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

Ethics statement and animal feeding regimens

The animal experimental procedures were approved by HuaZhong Agricultural University Institutional Animal Care and Use Committee, Wuhan, China (Approval ID: SCXK (Hubei) 2008_0005). Kunming mice (3–4 weeks old) were obtained from the Centre of Laboratory Animals of Hubei Province (Wuhan, PR China). Mice were housed under controlled temperature (25 °C) and lighting (12 h light/12 h darkness) with food and water ad libitum and sacrificed with cervical dislocation method.

Isolation and culture of DOs, COCs and follicles

Ovaries were collected from 3 to 4 weeks KM mice, and were minced properly. Later culture medium was added to be convenient for collecting oocytes by straws. A total of 50 fully grown and immature oocytes arrested at the GV stage were cultured in 500 µl M199 medium (Life Technologies Corporation, Rockville, MD) with 4 mM HX or not.

In the same way, ovaries were collected from 3 to 4 weeks KM mice injected with 10 IU PMSG (Sansheng Pharmaceutical Corporation, China) for 44 h, and were punctured by the needles of 1 ml injector. Then COCs with at least two or three layers of granulosa cells or antral follicles (FOs) with a diameter about 300–400 µm were collected from the medium by straws. 50 COCs or FOs were cultured in 500 µl M199 medium with 4 mM HX.

RT-PCR analysis

RNA from oocytes and granulosa cells was isolated using Rneasy Plus Micro Kit (QIAGEN, Germany) and RNAprep pure cell kit (Tiangen, Beijing China), respectively. Total RNA was transcribed to cDNA with first strand cDNA synthesis kit (Therm, USA). And quantitative real-time PCR was carried out using SYBR Green (QuantiFast SYBR Green PCR kit (QIAGEN, Germany)) on a Bio-Rad LC480 real-time PCR system. All primer pairs used for real-time PCR are summarized in Table 1. The amplification efficiencies of primers were obtained by making standard curves and efficiencies of PCR amplification were between 90 and 105% and the variation was less than 5%. The relative quantification of mRNA expression for each calcineurin isoform was estimated using formula 2^−DDCt, according to the comparative Ct method, normalized to the housekeeping gene (Actb) in each sample.

Immunohistochemistry

Mouse ovaries were collected and fixed in Borne’s solution for 12–16 h at RT. The fixed mouse ovaries were dehydrated in graded ethanol, dealcoholized with xylene, soaked in methyl salicylate for 12–16 h, and embedded in paraffin. The paraffin-embedded tissues were sectioned into 4-µm thick slices, mounted onto poly-L-lysine-coated slides and dried at 42 °C for 2–4 h in air, subsequently transferred into 60 °C overnight in incubator. The sections were deparaffinized and rehydrated in graded ethanol, soaked in 3% hydrogen peroxide for 30 min to remove endogenous peroxidase and washed in ddH2O two times for 5 min each followed by PBS two times for 5 min each. The sections were boiled in 0.01 M sodium citrate (pH 6.0) three times for 2 min each at 5-min intervals for antigen retrieval. Then the samples were blocked by donkey serum for 30 min at RT, then incubated overnight at 4 °C with anti-Cacineurin A antibody (SC9070; Santa Cruz Biotechnology, USA) (1:100 dilution) or rabbit IgG (Santa Cruz Biotechnology, USA) (1:100 dilution) as control in blocking solution. The sections washed three times for 5 min each in 0.1% Tween-20 in PBS and the samples were incubated with goat anti-rabbit biotin-SP-conjugated antibody (Guge Biotechnology Corporation, Wuhan, China) for 30 min at RT. The sections washed three times for 5 min each in 0.1% Tween-20 in PBS and the samples were incubated with SABC for 30 min at 37 °C. After washing 4–5 times for 5 min each in PBS, the immune reactive signals were detected using DAB Map Kit (Guge Biotechnology Corporation, Wuhan, China) for 3–5 min. The sections washed four times for 5 min each in ddH2O. The samples were stained by hematoxylin for 2 min, differentiated by 5% hydrochloric acid for 8 s, washed by ddH2O three times for 5 min each and washed by PBS three times for 5 min each to make the stain of nucleus return to blue. The sections were dehydrated in graded ethanol, dealcoholized with xylene, and sealed with neutral resin and cover glass. The sections were put in 37 °C for 24 h–48 h and were observed by microscope with Nomarski optics and digital camera.

Immunofluorescence and confocal microscopic techniques

Oocytes cultured for 0, 2 or 12 h, at which point most of the oocytes had reached the GV, GVBD or M II stages, were collected for immunofluorescent staining. Oocytes were fixed in 4% paraformaldehyde in PBS (Phosphate Buffered Saline) for 1 h at RT (room temperature). After washing three times in wash buffer (PBS containing 2% BSA), oocytes were treated with 0.5% Triton X-100 in PBS for 1–1.5 h at RT, blocked in PBS containing 5% BSA for 1 h at RT and incubated overnight at 4 °C  with anti-Cacineurin A antibody (Santa Cruz Biotechnology, USA, SC9070) (1:100 dilution) or rabbit IgG (Santa Cruz Biotechnology, USA) (1:100 dilution) as control in blocking solution. Then, oocytes were incubated with a FITC-conjugated goat anti-rabbit antibody (Boster Co., Wuhan, China) (1:100 dilution) in dark after washing three times in 15 min, nuclei were stained in washing buffer containing 10 µg/ml PI (Propidium Iodide) for 10 min. After immunofluorescent staining, oocytes were mounted on slides in DABCO and fluorescent signals were detected using confocal microscope.

Granulosa cells adherent to coverslip were fixed in 4% paraformaldehyde in PBS for 30 min at RT and washed for three times. After treating with 0.5% Triton X-100 in PBS for 15–20 min at RT, granulosa cells were blocked in 5% BSA with PBS for 30 min at RT and incubated overnight at 4 °C with anti-Calcienurin A antibody (Santa Cruz Biotechnology, USA, SC9070) (1:100 dilution) or rabbit IgG (1:100 dilution) as control in blocking solution, then incubated with a FITC-conjugated goat anti-rabbit antibody (1:100 dilution) in dark and washed 3 times in 15 min, later nuclei were stained in washing buffer containing 10 µg/ml PI for 10 min. After immunofluorescent staining, coverslips were mounted on slides with DABCO and fluorescent signals were detected using confocal microscope.

Western blot analysis

A total of 200 fully grown GV, GVBD and MII oocytes were collected and lysed in 2X SDS sample buffer and boiled in water for 5 min. Total protein was concentrated by 5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis) for 30 min at 90 V, and then was separated by 12% SDS-PAGE for 90 min at 120 V and electrophoretically transferred to a PVDF membrane for 1 h at 200 mA. After washing in TBS (Tris–HCl Buffer Saline) for 3 times, the membrane was blocked in TBST (TBS with 0.1% Tween 20) containing 5% skim milk for 1–2 h, and then incubated with a rabbit anti-Calcineurin A antibody (Santa Cruz Biotechnology, USA, SC9070) (1:200 dilution) or a mouse anti-β-actin (Santa Cruz Biotechnology, USA, SC69879) (1:1000 dilution) in TBST overnight at 4 °C. After washing 3 times in TBST, the membrane was incubated with an HRP-conjugated anti-rabbit or mouse secondary antibody (Boster Corporation, Wuhan, China) (1:2000 dilution) in TBST for 1 h at RT. The membrane was washed 3 times with TBST and processed with the ECL (Enhanced Chemilumidescence) detection system. ACTB was detected as an internal control.

MPF activity assay

After treating with or without FK506 for 12 h, 100 oocytes were lysed with 30 µl of radio immune precipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail (Sigma). Samples were lysed on ice for 5–10 min, and then centrifuged at 4 °C at 12,000 rpm for 15 min. The supernatant was collected and stored at −80 °C until use. The level of MPF was determined using the Mouse MPF ELISA Kit (Kexing Biotechnology Company, China) following the manufacturer’s protocol and each sample was tested with three repeats. All other chemicals were purchased from Sigma Aldrich, unless stated otherwise.

Statistical analysis

Each experiment was repeated at least three times and data were presented as mean ± SEM (standard error of the mean). Differences between groups were analyzed using one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) test using SPSS (Version 17.0; SPSS, Chicago, IL). P values < 0.05 were considered to be statistically significant.

Identification of the mRNA expression level for isoforms of calcineurin subunits

To identify the different expression pattern of calcineurin’ subunits during oocytes meiotic maturation, three developmental stages (GV, GVBD and PB1 extrusion) oocytes and granulosa cells were isolated for detection.

Three isoforms (α, β and γ) of calcineurin A subunit mRNA were detected in oocytes and the mRNA level of β isoform was significantly higher than α and γ during meiotic maturation. While only B1 isoform of calcineurin B was found in oocytes and expressed during oocyte maturation constantly (Fig. 1A). Similar with oocytes, three isoforms of calcineurin A subunit were detected in granulosa cells and the mRNA level of Cn Aβ was significantly higher than Cn Aα and Cn Aγ. However, mRNAs of two isoforms of calcineurin B (B1 and B2) subunit were both detected in granulosa cells and the mRNA level of B1 was significantly higher than B2 (Fig. 1B).

Localization and protein expression of calcineurin A in oocytes and granulosa cells

The above study showed that Cn Aβ mRNA level decreased significantly after GVBD, which suggested Cn A might play important roles during GVBD. Thus, we examined the localization and expression of Cn A in mouse ovaries. Positive reaction of calcineurin A was found in different stages of follicles (Fig. 2A). Furthermore, immunocytochemical evaluation showed the present of calcineurin A subunit in different stages of oocytes and granulosa cells and was expressed in the nucleus of fully grown GV stage oocytes and granulosa cells, while homogenously dispersed into cytoplasm after GVBD (Figs. 2B and 2E). Western blot analysis showed that the expression of calcineurin A subunit at GV and PB1 extrusion stages of oocytes was significantly higher than GVBD stage oocytes (Figs. 2C and 2D), which was consistent with the mRNA expression of calcineurin A.

Inhibition of calcineurin by FK506 could induce the GVBD of oocytes

To further study the functional role of calcineurin in the development of oocyte, DOs (denuded oocytes) were incubated in basic medium (without HX) containing different concentration of FK506. Since 50 µM FK506 could lead to abnormal cell morphology of oocytes (data not showed), we used 5 µM and 25 µM FK506 to culture DOs and examined the GVBD rate after 2 h. The results showed that 5 µM and 25 µM could not disturb the GVBD of oocytes (Fig. 3A) while, when DOs were cultured in medium containing 4 mM HX for 12 h, 25 µM FK506 could significantly increase the GVBD% (54.91 ± 1.47%) compared to the control group (37.93 ± 2.31%), as well as 5 µM FK506 (45.81 ± 1.59%) (Fig. 3B).

As calcineurin was expressed both in the oocytes and granulosa cells, we supposed that calcineurin may participate in the communication between oocyte and cumulus cells. To confirm the hypothesis, COCs (cumulus oocyte complexes) were cultured in medium with HX and FK506, and GVBD rates were examined after 22 h. COCs cultured with FK506 and HX showed an significantly increase of GVBD% (83.15 ± 3.28%) compared to control group (58.13 ± 6.74%), and showed no significant difference with FSH group (88.85 ± 6.16%) (Fig. 3C). However, inhibition of calcineurin by FK506 had no effect on cumulus expansion (Fig. 3D). These results suggested that calcineurin might play an important role in maintaining the oocytes stayed at GV stage. Antral follicles, which had a diameter about 300–400 µm and contained about the same condition of COCs as we cultured, were cultured by 25 µM FK506 to confirm that 25 µM FK506 also could induce the GVBD of oocytes from the antral follicles after culture for 22 h (Fig. 3E).

FK506 increased MPF level in oocyte which could be reduced by U0126

It has been well established that MPF and MPAK are important pathways in meiotic maturation. To further test whether there was feedback regulation between MPF and calcineurin in oocytes, MPF concentration was detected by ELISA. The results showed that the MPF concentration increased significantly after 25 µM FK506 treating for 12 h and partially decreased by MAPK inhibitor U0126 (Fig. 4). These results suggest that both MAPK and MPF are associated with the regulation of calcineurin in oocytes.