Recent reports have demonstrated that follicular atresia is initiated or caused by granulosa cell apoptosis followed by theca cell degeneration in mammalian ovaries, but the mechanism of follicular atresia is still to be elucidated. Therefore, our present study was designed to examine our hypothesis that the changes of follicular microenvironment induce the granulosa cell apoptosis during pocrine follicular atresia in vivo. We fi rstly isolated intact porcine antral follicles and identifi ed them into three groups, healthy follicles(HF), early atretic follicles(EAF) and progressed atretic follicles(PAF) through morphology and histology. To further confi rm their status, we detected hormone levels in follicular fl uids and the expression level of apoptosis gene Bax in granulosa cells. The rate of progesterone(P) and estradiol(E2) was increased with the expression of Bax, indicating hormone can be used as a marker of granulosa cell apoptosis or follicular atresia. Finally, we analyzed the expression level of hormone receptor genes in granulosa cells and their relationship with follicular atresia. In PAF, the expression of Progesterone receptor(PGR) was increased signifi cantly while estradiol receptor(ER) had no notable changes, which suggesting the increased-PGR accelerated the effect of P-stimulated granulosa cell apoptosis. The dramatic increasing of androgen receptor(AR) expression in PAF and the obvious increase of tumor necrosis factor-α receptor(TNFR) in EAF indicated that there are different pathways regulating granulosa cell apoptosis during follicular atresia. Together, our results suggested that different pathways of granulosa cell apoptosis was induced by changing the follicular microenvironment during follicular atresia.
Eight ewes of Hu sheep which bred multi-lamb were used as the high-fecundity group and the other eight ewes of Hu sheep which bred single lamb were used as the control group to investigate the relationship between the mRNA expression level of TGF-β receptor genes in tissues and ovulation rate in Hu sheep. Cloprostenol sodium was injected to make the synchronization of estrus treatment, then all ewes were slaughtered within 24-36 h after empathema and the ovaries were collected. Furthermore, the number of ovulation points was counted to determine ovulation rate for each sheep. Tissue expression analysis was conducted by RT-PCR for one ewe form the high-fecundity group and the relationship between the mRNA expression of genes encoding TGF-β receptors and ovulation rate was detected by real-time fluorescent quantitative PCR. The results showed that the relative expression level of TGF-βR I gene in the reproductive organ was significantly higher than in the lung and muscle (P < 0.01), while relative expression level of TGF-βR II in reproductive organ was significantly higher than that of other tissues (P < 0.01), indicating that these are highly expressed genes in the ovary. In addition, mRNA expression level of TGF-βR I and TGF-βRII in the ovaries of the high-fecundity group were significantly higher (P < 0.01) and obviously higher (P = 0.011) than the control group, respectively. The mRNA expression level of TGF-βR I and TGF-βR II had a positive correlation with ovulation rate and the correlation coefficients were 0.562 (P > 0.05) and 0.711 (P < 0.05), respectively. It is suggested that TGF-β receptors have close relationship with highfecundity in Hu sheep.
LI Er-linXIE Xin-huaXU Ye-fenXIE ZhuangCHEN LingLIU Hong-linLI Qi-fa