Alterations in CXCR4 and CXCL12 in Ovine Placental Tissues During Early Pregnancy: Natural Breeding and Assisted Reproductive Technologies (ART).

Abstract

Implantation and placentation is a complex process, involving various signaling molecules, many of which have immune function. During this process, an increase in immune cells at the fetal/maternal interface occurs, which may immunoprotect the fetus as well as promote placental vascularization. Chemokines and their receptors are pivotal factors in implantation and vascularization of the placenta. Chemokine receptor 4 (CXCR4) is up regulated in endometrium during implantation and has only one recognized ligand, CXCL12. In human trophoblast cells, CXCL12/CXCR4 signaling suppresses apoptosis, promotes cell survival, and stimulates proliferation and invasion. We have recently shown increased CXCL12 and CXCR4 mRNA in ovine conceptuses on days 17-30, with complementary elevated CXCR4 mRNA and protein in the endometrium on day 35. This elevation prompted investigation of CXCL12/CXCR4 signaling during earlier days of gestation, specifically during placenta development. Further, because placental defects appear during this time frame (first few weeks of gestation) with ART, we also examined placental CXCL12/CXCR4 expression in sheep undergoing ART. We hypothesized that during early pregnancy CXCL12 and CXCR4 increase in placental tissue to assist placentation and that ART compromises the CXCL12/CXCR4 system. To test this hypothesis, caruncle (CAR) and fetal extraembryonic membrane (FM) tissues were collected on days 0 (non-pregnant), 18, 22, 26 and 30 of pregnancy. For ewes exposed to ART, samples were collected on day 22 of pregnancy after transfer of embryos generated through natural breeding (ET), in-vitro fertilization (IVF) or in-vitro activation (IVA; parthenogenetic clones) and compared to control pregnancies resulting from natural breeding (NAT). Real time PCR was used to assess relative mRNA levels, and Western blot analysis was conducted for protein expression. Differences described are P < 0.05. During early pregnancy in CAR, the greatest levels of CXCR4 mRNA were on days 18 and 22 compared to day 0. Interestingly, corresponding peak levels of CXCL12 mRNA were noted in FM on day 22 compared to day 26. In FM, CXCR4 mRNA increased as gestation advanced with the greatest levels on day 26. In sheep subjected to ART, CXCR4 mRNA was less in CAR in the IVA compared to NAT and ET; in FM, CXCL12 protein was elevated with every type of ART compared to NAT. The increase of CXCL12/CXCR4 in CAR and FM tissues during early pregnancy is intriguing and suggestive of this signaling system playing a role in immune cell recruitment and selection and further promoting implantation and placentation. Because CXCL12 was greater in FM from ART pregnancies, it may function in immune cell migration, which may be compromised during ART. The decline in CXCR4 in CAR from IVA is interesting as this group is the parthenote, which is a maternal genome-only clone. We believe this supports two hypotheses; first that clones are defective very early, and second that maternal genes tend to limit placental development. We interpret this data to mean the CXCL12/CXCR4 signaling system must be properly regulated during early pregnancy and dysregulation in this system may compromise placental development, possible leading to embryonic mortality. Supported by USDA-NIFA-AFRI 2009-65203-05717 to RLA, and by USDA 2007-01215 to LPR and ATGB.