Effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation implantation dysfunction.

Abstract

The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation (COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG), followed by 7.5 IU human chorionic gonadotrophin (HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group (NC), COH implantation dysfunction model group (COH), low dosage of Bu-Shen-An-Tai recipe group (LOW), middle dosage of Bu-Shen-An-Tai recipe group (MID) and high dosage of Bu-Shen-An-Tai recipe group (HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17β-estradiol (E₂) and progesterone (P₄) were determined by radioimmunoassay (RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope (TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis- related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of E2 and P4, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It's helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.