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Abstract：
OBJECTIVE: Kidney-tonifying formulas are frequently used in clinical practices to enhance follicular development and maturation. This research explored the impacts of the Bushen Tiaojing formula (BSTJF) on the development of mouse preantral follicles in vitro and its relationship with granulosa cells and gonadotropins.
METHODS: Preantral follicles were extracted from mice and cultured with or without serum from rats that were previously treated with or without BSTJF. During cultivation, the follicles were monitored for morphological changes and developmental maturation. Exhausted medium was collected every other day for the measurement of progesterone and estradiol (E2) levels by ELISA. Granulosa cells in in-vitro medium were collected on days 8, 10, and 12 and analyzed for determining the expressions of apoptosis-associated genes (Bax, Bcl-2, and Caspase-3). Propagation and apoptosis rates of collected granulosa cells were measured by CCK-8 assay and flow cytometry.
RESULTS: Compared with control follicles, follicles cultured with serum from BSTJF-treated rats had a higher survival rate, larger follicle diameter, higher Bcl-2 expression, and lower Bax and Caspase-3 expressions (all P ≤ 0.05). In addition, their granulosa cells presented substantially elevated proliferation (P ≤ 0.05) and a lower rate of apoptosis (P ≤ 0.05) compared with granulosa cells from control follicles. The level of E2 in the culture media of all groups increased slowly in the first 6 days. Subsequently, after formation of the antrum, the levels of E2 and progesterone were enhanced in the medium of follicles cultured with serum from BSTJF-treated rats compared with those in the media of control follicles (all P ≤ 0.05).
CONCLUSIONS: Serum from BSTJF-treated rats facilitated the in vitro development and maturation of mouse follicles by increasing the expression of anti-apoptotic gene Bcl-2, reducing the expressions of pro-apoptotic genes Bax and Caspase-3 as well as the apoptosis of granulosa cells, promoting the proliferation of granulosa cells and increasing the secretion of E2 and progesterone in the cells.
METHODS: Preantral follicles were extracted from mice and cultured with or without serum from rats that were previously treated with or without BSTJF. During cultivation, the follicles were monitored for morphological changes and developmental maturation. Exhausted medium was collected every other day for the measurement of progesterone and estradiol (E2) levels by ELISA. Granulosa cells in in-vitro medium were collected on days 8, 10, and 12 and analyzed for determining the expressions of apoptosis-associated genes (Bax, Bcl-2, and Caspase-3). Propagation and apoptosis rates of collected granulosa cells were measured by CCK-8 assay and flow cytometry.
RESULTS: Compared with control follicles, follicles cultured with serum from BSTJF-treated rats had a higher survival rate, larger follicle diameter, higher Bcl-2 expression, and lower Bax and Caspase-3 expressions (all P ≤ 0.05). In addition, their granulosa cells presented substantially elevated proliferation (P ≤ 0.05) and a lower rate of apoptosis (P ≤ 0.05) compared with granulosa cells from control follicles. The level of E2 in the culture media of all groups increased slowly in the first 6 days. Subsequently, after formation of the antrum, the levels of E2 and progesterone were enhanced in the medium of follicles cultured with serum from BSTJF-treated rats compared with those in the media of control follicles (all P ≤ 0.05).
CONCLUSIONS: Serum from BSTJF-treated rats facilitated the in vitro development and maturation of mouse follicles by increasing the expression of anti-apoptotic gene Bcl-2, reducing the expressions of pro-apoptotic genes Bax and Caspase-3 as well as the apoptosis of granulosa cells, promoting the proliferation of granulosa cells and increasing the secretion of E2 and progesterone in the cells.
摘要：
目的：补肾方经常用于临床实践，以促进卵泡发育和成熟。本研究探讨了补肾调经方(BSTJF)对体外培养小鼠腔前卵泡发育的影响及其与颗粒细胞和促性腺激素的关系。
方法：从小鼠中提取腔前卵泡，并在有或没有血清的情况下培养，这些血清来自先前用或没有用BSTJF治疗的大鼠。在种植期间,监测卵泡的形态变化和发育成熟。每隔一天收集耗尽的培养基用于通过ELISA测量孕酮和雌二醇(E2)水平。在第8、10和12天收集体外培养基中的颗粒细胞，并进行分析以确定凋亡相关基因的表达（Bax，Bcl-2和Caspase-3）。通过CCK-8测定和流式细胞术测量收集的颗粒细胞的增殖和凋亡率。
结果：与对照卵泡相比，用BSTJF处理的大鼠血清培养的卵泡具有较高的存活率，较大的卵泡直径，高Bcl-2表达，Bax和Caspase-3表达降低(P均≤0.05)。此外,与对照卵泡的颗粒细胞相比,其颗粒细胞的增殖明显升高(P≤0.05),凋亡率降低(P≤0.05).所有组的培养基中的E2水平在前6天缓慢增加。随后，在胃窦形成后，与对照卵泡培养基相比，用BSTJF治疗的大鼠血清培养的卵泡培养基中E2和孕酮的水平均提高（均P≤0.05）。
结论：BSTJF治疗的大鼠血清通过增加抗凋亡基因Bcl-2的表达，减少促凋亡基因Bax和Caspase-3的表达以及颗粒细胞的凋亡，促进小鼠卵泡的体外发育和成熟。促进颗粒细胞的增殖，增加细胞中E2和孕酮的分泌。
方法：从小鼠中提取腔前卵泡，并在有或没有血清的情况下培养，这些血清来自先前用或没有用BSTJF治疗的大鼠。在种植期间,监测卵泡的形态变化和发育成熟。每隔一天收集耗尽的培养基用于通过ELISA测量孕酮和雌二醇(E2)水平。在第8、10和12天收集体外培养基中的颗粒细胞，并进行分析以确定凋亡相关基因的表达（Bax，Bcl-2和Caspase-3）。通过CCK-8测定和流式细胞术测量收集的颗粒细胞的增殖和凋亡率。
结果：与对照卵泡相比，用BSTJF处理的大鼠血清培养的卵泡具有较高的存活率，较大的卵泡直径，高Bcl-2表达，Bax和Caspase-3表达降低(P均≤0.05)。此外,与对照卵泡的颗粒细胞相比,其颗粒细胞的增殖明显升高(P≤0.05),凋亡率降低(P≤0.05).所有组的培养基中的E2水平在前6天缓慢增加。随后，在胃窦形成后，与对照卵泡培养基相比，用BSTJF治疗的大鼠血清培养的卵泡培养基中E2和孕酮的水平均提高（均P≤0.05）。
结论：BSTJF治疗的大鼠血清通过增加抗凋亡基因Bcl-2的表达，减少促凋亡基因Bax和Caspase-3的表达以及颗粒细胞的凋亡，促进小鼠卵泡的体外发育和成熟。促进颗粒细胞的增殖，增加细胞中E2和孕酮的分泌。
