The expression of genes encoding zona pellucida glycoproteins in canine cumulus-oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol.

The role of progesterone (P4) and estradiol-17beta (E2) on the efficiency of canine oocyte maturation in vitro is recognized, but little is known about the influence of both steroids on the expression of zona pellucida (ZP) glycoproteins. It has been shown that E2 and P4 used in the IVC significantly influenced canine oocytes meiotic competence, although the effect is specifically related to the combination of hormones used in the experiment. Because both of these steroids may stimulate or inhibit maturation competence of oocytes in a dose-dependent manner, there is a high possibility that they also influence the fertilization ability of canine oocytes. Our study was aimed to analyze whether genes, encoding ZP glycoproteins, are regulated by P4 or E2. Canine cumulus oocyte complexes (COCs) were recovered from anestrous mongrel bitches after ovariohysterectomy and cultured in serum-free tissue culture medium 199. The expression pattern of ZP glycoproteins 2 and 3 (ZP2 and ZP3) mRNAs, using quantitative real-time polymerase chain reaction (RQ-PCR), and of ZP3 and ZP4 proteins, using Western blot analyses, was examined in oocytes after the supplementation of the culture medium with (1) 0.5 μg/mL, 1.0 μg/mL, and 2.0 μg/mL of P4 (experiment 1), or with (2) 2.0 μg/mL E2, and with (3) a combination of E2 (2.0 μg/mL) and P4 (0.5, 1.0, or 2.0 μg/mL, respectively; experiment 2). The analysis revealed an inhibited expression of ZP2 mRNA in oocytes after in vitro maturation (IVM) with different P4 supplementations as compared with oocytes before IVM. The expression of ZP3 mRNA was stimulated (P < 0.01) by the supplementation of 1.0 μg/mL P4. The expression of both ZP3 and ZP4 proteins was also stimulated after the treatment with 1.0 μg/mL P4. On the other hand, the level of ZP2 mRNA was inhibited (P < 0.01) after the supplementation with E2 or with combinations of E2 and P4 as compared with control oocytes. The expression of ZP3 mRNA was significantly higher after the supplementation with E2 and 0.5 μg/mL P4. Similarly, ZP3 and ZP4 proteins were highly expressed (P < 0.01) after such hormone supplementation. The results clearly show that in vitro, P4 regulates the expression of ZP glycoproteins in a dose-dependent manner. We demonstrated that E2 used alone and in combination with P4 upregulates the expression of ZP3 mRNA as well as ZP3 and ZP4 protein in canine oocytes. ZP2 mRNA is downregulated by E2 alone and in combination with E2 and P4. Furthermore, ZP glycoproteins expression is regulated by E2 alone or in combination with P4, and such synergistic or adverse effect is P4 concentration-dependent.