Effects Of Inhibin A On Porcine Granulosa Cell Proliferation And Oocyte Maturation In Vitro

Inhibins, including inhibin A and inhibin B, are glycoprotein hormones secreted by ovaries infemale and testes in male, and belong to the TGFβ superfamily. In female, inhibin primarily inhibits thesynthesis and secretion of FSH from pituitary gland, regulates the development of the ovarian folliclesand oocyte. Inhibin can regulate the synchronous development of oocyte and granulosa cell throughautocrine and paracrine at local ovary. The level of inhibin is one of an indicator reflecting the numberof growing follicles in ovary during estrous cycle, which is a key factor in determining the developmentof follicles and ovulation. Yet few studies have looked into the effects of inhibin on the maturation ofoocytes, especially in sows. In this study, a series of experiments were taken in order to find out theeffects of inhibin A on the synthesis of steroid hormone, proliferation or apoptosis of the granulosa cell(GCs) and the maturation of oocytes. In addition, the association between polymorphisms of the inhibinα subunit and follicular cyst was studied. The contents of the study are as follows.1. Effects of inhibin A on the synthesis of steroid hormoneGCs were separated from ovarian follicles, at the purity of more than90%identified byimmunofluorescence technique with the FSHR antibody. After that, the GCs were treated with differentconcentrations of inhibin A (0,10,100,200ng/mL respectively) and the antibody against inhibin α(400ng/mL) for4different phases,12h,24h,48h, and72h. Effects of inhibin A on mRNA expression inGCs for FSHR, LHR, STAR and CYP11a1were detected using RT-PCR. The results showed that themRNA expression of FSHR, LHR, CYP11a1and STAR was significantly elevated (p<0.05) aftertreatment with200ng/mL inhibin A for48h. The concentrations of progesterone and estradiol in theculture supernatant were detected by ELISA. The results showed that200ng/mL inhibin A stimulatedestradiol secretion after treatment for48h (p<0.05). The concentrations of progesterone decreased aftertreatment with200ng/mL inhibin A for24h-48h. However, the secretion of progesterone wasincreased after treatment with200ng/mL inhibin A for72h. and remarkably inhibited by inhibin αantibody. 2. Effects of inhibin A on the GCs proliferation and apoptosisIn order to study the effects of inhibin A on the proliferation and apoptosis of GCs, GCs weretreated with inhibin A at different concentrations (0,20,50,100ng/mL) for24h. The cell viability wasdetected with MTS, the result showed that high concentration (50,100ng/ml) of inhibin A significantlyenhanced the GCs viability (p<0.05) with a dose dependent manner. Meanwhile, the MMP remarkablyboosted after treated with100ng/mL inhibin A for48h. The influence of inhibin A on the expression ofproliferation and apoptosis genes was studied, the results displayed that incubating the cell for48h withinhibin A promoted the expression of genes PCNA and CCNB1(p<0.05), but inhibited the expression ofCaspase-3(p<0.05) with dose dependent manner. The flow cytometry results showed that S phase cellsincreased from8.50±0.25%to12.36±0.61%after treated with200ng/mL inhibin A for48h (p<0.05).Then, the gene of α subunit of inhibin was silented by using Ri-RNA. In consequences, the expressionof gene Caspase-3(p<0.05) was increased while the expression of the cell cycle gene CCNB1(p<0.05)was inhibited. All the results pointed to the conclusion that inhibin A promoted the proliferation of GCswhile inhibited apoptosis.3. Effects of inhibin A on the maturation of the oocytes in vitroInhibin A had a positive effects on oocytes maturation in vitro. IVM culture medium supplied with200ng/mL inhibin A increased the expression of GDF9and COX2(p<0.05) at stage of germinal vesicle(GV), and promoted cumulus cell expansion after44h (p<0.05). The inhibin A affected the kinetics ofthe early cleavage divisions, but had no effect on the development of blastocysts of PA (p>0.05).4. Association of inhibin α subunit gene polymorphisms with ovarian cystsThe polymorphisms of c.-42G>A and c.3222G>A were analysed by PCR-SSCP and PCR-RFLP.The results showed that the c.-42G>A and c.3222G>A polymorphisms were significantly associatedwith follicular cysts, and that sows with c-42GG and c.3222GG genotypes had lower risk of developingcysts. Also, the result of ELISA demonstrated that follicular fluid from the cystic follicle containedhigher level of inhibin A compared to the large follicle (p<0.05).To conclude, the study demonstrated that inhibin A stimulated the synthesis and secretion of steroidhormones, contributed to GCs proliferation and oocytes maturation in vitro. The inhibin A affected thekinetics of the early cleavage divisions, but had no effect on the development of blastocysts of PA. Iaddition, the c.-42G>A and c.3222G>A polymorphisms of inhibin α subunit gene were significantly associated with follicular cysts.