RESUMO
BACKGROUND: In mammalian ovaries, loss of over two-thirds of germ cells happens due to cell death. Nonetheless, the exact mechanism of cell death has yet to be determined. The present basic practical study was designed to detect 3 types of programmed cell death, namely apoptosis, autophagy, and necrosis, in murine embryonic gonadal ridges and neonatal ovaries. METHODS: Twenty gonadal ridges and ovaries from female mouse embryos 13.5 days post coitum and newborn mice 1 day postnatal were collected. The TUNEL assay was performed to evaluate apoptosis. The interplay of autophagy was evaluated by immunohistochemistry for beclin-1. Necrotic cell death was analyzed by propidium iodide (PI) staining. The count and percentage of the labeled oocytes in the gonadal ridges and ovaries were evaluated and compared using the independent t test and one-way ANOVA. A P value less than 0.05 was considered statistically significant. RESULTS: We detected TUNEL-positive reaction in the embryonic germ cells and in the small and large oocytes of the neonatal ovaries. The germ cells and small oocytes reacted to beclin-1. PI absorption was detected in the embryonic germ cells and the large oocytes of the neonatal ovaries, but not in the small oocytes. The percentage of the TUNEL-positive and PI-labeled oocytes in the gonadal ridges was significantly higher than that in the neonatal ovaries (P<0.01 and P=0.01). In the neonatal ovaries, the percentage of the beclin-1-labeled oocytes was significantly higher than that in the embryonic phase (P<0.01). CONCLUSION: We showed that all 3 types of programmed cell death, namely apoptosis, autophagy, and necrosis, accounted for embryonic and neonatal germ-cell loss. Our observations demonstrated a potential role for necrosis, particularly in the embryonic gonadal ridge in comparison to the neonatal ovary, in mice.
RESUMO
BACKGROUND: Bone morphogenetic protein 15 (BMP15) is a growth factor derived from oocyte and is essential for in vivo ovarian follicular growth and in this study, its effects on the improvement of growth and development of follicles during in vitro culture of neonatal mouse ovaries was investigated. METHODS: Two week old mice were cultured for 7 days in the basic culture media with or without follicle stimulating hormone (FSH) and BMP15 as four experimental groups; FSH-/BMP15-, FSH+/BMP15-, FSH-/BMP15+ and FSH+/BMP15+. The ovarian follicles at different developmental stages in paraffin embedding sections of cultured and non-cultured ovaries were counted and compared. The 17-ß estradiol (E2) and progesterone (P4) levels were analyzed in collected culture media. The expression ratio of developmental genes (PCNA, BMPR-IB, BMPR-II, FSH-R, CYP17 and ZP3) to housekeeping gene (GAPDH) was analyzed by real time PCR (RT-PCR) in comparison with non-cultured control ovaries. The data was compared by independent t-test and one-way ANOVA (with Tukey's Post Hoc test). The p<0.05 was considered significant. RESULTS: The percentage of antral follicles, ovarian size, concentration of E2 and P4 and the expression ratio of PCNA and ZP3 genes in the ovaries cultured in medium supplemented with BMP15 and FSH increased significantly in comparison with other cultured groups (p<0.05). The BMPR-IB, BMPR-II and FSH-R mRNA level was significantly lower (p<0.05) and CYP 17 mRNA level did not change in the FSH+/BMP15+ group than other cultured groups. CONCLUSION: This study demonstrated a favorable effect of BMP15 in combination with FSH on in vitro development of small size mouse follicles to antral stage.