assess the function of LRH-1 on GCs and the mechanisms involved. Here, LRH- was highly expressed in the bovine GCs of atretic follicles. Treatment with 50 µM of LRH-1 agonist (DLPC) significantly induced the expression of LRH-1 (p < 0.05). In particular, LRH-1 activation blocked the progestogen receptor signaling pathway via downregulating progesterone production and progestogen receptor levels (p < 0.05), but had no effect on 17 beta-estradiol synthesis. Meanwhile, LRH-1 activation promoted the apoptosis of GCs and increased the activity of caspase 3 (p < 0.05). Importantly, upregulating the progestogen receptor signaling pathway with progestogen could attenuate the LRH-1-induced proapoptotic effect. Moreover, treatment with progestogen decreased the activity of the proapoptotic gene caspase 3 and increased the expression of antiapoptotic gene Bcl2 in LRH-1 activated GCs (p < 0.05). Taken together, these results demonstrate that LRH-1 might be dependent on the progestogen receptor signaling pathway to modulate bovine follicular atresia.