Inhibition of calcineurin by FK506 stimulates germinal vesicle breakdown of mouse oocytes in hypoxanthine-supplemented medium

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.

Keywords: Calcineurin; FK506; GVBD; MAPK; MPF.

Figure 1. Relative mRNA expression of calcineurin isoforms in mouse oocytes (A) and granulosa cells (B).

The relative mRNA levels represent the amount of mRNA expression normalised to Actb. The comparison of calcineurin mRNA expression was made between each oocytes’ stage and oocytes in one stage, so was in granulosa cells. The statistical difference between two different lowercase letters represents P < 0.05, such as “a” and “b,” “a” and “c,” or “b” and “c” represents P < 0.05.

Figure 2. Expression and location of calcineurin A subunit in follicles, oocytes and granulosa cells.

(A) Calcineurin A subunit was detected by immunohistochemistry in different stage of follicles (1, primordial follicle; 2, primary follicle; 3, small antral follicle; 4, big antral follicle), which were pointed out by red arrows. (B) Cellular localization of calcineurin A subunit was detected by immunofluorescence in GV, GVBD and PB1 extrusion oocytes. (C, D) Calcineurin A subunit protein expression was confirmed by Western blot in GV, GVBD and PB1 extrusion oocytes and the protein ratios were analyzed according to the western blot results. “a” and “b” represents P < 0.05. (E) Cellular localization of calcineurin A subunit was detected by immunofluorescence in granulosa cells. Scale bar, 20 µm.

Figure 3. The effects of FK506 on GVBD rate of oocytes and cumulus expansion.

(A, B) The percentage of GVBD in oocytes cultured with different concentration of FK506 in medium with or without 4 mM HX. (C, D) The GVBD percentage of oocytes from COCs cultured with FSH, 25 µM FK506 or U0126 + 25 µM FK506 in medium with 4 mM and the cumulus expansion was examined. (E) The GVBD percentage of oocytes from FOs cultured with 25 µM FK506 in medium contain 4 mM. Data presented as mean ± standard error of the mean, from three independent experiments. Different lowercase letters indicated statistical difference (P < 0.05).

Figure 4. Inhibition of calcineurin by FK506 increased the concentration of MPF in oocytes.

Concentration of MPF was detected after culturing in 25 µM FK506 for 12 h. Data presented as mean ± SEM, from three independent experiments. “a” and “b,” “a” and “b” or “b” and “c” represents P < 0.05.