MiR-29c-5p regulates the function of buffalo granulosa cells to induce follicular atresia by targeting INHBA.

MicroRNAs (miRNAs) are involved in many physiological processes such as signal transduction, cell proliferation and apoptosis. Many studies have shown that miRNAs can regulate the process of follicular development. Our previous studies found that the expression of miR-29c-5p in buffalo atretic follicles was much higher than that in healthy follicles, suggesting that this miRNA may participate in the process of buffalo follicular atresia. In this study, we aim to explore to the role and molecular mechanisms of miR-29c-5p on the functions of buffalo granulosa cells (GCs). GCs cultured in vitro were transfected with miR-29c-5p mimics and its inhibitor, respectively, and it was found that the mimics significantly increased the apoptotic rate of GCs. They also inhibited the proliferation of GCs and the secretion of steroid hormones. The effect of the inhibitor was opposite to that of the mimics. MiR-29c-5p was subsequently shown to target the inhibin subunit beta A, (INHBA). Overexpression of INHBA could promote the production of activin A and inhibin A, and then reverse the effect of miR-29c-5p on buffalo GCs. In conclusion, these results suggest that miR-29c-5p promotes apoptosis and inhibits proliferation and steroidogenesis by targeting INHBA in buffalo GCs. This may ultimately promote atresia in buffalo follicles.

Targeted metabolomics analysis of bile acids and cell biology studies reveal the critical role of glycodeoxycholic acid in buffalo follicular atresia.

The follicular fluid of mammals has a high abundance of bile acids and these have proven to be closely related to the follicular atresia. However, the origin and content of bile acids in follicular fluid and its mechanisms on follicular atresia remain largely unknown. In this work, we analyzed the origin of bile acids in buffalo follicles by using cell biology studies, and quantified the subspecies of bile acids in follicular fluid from healthy follicles (HF) and atretic follicles (AF) by targeted metabolomics. The function of differential bile acids on follicular granulosa cells was also studied. The results showed that the bile acids transporters were abundantly expressed in ovarian tissues, but the rate-limiting enzymes were not, which was consistent with the inability of cultured follicular cells to convert cholesterol into bile acids. Targeted metabolomics analysis revealed thirteen differential subspecies of bile acids between HF and AF. The free bile acids were significant down-regulated and their conjugated forms were significantly up-regulated in AF as compared to HF. Finally, cell biological validation found a specific differentially conjugated bile acid, glycodeoxycholic acid (GDCA), which could promote follicular granulosa cell apoptosis and reduce steroid hormone secretion. In summary, our studies suggest that bile acids in buffalo follicles are transported from the blood rather than being synthesized within the follicles. The conjugated bile acids such as GDCA, accumulate in buffalo follicles, and may accelerate atresia by promoting apoptosis of granulosa cells and inhibiting steroid hormone production. These results will provide new clues for studying the physiological role and mechanism of bile acids involved in buffalo follicular atresia.

Non-targeted Metabolomics Reveals Metabolic Characteristics of Porcine Atretic Follicles.

Follicular atresia is one of the main factors limiting the reproductive power of domestic animals. At present, the molecular mechanisms involved in porcine follicular atresia at the metabolic level remain unclear. In this study, we divided the follicles of Bama Xiang pigs into healthy follicles (HFs) and atretic follicles (AFs) based on the follicle morphology. The expression of genes related to atresia in granulosa cells (GCs) and the concentration of hormones in the follicular fluid (FF) from HFs and AFs were detected. We then used liquid chromatography-mass spectrometry-based non-targeted metabolomic approach to analyze the metabolites in the FF from HFs and AFs. The results showed that the content of estradiol was significantly lower in AFs than in HFs, whereas that of progesterone was significantly higher in AFs than that in HFs. The expression of BCL2, VEGFA, and CYP19A1 was significantly higher in HFs than in AFs. In contrast, the expression of BAX and CASPASE3 was significantly lower in HFs. A total of 18 differential metabolites (DMs) were identified, including phospholipids, bioactive substances, and amino acids. The DMs were involved in 12 metabolic pathways, including arginine biosynthesis and primary bile acid biosynthesis. The levels of eight DMs were higher in the HF group than those in the AF group (p < 0.01), and those of 10 DMs were higher in the AF group than those in the HF group (p < 0.01). These findings indicate that the metabolic characteristics of porcine AFs are lower levels of lipids such as phospholipids and higher levels of amino acids and bile acids than those in HFs. Disorders of amino acid metabolism and cholic acid metabolism may contribute to porcine follicular atresia.

Integration of transcriptomics and non-targeted metabolomics reveals the underlying mechanism of follicular atresia in Chinese buffalo.

Follicular atresia is a complex physiological process, which results in the waste of follicles and oocytes from the ovary. Elucidating the physiological mechanism of follicular atresia will hopefully reverse the fate of follicles, thereby improve the reproductive efficiency of female animals. However, there are still many gaps to be filled during the follicular atresia process. In this study, we first comprehensively summarized and compared a variety of methods to classify Chinese buffalo follicles with different extent of atresia. Then follicular fluid and granulosa cells from the corresponding follicles with different extent of atresia were collected for non-targeted metabolomics and transcriptomics analysis, respectively. After the detection and analysis of 129 follicles, a reasonable classification standard was formed: on the basis of morphological classification, the relative concentrations of estradiol (E2) and progesterone (PROG) in the follicular fluid were determined, follicles with an estradiol-to-progesterone (E2/PROG) ratio >5 were classified as healthy follicles (HF), 1≤ E2/PROG ≤5 as early atretic follicles (EF) and E2/PROG <1 as late atretic follicles (LF). Correspondingly, follicles with granulosa cells apoptosis rate less than 15 % were divided into HF, 15%-25% were classified as EF and more than 25 % were classified as LF. The integration analysis of non-targeted metabolomics and transcriptomics highlights the following three aspects: (1) Atresia seriously damaged the lipid metabolism homeostasis of follicle, in which PPARγ play important roles. (2) Energy metabolism and nucleotide metabolism of atretic follicles were inhibited. (3) Bilirubin is involved in follicular atresia, and it may be the main force to prevent lipid peroxidation in follicular cells. In summary, results of this study provide new understanding of the molecular mechanisms of Chinese buffalo follicular atresia.