Abstract
The aim of this study was to investigate the effect and mechanism of bone morphogenetic protein-6 (BMP-6) on the growth and maturation of mouse follicles in vitro. Preantral follicles isolated from mice were incubated with recombinant human BMP-6 (rhBMP-6) before analysis. BMP-6 expression was detected by immunofluorescence and western blot. Maturation of oocytes was observed microscopically. Estradiol (E2) and progesterone (P4) levels were measured by enzyme-linked immunosorbent assay. Expression of steroidogenesis-related genes was detected by reverse transcription quantitative polymerase chain reaction. There was a marked increase in the preantral follicles maturation in cells incubated with 50 ng/mL of rhBMP-6 for eight days, compared with the control. The levels of E2, P4 and steroidogenesis-related genes were also significantly increased in granulosa cells and theca cells cultured for 6, 10 and 11 days, respectively. Conversely, the preantral follicle maturing rate was remarkably decreased in cells incubated with 50 ng/mL of rhBMP-6 for day 11, accompanied with reduction in E2, P4 levels and steroidogenesis-related genes levels. Meanwhile, compared with the control, the maturing rate was not significantly different in cells incubated with 100 ng/mL of rhBMP-6 for day 8 or day 11. However, the E2 levels and its relevant regulation gene expression all increased significantly, while the P4 content and its relevant regulation gene expression decreased. The results indicate that BMP-6 can promote the maturation of preantral follicles in vitro in a concentration and time-dependent manner and may play a role in the regulation of steroid hormone synthesis and/or secretion.
Highlights
Development and maturation of follicles are important for mouse reproduction
bone morphogenetic protein-6 (BMP-6) protein expression can be detected in oocytes and granulosa cells in humans, cattle, sheep, swine and rats.[1À6] The expression of BMP-6 protein undergoes a dynamic change during development of follicle and luteinization
BMP-6 is regarded as the corpus luteum inhibitor, since it inhibits progesterone production to prevent early maturation of follicles.[3,7À9] The effects of BMP-6 on estradiol synthesis and proliferation of the granulosa cells and theca cells vary among different species.[7À11] In addition, it has been demonstrated that the reproductive capacity of BMP-6 knockout female mice is not notably reduced.[12]
Summary
Follicle development and maturation are regulated by the hypothalamicÀpituitaryÀgonadal axis, and are dependent on the interaction between oocytes, granulosa cells and theca cells. The latter cells may produce many regulatory factors, such as epidermal growth factor (EGF), insulin growth factor, factors of the transforming growth factor-b (TGF-b) superfamily, etc. These autocrine or paracrine factors regulate follicle development through acting on gonadotropin and steroid hormones. Koji et al [13] reported that the litter size, natural ovulation rate and development of zygote after fertilization were markedly decreased in BMP-6 knockout mice
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