144 ROLE OF GnRH ON MOUSE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT IN VITRO

Abstract

The interaction between GnRH and its receptor on gonadotropes within the anterior pituitary gland represents a key point for regulation of the reproduction. In addition, GnRH can act in multiple extrapituitary tissues via autocrine/paracrine mechanisms. Protein for GnRH and mRNA for both GnRH and its receptor have been detected in human uterine endometrium and oviduct as well as in embryos at the morula/blastocyst stage in the mouse and human. Therefore, we hypothesized that GnRH may have a critical role in the development of pre-implantation embryos. To address this question, we examined the effect of a GnRH agonist and antagonist on the development of mouse embryos in vitro. For these studies, 1-cell embryos were randomly allocated to culture in KSOM containing the appropriate treatment for 144 h at 37°C in a 5% CO₂ in air environment. The medium was changed every 12 h and embryos were scored daily for development. The data were compared using a χ² test. First, we wanted to determine if a GnRH agonist, histrelin, could enhance embryonic development. Embryos were cultured with (n = 35) or without (n = 36) 10 μM histrelin. The addition of histrelin did not increase morula or blastocyst formation v. control. Second, we cultured embryos in the presence of different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of the GnRH antagonist, SB-75 (cetrorelix; n = 22/treatment) in order to determine its effect on embryonic development. The 10 μM SB-75 treatment blocked embryo development beyond the compact morula stage (P < 0.001). To determine if this was a receptor mediated effect, we attempted to rescue development of SB-75 treated embryos with a histrelin challenge. Our treatments consisted of control (n = 30), 10 μM histrelin (n = 27), 10 μM SB-75 (n = 29), and 10 μM SB-75 in combination with either 1 μM (n = 27) or 10 μM (n = 25) histrelin. Both levels of histrelin partially rescued the inhibition of blastocyst formation by SB-75 (P < 0.01). Next, we were interested in examining the signaling cascade activated following binding of GnRH to its receptor in pre-implantation embryos. Toward this end, we treated embryos with inhibitors of either PKC or PKA. First, embryos were cultured in the presence of 0 (n = 33), 0.1 (n = 35), 1 (n = 35), or 10 (n = 35) μM GF109203X (GFX), a PKC inhibitor. Similar to the results obtained with SB-75, treatment with 10 μM GFX significantly reduced development to the compact morula stage and completely blocked blastocyst formation. Second, we treated embryos (n = 15 to 17/treatment) with different concentrations (0, 0.01, 0.1, 0.5, or 1 mM) of the PKA inhibitor, SQ22536. In contrast to treatment with GFX, rates of blastocyst formation were decreased only by 35% (P < 0.05) at the highest concentration of SQ22536. The percentage of embryos developing to the hatched blastocyst stage was decreased in a dose-dependent manner following SQ22536 treatment (P < 0.05); however, this effect was not consistent with SB-75 inhibition of blastocyst formation. We suggest that GnRH has an important autocrine effect on early embryonic development, potentially signaling via PKC.

Funding for M Montagner was provided by CAPES, Brazil.