Objective To investigate the effects of Bushen Tiaojing Decoction (补肾调经汤, BSTJD) on oocyte meiotic progression and reproductive function in aged mice, and to explore the potential involvement of the anaphase-promoting complex/cyclosome (APC/C)-cell division cycle 20 (CDC20)-related signaling pathway in these effects.
Methods A total of 105 female Kunming mice were used, including 35 young mice aged 8 – 10 weeks and 70 old mice aged 36 – 40 weeks. The young mice constituted the young group, whereas the old mice were randomly assigned to the old group or the old + BSTJD group (n = 35 per group). In addition, 10 male Kunming mice were used as mating partners for fertility assessment. Mice in the old + BSTJD group received BSTJD by oral gavage at 20 g/(kg·d), divided into two equal doses, for 10 consecutive weeks, whereas mice in the young and old groups received equal volumes of sterile distilled water. Body weight and body length of mice were measured to calculate Lee’s index, and vaginal cytology was performed to evaluate estrous cyclicity. Serum anti-Müllerian hormone (AMH) level, ovarian morphology, preantral and antral follicle numbers, germinal vesicle (GV)- and metaphase II (MII)-stage oocyte numbers, and litter size were evaluated to assess reproductive function. Immunofluorescence staining and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to determine the protein and mRNA expression levels of CDC20, cell division cycle 26 (CDC26), and extra spindle pole bodies like 1 (ESPL1) in GV-, metaphase I (MI)-, and MII-stage oocytes, and spindle/chromosomal morphology and abnormal spindle rates were assessed. To further evaluate the involvement of APC/C-related signaling, GV-stage cumulus-oocyte complexes (COCs) from the mice of the young, old, and old + BSTJD groups were subjected to in vitro maturation (IVM) in the presence or absence of the APC/C inhibitor tosyl-L-arginine methyl ester (TAME), followed by assessment of oocyte maturation and spindle morphology. In a complementary experiment, 10 female Sprague-Dawley (SD) rats were used to prepare blank serum and BSTJD-containing serum. GV-stage COCs from the old group were cultured with blank serum or BSTJD-containing serum, with or without TAME, and the MII maturation rate was determined.
Results Lee’s index did not differ significantly among the young, old, and old + BSTJD groups (P > 0.05). Compared with the old group, the old + BSTJD group showed improved estrous cyclicity and significantly increased numbers of preantral follicles, antral follicles, GV-stage oocytes, and MII-stage oocytes, as well as increased litter size (P < 0.05). Although serum AMH level was higher in the old + BSTJD group than in the old group, the difference was not statistically significant (P > 0.05). Compared with the old group, the old + BSTJD group showed significantly increased CDC20 and CDC26 protein and mRNA expression levels in GV-stage oocytes and decreased ESPL1 expression level in MII-stage oocytes (P < 0.05). These changes were accompanied by improved spindle/chromosomal morphology and a lower abnormal spindle rate. No significant differences in CDC20, CDC26, or ESPL1 expression levels were observed between the old and old + BSTJD groups at the MI stage (P > 0.05). In the first IVM experiment, TAME treatment reduced oocyte maturation and increased spindle abnormalities, whereas oocytes obtained from the old + BSTJD group showed an increased maturation rate and a reduced abnormal spindle rate (P < 0.05). In the complementary serum experiment, BSTJD-containing serum increased oocyte maturation, including in the presence of TAME (P < 0.05).
Conclusion BSTJD improves reproductive function and oocyte meiotic competence in naturally aged mice. These protective effects are associated with stage-specific regulation of CDC20, CDC26, and ESPL1, with pharmacological inhibition experiments further supporting the involvement of the APC/C-CDC20 signaling pathway. These findings provide preclinical evidence for BSTJD as a potential intervention for age-related decline in oocyte quality and female fertility.
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