Exploring the genetic mechanism of ovulation rate is an important way to improve the kidding rate, which is fundamental to goat production. The greater number of large follicles that stimulate ovulation are believed to be a primary reason for higher ovulation rate . Despite many mRNAs and miRNAs have been found in playing critical roles in ovarian biological processes, the major genes and miRNAs related to ovulation rate and kidding rate have not been identified in goats. In addition, the interaction between mRNAs and miRNAs in follicular development is not yet completely understood. To identify the key genes and miRNAs as well as interactions between them involved in ovulation rate and kidding rate, we compared DEmRNAs and DEmiRNAs from different sizes of follicles between uniparous and multiple CZ black goats during estrus phase by using RNA-Seq.
The result showed that there were more large follicles in multiple than in uniparous goats (P <0.05), while no difference was observed in small follicles between multiple and uniparous goats, verifying that the higher number of large follicles was related to higher ovulation rate . Based on the RNA sequence data, we identified 119 and 37 DEmRNAs in Uni-S vs Mul-S and Uni-L vs Mul-L, respectively (FPKM >1 of at least three samples per group). These DEmRNAs were involved in the ovarian development related pathways, such as ovarian steroidogenesis, steroid hormone biosynthesis, metabolism of xenobiotics by cytochrome P450 and so on (Figure 2e-f). And many genes (about 37% in Uni-S vs Mul-S and about 41% Uni-L vs Mul-L) had been reported to be associated with reproduction in mammal (data not showed), such as TNFAIP6, MMP9, INSL3, LEPR, 3BHSD, LHCGR, ARL4C, CD36, CYP11A1, AMDHD1, SPOCK2, AMDHD1, MFAP5, CCL21, PTGFR, SERPINA5, and so on [19, 41–54]. Of these genes, TNFAIP6, CYP11A1, CD36, PTGFR and SERPINA5 had been reported to be associated with ovulation rate, and TNFAIP6, CYP11A1 and CD36 were differentially expressed in Uni-S vs Mul-S, PTGFR and SERPINA5 were in Uni-L vs Mul-L.
TNFAIP6 is a secretory protein of the hyaluronan-binding protein family, which plays a role in cumulus cell stabilisation and expansion, being upregulated in bovine granulosa cells (GCs) during ovulation [41, 55–57]. And TNFAIP6-deficient females were sterile in mice. The present study reported that TNFAIP6 gene expression was 8-fold higher in Mul-S than in Uni-S, suggesting a possible role of TNFAIP6 gene in the cumulus cells (CCs) expansion in small follicles of multiple goats at estrus phase. CYP11A1 played a key role in the regulation of steroid-producing pathways in GCs . The first step of steroid biosynthesis pathway was to convert cholesterol into pregnenolone through the action of CYP11A1 gene in mitochondria, and pregnenolone acted as a substrate for progesterone synthesis through the mediation of 3BHSD gene [59, 60]. In this study, CYP11A1 gene was up-regulated in small follicles from multiple goats, which was consistent with the reports in goat ovary , demonstrating that CYP11A1 gene might perform multiple roles in ovarian development in goat. CD36 is a multifunctional receptor-binding autocrine growth factor that can regulate angiogenesis, cell growth and adhesion. The expression of CD36 genewas follicle-type dependent with the greatest expression in atretic follicles, and the lowest in healthy follicles[61–64]. Knockdown of CD36 gene had been shown to increase proliferation and expression of survival and angiogenic in granulosa, endothelial and tumor cells . In this study, CD36 gene was up-regulated in uniparous goats and participated in hematopoietic cell lineage, speculating that CD36 gene may affect litter size by affecting the proliferation of GCs or angiogenesis. Overall, TNFAIP6 and CYP11A1 genes played a positive role in the regulation of ovulation, while CD36 gene was contributed to follicular atretic. Accordingly, TNFAIP6 and CYP11A1 genes was upregulated in small follicles of multiple goats in our study, and CD36 gene was downregulated, indicating that there were more small follicles could grow up to dominant follicle in multiple goat. Thus, these genes might play a key role in ovulation rate or kidding rate in goat.
In GCs of periovulatory follicles and corpus luteum (CL) of mice, the expression of PTGFR was drastically reduced . For human and primates, PTGFR stimulation promotes functional luteolysis only when PTGFRs relocated from the cytoplasm to the perinuclear region, which may be a necessary step in the initiation of luteolysis in monkey. Our result showed that PTGFR gene was downregulated in large follicles from multiple goats, suggesting that the greater reduction of PTGFR gene expression in multiple goats may contribute to ovulate more. The previous studies reported that SERPINA5, as a protease inhibitor, was expressed in the reproductive tract of adult mice and in the GCs of bovine follicles, and was highly expressed in bovine healthy follicles [54, 67]. SERPINA5 gene was down-regulated in ovarian cancer (OC) studies [68–70]. We found that SERPINA5 gene was up-regulated in large follicles from multiple goats, suggesting that SERPINA5 gene may affect follicular development and kidding ratein goat. Take it together, the downregulation of PTGFR gene and upregulation of SERPINA5 gene in large follicles would be helpful to ovulate more in multiple goats.
We identified seven and sixteen differentially expressed miRNAs in Uni-S and Mul-S and Uni-L and Mul-L groups, respectively. And miR–200a, miR–451–5p, miR–141, miR–182, miR–206 and miR–122 was highly expressed miRNAs in Uni-S and Mul-S groups, miR–1, miR–206, miR–133a–3p, miR–133b, miR–182, miR–215–5p, miR–122 and miR–451–5p was highly expressed miRNAs in Uni-L and Mul-L groups. However, only miR–200a were reported highly expressed in the ovaries of goats , demonstrating that the expression of miRNAs in whole ovary, small follicles and large follicles were independent of each other. Of these 14 highly expressed DEmiRNAs, miR–200a, miR–141, miR–1, miR–206, miR–133b, miR–133a–3p, miR–182 and miR–122 had been reported to play important roles in basic reproductive activities. miR–200a was more abundant in ovarian tissue during the luteal phase and may have an important role in the follicular-luteal transition by binding to LHR mRNA directly in sheep . miR–200a was frequently overexpressed and was closely related to the cell migratory, cell proliferation and invasive abilities in ovarian [72–74]. miR–141 was significantly upregulated in OC cell lines and advanced metastatic ovarian cancers [75, 76]., and it might inhibit granulosa cell apoptosis by targeting DAPK1 through MAPK signaling pathway and further lead to the development of polycystic ovary syndrome . In this study, miR–200a and miR–141 were upregulated in small follicles of uniparous goats, suggesting that they might affect the normal development of granulosa cells, thereby affected maturation of the oocyte, and finally reducing ovulation rate. miR–206 and miR–1 were the potential tumor suppressor and down-regulated in OC tissues, and they both can inhibit c-Met expression and regulate cell proliferation, migration and invasion [78, 79]. miR–206 inhibited OC cell proliferation, migration and invasion, and induced apoptosis [78, 80, 81]. miR–206 and miR–1were downregulated in large follicles from multiple goats in our study, which may be related to the maturation of the oocyte and ovulation in goat. miR–133 family (miR–133a–3p and miR–133b) had been involved in regulation of many cellular processes such as cell proliferation, apoptosis, migration and invasion [82, 83]. miR–133a–3p was the target of LncRNA HOXD-AS1, whcih promoted the proliferation, invasion, and EMT process of EOC cells and activated Wnt/β-catenin signaling pathway . Foxl2 is a conserved, early-acting gene in vertebrate ovarian development, and play an important role on proliferation of GCs and maturation of the oocyte [61, 62]. Recently, the Foxl2 gene has been reported to be regulated by miR–133b. miR–133b can bind to the Foxl2–3’UTR in GCs to inhibit the expression of the downstream genes STAR and CYP19A1, which can promote estrogen secretion in granulosa cells simultaneously . miR–133b was up-regulated more than 30-fold in MI oocytes after IGF–1 treatment, and may play important roles in the growth and maturation of oocytes by regulating its potential target gene TAGLN2 .
Compared with Uni-S vs Mul-S and Uni-L vs Mul-L, miR–182 and miR–122 were differentially expressed in both groups. miR–182 was up-regulated in follicular fluid of polycystic ovary syndrome (PCOS) patients , and it’s expression was significantly increased in OC cell lines and tissues . miR–122 inhibited epithelial mesenchymal transition by regulating P4HA1 in OC cells . miR–122 played a regulatory role in LHCGR expression, which were crucial for mediating LH action in growing follicles, by modulating LRBP levels during FSH-induced follicle growth . miR–122 mediated LHR mRNA levels by modulating the expression of LRBP through the regulation of SREBP activation, which were crucial for supporting key reproductive processes such as ovulation and CL function . In this study, miR–182 and miR–122 both were upregulated in uniparous goat, suggesting that they may affect ovulation rate by affecting follicular growth in uniparous goats.
Taken together, this study showed that CD36, TNFAIP6, CYP11A1, SERPINA5 and PTGFR would be related to ovarian follicular development and luteinization in goat, but their upstream determinants were still uncertain. By predicting the target genes of DEmiRNAs, high expression of CD36 (miR–122, miR–200a), TNFAIP6 (miR–200a, miR–182), CYP11A1 (miR–122), SERPINA5 (miR–1, miR–206, miR–133a–3p, miR–133b) and PTGFR (miR–182, miR–122) were screened (Figure 6), which may be related to ovulation and kidding rate.