Unlike many other chemokine receptors, CXCR4 has only one recognized ligand, CXCL12 . The CXCL12/CXCR4 system is implicated in cross talk between trophoblasts and endometrium, recruitment of lymphocytes into the uterus of pregnant females and vascularization. Based on known critical functions of CXCR4 activation during early pregnancy in other species, we hypothesized that CXCL12 and CXCR4 in the endometrium and conceptus increased in response to implantation and placentation in ewes to facilitate communication between trophoblast cells and maternal endometrium. To test this hypothesis, our first study investigated the expression of CXCL12 and CXCR4 in endometrium from pregnant and non-pregnant sheep. CXCR4 was consistently expressed in pregnant ewes across days tested, with decreasing CXCR4 in the endometrium from non-pregnant ewes with advancing days of the estrous cycle. On Day 15 of pregnancy we observed greater (P < 0.05) mRNA and protein for CXCR4 in the endometrium from pregnant compared to non-pregnant ewes. By Day 15 of gestation initial attachment of the conceptus to maternal endometrium begins in sheep . As signal transduction pathways activated by the CXCL12/CXCR4 axis are known to regulate adhesion, as reviewed by Kucia and colleagues , it is plausible that CXCR4 may play a role during attachment. However, further study needs to be conducted to ascertain the role of CXCL12/CXCR4 signaling during implantation.
Coupled with initial attachment of the conceptus to maternal endometrium, recent studies reported by Grazul-Bilska and co-workers have demonstrated that angiogenesis is initiated very early in gestation as reflected by proliferation of capillary endothelial cells, as early as Day 16 of pregnancy in sheep . The most potent inducer of both angiogenesis and microvascular permeability identified so far is vascular endothelial growth factor (VEGF). Gene-knockout (KO) studies have provided convincing evidence for a central role of VEGF in fetal and placental angiogenesis. A lack of even a single allele of VEGF or VEGFRs results in defective fetal and placental vasculogenesis and angiogenesis, culminating in embryonic death by mid-gestation [24–27]. Another potent angiogenic factor, basic fibroblast growth factor (bFGF, or FGF2), stimulates proliferation of both uterine arterial and fetal placental arterial endothelial cells [28–31] and is produced by both fetal and maternal placental tissues [32–35]. Thus, bFGF may function as an angiogenic factor during placentation. Interestingly, both VEGF and bFGF increase expression of CXCR4, but not other chemokine receptors . Subcutaneous injection of CXCL12 into mice induces formation of local small blood vessels accompanied by leukocyte infiltration . CXCL12 binding with CXCR4 on endothelial cells further amplifies angiogenesis by inducing more VEGF release [37, 38]. VEGF and bFGF not only induce CXCR4, but also enhance CXCL12 production by endothelial cells , establishing a positive-feedback loop in which VEGF induces CXCR4 and CXCL12 expression, and conversely CXCL12/CXCR4 interactions enhance VEGF expression by these cells, consequently linking classic angiogenic factors to chemokine-induced angiogenesis. CXCL12/CXCR4 interactions induce expression of other angiogenic signals as well, specifically prostacyclin production by endothelial cells . It should be emphasized that despite expression of multiple functional chemokine receptors on endothelial cells, only CXCL12/CXCR4 interactions are necessary for some, but not all types of angiogenesis . Further, only CXCL12 and CXCR4 KO mice exhibit vascular abnormalities compared to other chemokines and their receptors . Defects in vascularization have not been observed in any other chemokine receptor or ligand KO mice, thus their angiogenic role is, if anything, redundant.
The above provides strong support for CXCL12/CXCR4 interactions during vascularization and it is conceivable similar functions exist during placental development, which is characterized by extensive vascularization. In the present study CXCR4 increases between Days 15 and 35 of pregnancy with peak expression noted on Day 35 in maternal endometrium and subsequently decreasing by Day 50 of gestation. Interestingly, CXCL12 mRNA in white blood cells from both jugular and uterine vein samples exhibited a similar expression pattern, with peak CXCL12 observed on Day 35 of pregnancy when the greatest expression of CXCR4 is observed in the endometrium. The combined data are curious with respect to the timeframe of early pregnancy in sheep as the process of attachment and placentation is a prolonged process with initial attachment beginning on Day 15-16 of gestation, endometrial vascularization increasing on Days 20-22 and placentation not complete until Days 50-60 of pregnancy [23, 42–44]. In our study, peak expression of CXCR4 in caruncle and intercaruncle tissue is observed on Day 35 compared to Day 50, when placentation should be close to completion. Given the strong role CXCR4 plays in vascularization in other systems, it is probable that activation of CXCR4 is also driving similar functions during placentation.
The present study demonstrated that mRNA for CXCL12 and CXCR4 in ovine conceptuses is increased from Day 17-21, which is intriguing as this time frame correlates with apposition of trophoblast cells with luminal epithelium of the endometrium where adhesion complexes are formed by Day ~21-22 [21, 45]. Also, from Day 21-30 of gestation, during placentation, a dramatic increase in trophoblast CXCR4 and CXCL12 is noted in conjunction with the increased CXCR4 (mRNA and protein) in endometrium of pregnant ewes from Day 15-35. Literature with respect to CXCL12 and CXCR4 in ruminants is limited, but in women, activation of CXCR4 causes recruitment of lymphocytes into decidua and stimulates trophoblast proliferation and invasion [14–16]. Trophoblasts secrete CXCL12, which binds to CXCR4 on decidual cells resulting in increased activity of matrix metalloprotease (MMP) 9 and MMP2 . MMP9 and MMP2 are critical determinants for trophoblast migration and invasion [47, 48]. Wu and co-workers  demonstrated increased viability of trophoblast cells and activation of MAPK ERK1/2 pathway in trophoblast cells treated with recombinant CXCL12, suggesting that CXCL12/CXCR4 interactions play an important role in early pregnancy. In support of these findings, Jaleel and co-workers  showed CXCL12/CXCR4 signaling suppressed apoptosis and enhanced trophoblast survival through the MAPK pathway and speculated that a lack of CXCL12/CXCR4 signaling may result in significant utero-placental pathology. Activation of CXCR4 by CXCL12 also promotes CD4+ T-cell survival and increases expression of cell survival genes while inactivating pro-apoptosis genes such as Bcl2 . In pregnant mice, CXCL12 causes migration of CXCR4+ Treg cells into the uterus and prevents embryo loss . Therefore, the CXCL12/CXCR4 system may affect migration of immunocompetent cells into the uterus and aid in establishment and maintenance of maternal tolerance to the fetal allograft. In support of this in the ovine model, the percentage of endometrial CD45R+ lymphocytes is greater in early pregnant ewes compared to cyclic ewes [52, 53], but whether CXCL12/CXCR4 activation is functioning to drive this migration in sheep is not known. To date, no research has been conducted on CXCL12/CXCR4 interactions in placenta during early pregnancy in sheep. We interpreted the data from the current study to mean the CXCL12/CXCR4 pathway is activated during implantation and placentation in sheep and is likely playing a role in the communication between trophoblast cells and the maternal endometrium.
In summary, we have described upregulation of a unique chemokine, CXCL12 and its receptor, CXCR4 during early pregnancy in sheep in both fetal and maternal tissues. Historically, study of chemokines and their respective receptors have focused specifically on immune functions, yet research into their biological roles in other systems has grown extending the functional implications of chemokine receptor activation. The unique relationship between VEGF, bFGF and the CXCL12/CXCR4 system may provide novel insights into the temporal changes in expression of select angiogenic factors and their functions during placentation. In ruminants, interferon tau (IFNτ) is produced by trophoblast cells and serves as the classic signal for maternal recognition of pregnancy. Pertinent to the current study, CXCL12 was recently identified as a new IFNτ induced gene in cattle . Whether similar gene regulation is observed in sheep is not known, but underscores the communication that exists between trophoblast cells and the maternal endometrium during early gestation with regards to the CXCL12/CXCR4 system. In conclusion, data from the current study provides a solid foundation for future study of CXCL12 and CXCR4 functions during implantation and placentation, specifically vascularization of the placenta and highlights the novel roles CXCL12/CXCR4 signaling may have during early pregnancy.