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

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It is reported that lanosterol 14alpha-demethylase (CYP51, LDM) is an immediate early response gene (Fink et al., 2005); exposure of JEG-3 cells to forskolin, a mediator of the cAMP-dependent signaling pathway, provokes an immediate early response of CYP51, and the CYP51 mRNA increases up to 4-fold in 2 h and drops to basal level after 4 h. In our present study, during 11 h to 15 h after hCG injection, oocytes may have enough time to further promote the plasma maturation for fertilization and early embryo development by accumulation of MAS under the activity of LDM. It is known that during normal fertilization in vivo, oocytes are not fertilized until a few hours after being released from the ovary into the oviduct (Ma et al., 2005). In addition, at the time of ovulation in vivo, oocytes are expelled together with the follicular fluid which contains high concentration of FF-MAS (Byskov et al., 1995; Byskov et al., 2002) and presumably still exposed to FF-MAS during the first part of its passage through the oviduct. All these suggest that FF-MAS which derived from lanosterol by the LDM may play some important role on fertilization.

To prove this suggestion, azalanstat, a specific inhibitor of LDM, was used in this study. It has been confirmed that azalanstat could inhibit synthesis of FF-MAS specifically and decrease the production of FF-MAS both in vivo and in vitro (Burton et al., 1995). Our results showed that azalanstat pre-culture dose-dependently inhibited fertilization of oocytes and reduced blastocyst development competence. Since the azalanstat was dissolved in ethanol as a stock solutions according to previous studies (Burton et al., 1995; Wang et al., 2006). Actually, studies on bovine oocytes have indicated that ethanol could influence both nuclear and cytoplasmic maturation (Avery and Greve, 2000). Plasma membrane outward currents and hyperpolarization of membrane potential were generated by treating oocytes with ethanol (Tosti et al., 2002); these changes may be harmful to embryos development. It is therefore important that caution be exercised when using ethanol as a vehicle in oocyte culture experiments. In addition, mouse oocytes are able to oxidize ethanol to acetaldehyde in the presence of NAD and alcohol dehydrogenase (ADH); the analysis of differentiation and cell number of mouse morulae and blastocysts shows that acetaldehyde is three orders of magnitude more toxic than ethanol, which indicate that the metabolite is responsible for the embryo toxicity of ethanol in preimplantation embryos (Lau et al., 1991). It is important to point out that although higher concentrations of ethanol (2%, V/V) significantly inhibited blastocyst development in our results (Figure 3B), the final concentration of ethanol in this 25 [micro]M azalanstat preparation was only 0.25%, a concentration alone that has no any effect on fertilization and further blastocyst development. Thus, the action of azalanstat on oocyte fertilization and early embryo development is specific for inhibiting lanosterol 14[alpha]-demethylase activity. Our results suggest that 5, 25 and 50 [micro]M azalanstat prevent oocyte development to the blastocyst stage, and the doses have been shown to effectively suppress FF-MAS synthesis (Xie et al., 2004).