Abstract
Calcineurin (CN) is a serine/threonine phosphatase which plays important roles in meiosis maturation in invertebrate oocytes; however, the role of CN in mouse oocytes is relatively unexplored. In this study, we examined the expression, localization and functional roles of CN in mouse oocytes and granulosa cells. The RT-PCR results showed that the β isoform of calcineurin A subunit (Cn A) expressed significantly higher than α and γ isoforms, and the expression of Cn Aβ mRNA obviously decreased in oocytes in which germinal vesicle breakdown (GVBD) occurred, while only B1 of calcineurin B subunit (Cn B) was detected in oocytes and stably expressed during oocytes maturation. The following fluorescence experiment showed that Cn A was mainly located in the nucleus of germinal vesicle (GV) stage oocytes and gruanlosa cells, and subsequently dispersed into the entire cytoplasm after GVBD. The decline of Cn A in oocytes suggested that it may play an important role in GVBD. To further clarify the role of calcineurin during meiotic maturation, FK506 (a calcineurin inhibitor) was used in the culture medium contained hypoxanthine (HX) which could keep mouse oocytes staying at GV stage. As expected, FK506 could induce a significant elevation of GVBD rate and increase the MPF level of denuded oocytes (DOs). Furthermore, FK506 could also play an induction role of GVBD of oocytes in COCs and follicles, and the process could be counteracted by MAPK kinase inhibitor (U0126). Above all, the results implied that calcineurin might play a crucial role in development of mouse oocytes and MPF and MAPK pathways are involved in this process.
Highlights
Mammalian oocytes arrest at prophase of meiosis I with low MPF activity in the ovarian follicle
Western blot analysis showed that the expression of calcineurin A subunit at germinal vesicle (GV) and PB1 extrusion stages of oocytes was significantly higher than germinal vesicle breakdown (GVBD) stage oocytes (Figs. 2C and 2D), which was consistent with the mRNA expression of calcineurin A
The results showed that calcineurin was expressed in oocytes and granulosa cells of all stages of follicles and inhibition of calcineurin could induce mouse oocytes GVBD, which indicated that calcineurin is an important regulator in mouse ovary
Summary
Mammalian oocytes arrest at prophase of meiosis I with low MPF (maturation promoting factor) activity in the ovarian follicle. How to cite this article Wang et al (2017), Inhibition of calcineurin by FK506 stimulates germinal vesicle breakdown of mouse oocytes in hypoxanthine-supplemented medium. Cultured with protein phosphatase inhibitor, okadaic acid could induce mouse oocytes to overcome the GVBD inhibition (Rime & Ozon, 1990), which occurs in mouse oocytes microinjected with okadaic or anti-PP1 antibody (Swain et al, 2003). Further study is still needed to identify whether other PP types are involved during prophase I arrest of mouse oocytes
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