Expression of Gap Junctional Proteins Connexin (Cx) in Ovine Utero-Placental Tissues During Early Pregnancy: Effects of Assisted Reproductive Technology.

Abstract

Compromised pregnancies may be caused by genetic, epigenetic, environmental and/or other factors. Assisted reproductive technology (ART) may have profound effects on placental and fetal development, leading eventually to compromised pregnancy. We have previously demonstrated that application of ART leads to decreased cell proliferation in maternal and fetal placenta, delayed development of blood vessels in fetal placenta, and decreased fetal size during early pregnancy. Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and Cx proteins have been detected in uterine and placental tissues in several species. Both, expression of gap junctions/Cx proteins and cell-cell communication mediated through gap junctional channels are likely involved in the regulation of utero-placental and fetal function and growth. Thus, we hypothesized that ART application will affect the expression of four Cx in placenta during early pregnancy in sheep. To determine the expression of Cx26, Cx32, Cx37 and Cx43 in fetal and maternal placenta after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF) or in vitro activation (IVA; parthenotes) compared to control natural breeding (NAT), uterine tissues (n=7 per group) were collected on day 22 of pregnancy. Cross sections of uterus were fixed for immunohistochemistry. Additionally, caruncular (CAR; maternal placenta) tissues and fetal membranes (FM; fetal placenta) were snap-frozen separately for RNA extractions followed by quantitative real time RT-PCR. Protein and mRNA for all four Cx were detected in uterine tissues of NAT. In NAT, Cx26 protein was localized to surface epithelium, endometrial glands and stroma and to FM; Cx32 protein was present in surface epithelium, endometrial glands, stroma and blood vessels and FM; Cx37 protein was expressed in surface epithelium, endometrial glands and FM; and Cx43 protein was expressed in endometrial stroma and FM. In FM, expression of mRNA for Cx26 was enhanced (P<0.05) 2.7-fold in IVA compared to NAT group, and expression of Cx26 mRNA was similar in NAT, NAT-ET and IVF groups. Expression of mRNA for Cx32, Cx37, and Cx43 in FM was similar in all pregnancy types. In CAR, mRNA expression for Cx32 was decreased (P<0.004) 2-4-fold in NAT-ET, IVF and IVA groups compared to NAT group. Expression of mRNA for Cx26, Cx37, and Cx43 in CAR was similar in all pregnancy types. These data demonstrate that Cx26, Cx32, Cx37 and Cx43 are expressed in utero-placental tissues, localization of these Cx in uterine compartments differs likely reflecting differing functions, and that application of ART affects mRNA expression for Cx26 and Cx32 but not Cx37 and Cx43. Thus, ART may have a negative effect on growth and function of utero-placental and fetal tissues in sheep possibly mediated through regulation of Cx expression and likely other factors. These data will help us to 1) better understand placental regulatory mechanisms in compromised pregnancies, and 2) in the future identify strategies for rescuing such pregnancies. Supported by Hatch Project ND01712; USDA grant 2007-01215 to LPR and ATGB, NIH grant HL64141 to LPR and DAR, and NSF-MRI-ARRA grant to ATGB. (poster)