Stage-specific expression of lanosterol 14[alpha]-demethylase in mouse oocytes in relation to fertilization and embryo development competence

Asian - Australasian Journal of Animal Sciences, March, 2009 by Xiaoming Song, Hong Ouyang, Ping Tai, Xiufen Chen, Baoshan Xu, Jun Yan, Guoliang Xia, Meijia Zhang

INTRODUCTION

To complete successful fertilization and embryo development, the nucleus and cytoplasm maturation of oocyte must be coordinated with intercellular events, including growth, meiotic resumption, and ovulation. Many studies indicated that early embryos produced by in vitro fertilization (IVF) can not achieve satisfactory implantation and embryo development (Abeydeera et al., 2001; Hamamah et al., 2005; Jang et al., 2008). To improve the quality of these early embryos, it is necessary to notice the in vivo fertilization and early embryo development environment.

Follicular fluid meiosis-activating sterols (FF-MAS) is a lipophilic molecule (4,4-dimethyl-5[alpha]-cholest-8,14,24-trien3[beta]-ol), which was found in high concentrations in the follicular fluid of mammals including humans and proved to be stimulatory to oocyte meiotic resumption (Byskov et al., 1995; Byskov et al., 2002). Subsequent data showed that the synthesization of MAS is significantly increased with response to gonadotropins (Grondahl et al., 2003; Xie et al., 2004; Yang et al., 2008). Meanwhile, recent studies indicated that FF-MAS dramatically promotes the later stages of meiotic maturation, namely the progression of MI to MII. The treatment of FF-MAS on mouse oocytes during meiotic maturation also increased their subsequent competence to complete early embryos development (Marin Bivens et al., 2004a). However, the positive effect of FF-MAS was still controversial with the following results. FF-MAS increased the rate of chromosomal abnormality and had detrimental effects on cleavage and human early embryos development (Loft et al., 2004; 2005).

In trying to understand the effect of FF-MAS during oocyte maturation, FF-MAS synthesis is often taken into consideration. Involved in the cholesterol biosynthesis, cytochrome P450 lanosterol 14[alpha]-Demethylase (LDM) is the crucial and rate-limiting enzyme in the synthesis of FF-MAS by removing the methyl group from lanosterol (Rozman et al., 2002). A 2.5-fold increase in LDM mRNA and protein in rat ovaries and follicles has been previously described after hCG stimulation (Vaknin et al., 2001). Otherwise, the activity of LDM is supported to be gonadotropin dependent (Wang et al., 2006). Therefore, up-regulation of LDM induced by gonadotropin maybe directly lead to MAS production. Yet, the expression profile of LDM in mouse oocytes during meiotic maturation is unknown.

In present study, two highly specific inhibitors (azalanstat and AY9944-A-7) related to the metabolism of FF-MAS were introduced. Azalanstat could specifically inhibit LDM function and therefore decrease the production of FF-MAS of cumulus enclosed oocytes (CEOs) (Burton et al., 1995); whereas AY9944 could specifically inhibit activities of the sterol [DELTA]14-reductase and [DELTA]7-reductase (Kim et al., 1995) to decrease the transform of T-MAS from FF-MAS, which leads to the accumulation of FF-MAS in CEOs (Leonardsen et al., 2000).

Our objectives were to: i) investigate the expression profile of LDM during mouse oocyte meiotic maturation from GV stage to MII stage and to validate the involvement of FF-MAS accumulation in fertilization and development of early embryos; ii) use specific inhibitors (azalanstat and AY9944-A-7) related to the metabolism of FF-MAS to determine the role of FF-MAS on mouse oocyte competence, this may validate the effect of endogenous FF-MAS on the quality of oocyte maturation.

MATERIALS AND METHODS

Chemicals

All the chemicals used in this experiment were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) except for those specifically mentioned. Azalanstat (also named RS-21607. gift from Dr. D. C. Swinney, Roche Bioscience, USA; (Swinney et al., 1994)) was prepared as 10 mM stock solution by dissolving in ethanol, 10 mM AY9944 (gift from Dr. Wyeth-Ayerst, Princeton, NJ, USA) stock solution was dissolved in M2 medium, both stock solutions stored in a dark box at -20[degrees]C.

Animals, superovulation and oocyte recovery

Kunming White female mice, 6-8 weeks after birth, were kept in a mouse keeping room with 14 h/10 h light-dark cycles, the dark starting from 8 PM. Animal care and handling were conducted in accordance with policies on the care and use of animals promulgated by the ethical committee of the China Agricultural University. To obtain in vivo maturated oocytes, mice were induced to superovulation by injection of eCG (10 IU/mouse, i.p.), 48 h later followed by injection of hCG (10 IU/mouse, i.p.). The superovulated mice were killed 11 or 15 h after hCG injection according to experimental design, and the mature follicles in the ovary or the oviductal ampullae were ruptured in M2 to release the CEOs. To analyze the expression of LDM during mouse oocytes meiotic maturation in vivo, CEOs were collected at each time point after hCG injection, and removed cumulus cells with 0.1% hyaluronidase in M2 medium.

Staining of LDM and confocal microscopy

After removing the zonae pellucidae in acid Tyrode solution (pH 2.1) (Nicolson et al., 1975), oocytes were fixed in 4% paraformaldehyde in PBS (pH 7.4) for at least 30 min at room temperature. To compare LDM expression of different development stages in the same immunostaining experiment, the samples were processed in parallel. Cells were permeabilized with 1% (v/v) Triton-X100 for 30 min at 37[degrees]C, followed by blocking in 1% (w/v) BSA for 1 h at room temperature, and then incubated with 1:300 rabbit anti-human LDM antibody (gift from Dr. M. R. Waterman, Vanderbilt University School of Medicine, Nashville, USA) overnight at 4[degrees]C. The oocytes were rinsed three times and incubated with 1:200 FITC-conjugated goat anti-rabbit IgG for 1 h. Following three washes, the nuclear status of oocytes was evaluated by staining with 10 mg/ml propidium iodide in PBS for 10 min. In negative control groups, the polyclonal rabbit anti-human LDM antibody was replaced with rabbit IgG. Following extensive washing, samples were mounted between a coverslip and a glass slide supported by four columns of a mixture of vaseline and paraffin (9:1). Cells were observed under a laser scanning confocal microscope (Leica Microsystems, Leica, Wetzlar, Germeny); all samples were visualized or pictured using the same laser power. A total of approximately 30 oocytes were collected at each time point after hCG injection, the same group was repeated for at least 3 times.