255 SONIC HEDGEHOG PROMOTES IN VITRO OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN GOATS

Abstract

The signalling of the Hh family peptides is mediated through a cell surface receptor system consisting of 2 proteins: patched (Ptc) and smoothened (Smo). In the absence of Hh ligand, the Hh receptor Ptc represses Smo, whereas in the presence of Hh, the suppression of Smo is lifted, leading to the activation of downstream transcriptional factors (Gli1, Gli2, and Gli3) in vertebrates. Previous studies have examined Sonic hedgehog (Shh) signalling pathways in developing and adult mouse ovaries and concluded that the Shh signalling pathway may be involved in granulosa cell proliferation and oocyte maturation. We investigated the effects of Shh protein on caprine oocyte maturation, embryo development, and embryo survival rate after transfer of vitrified/thawed in vitro-produced (IVP) embryos to recipients. Cumulus-oocyte complexes (COC) were collected by slicing ovarian follicles (1–5 mm in diameter). On average, 40 to 50 oocytes were randomly allocated to each well containing 500 μL of IVM medium and supplemented with 0 (control), 0.125, 0.25, 0.5, or 1.0 μg mL–1 recombinant mouse Shh protein. After 24 h of IVM, cumulus cells were partially removed. Oocytes were washed and transferred into a droplet of 80 μL of fertilization medium and were fertilized with frozen-thawed sperm for 18 h at 38.8°C. After IVF, presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 for 9 days. The 2 frozen-thawed selected embryos were transferred to one recipient. All data were subjected to ANOVA, using the general linear model procedure in SAS (version 9), followed by Tukey's test. Embryo survival rates were compared by using the chi-square test. The RT-PCR analyses showed that the expressions of Shh, SMO, Ptch1, and Gli1 were detected in whole ovaries, granulosa cells, COC, cumulus cells, oocytes, and oviduct epithelia except for Ptch1 in cumulus cells. Supplementation of Shh (0.25 or 0.5 μg mL–1) enhanced oocyte maturation as opposed to the control group (92.4%, n = 67 and 95.0%, n = 62 v. 86.2%, n = 64, respectively, P < 0.05). This effect could be reversed by the simultaneous addition of cyclopamine (0.5–1.0 μm), a Shh inhibitor. Similar to intact COC, denuded oocytes showed enhanced maturation (72.0%, n = 94 v. 60.5%, n = 126) with Shh supplementation. For subsequent embryo development, an improved blastocyst rate (P < 0.05) was 66.3 ± 10.9 (n = 135) when embryos were derived from the oocytes matured in the presence of 0.5 μg mL–1 Shh rather than 41.4 ± 12.9 (n = 137) of the control group. After embryo transfer, the kidding and embryo survival rates of vitrified embryos derived from the Shh-supplemented group were 56 (16 recipients) and 31% (48 embryos) higher than that 38 (16 recipients) and 15% (54 embryos) without Shh supplementation (P < 0.05). The present study suggests that Shh signalling is active in caprine ovaries during folliculogenesis and beneficial to oocyte maturation and subsequent embryo development to the blastocyst stage (in vitro) and to term.