Role of chemokines in regulating luteal and uterine functions in pregnant cows.

In mammals, successful pregnancy requires proper communication between the embryo, endometrium, and corpus luteum (CL). When animals become pregnant, the CL remains functional, and prostaglandin (PG) F2α secretion from the uterus during early pregnancy decreases from those in comparable stages of the estrous cycle [ 1 , 2 ]. During the maternal recognition of pregnancy, the bovine conceptus produces interferon-τ (IFNT) to prevent luteolysis, which is induced by the pulsatile release of PGF2α from the uterus [ 2 ]. One mechanism through which IFNT inhibits luteolysis is the downregulation of the oxytocin (OT) receptor, which prevents OT-stimulated PGF2α secretion [ 3 ]. In cattle, IFNT inhibits OT-induced PGF2α secretion from the endometrium not only by downregulating the OT receptor but also by decreasing the expression of cyclooxygenase-2 (COX2) and PGF synthase [ 4 ]. The CL is a transient ovarian organ established by follicle cells following ovulation. The primary product of CL, progesterone (P4), is required for the establishment and maintenance of pregnancy in many mammals. In the absence of pregnancy, the CL degenerates. When pregnancy is established, the luteal lifespan is prolonged, and the CL continues to produce P4 during gestation [ 5 ]. The mammalian CL comprises a heterogeneous mixture of cell types that consist of not only steroidogenic luteal cells but also non-steroidogenic cells, i.e. , vascular endothelial cells, fibroblasts, and immune cells such as lymphocytes, granulocytes, and monocytes [ 6 , 7 , 8 ]. Cells composing the bovine CL produce various intraluteal factors, including PGs, growth factors, cytokines, and chemokines [ 5 ]. During the early phase of pregnancy, the CL is a prerequisite for all mammals. In some species ( e.g. , cows, pigs, goats, and dogs), it is also required in the later stages of pregnancy [ 9 ], whereas in other species ( e.g. , primates and sheep), it is not required because luteal P4 secretion is replaced by placental P4 secretion [ 10 ]. These findings suggest that the physiological role and function of the CL are different between pregnancy stages and between species. However, extensive research conducted over the last two decades, including global transcriptome studies, has demonstrated the enrichment of immune-related genes, including interferon-stimulated genes (ISGs), in the endometrium of pregnant cows compared with non-pregnant cows [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. The physiological roles of these pregnancy-dependent regulatory genes in maternal recognition are not yet fully understood, although understanding these roles during early pregnancy may help improve reproductive efficiency. Recently, we examined differences in comprehensive gene expression profiles in the bovine CL and endometrium during pregnancy and non-pregnancy periods using microarrays and showed that the expression of many genes, including those encoding several chemokines, fluctuates with pregnancy. In this review, we focus on studies on the roles of the CL and uterus during the gestational period, with an emphasis on the differences in the gene and protein expression profiles of chemokines in cows.

Numerous studies have been conducted on the mechanisms underlying bovine maternal recognition of pregnancy [ 1 , 2 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ], and more recently, comprehensive reviews have been published [ 53 , 54 ]. Our previous study also demonstrated that gene expression in the bovine endometrium during early pregnancy differs from that during the estrous cycle [ 48 ]. In particular, the gene expression of the chemokines listed in Table 3 Table 3. Comparison of mRNA levels for selected chemokines in the endometrium of pregnant vs. non-pregnant cows as determined by microarray analysis (fold > 2.0; P < 0.05) * Gene (Synonym) Day 15 (344 genes) Day 18 (1336 genes) CCL2 ( MCP-1 ) 3.58 – CCL3 ( MIP-1α ) 2.02 – CCL5 ( RANTES ) – 2.44 CCL8 ( MCP-2 ) 4.91 12.9 CCL11 ( Eotaxin-1 ) – 61.1 CCL14 ( HCC-1 ) 2.67 – CCL16 ( HCC-4 ) 2.55 – CCL19 ( MIP-3β ) 4.41 – CXCL10 ( IP-10 ) 2.10 12.1 –: Less than 2-fold change. * Modified from the data in our previous study [ 48 ]. is higher in the endometrium on days 15 and 18 of pregnancy than in the non-pregnant stage. Because IFNT mRNA expression begins to increase around 12 days after artificial insemination (AI) and plateaus on days 15–19, and IFNT protein levels also begin to increase around 12 days after AI, peak on days 19–20, and rapidly decrease by day 24 in cattle [ 55 ], we focused on the expression of endometrial genes on days 15 and 18 of pregnancy. –: Less than 2-fold change. * Modified from the data in our previous study [ 48 ]. CCL3 , CCL5 , and CCL19 are expressed in the human endometrium and are thought to be involved in decidualization and parturition [ 56 , 57 , 58 ]. CXCR4 and its ligand CXCL12 (SDF-1) have important functions in placentation and inhibition of CXCR4 during early pregnancy in sheep by increasing endometrial autophagy and reducing endometrial angiogenesis [ 59 , 60 ]. Because there are few reports regarding the involvement of these chemokines in the bovine endometrium, especially during pregnancy, the focus is placed next to the remaining six chemokines (CCL2, CCL8, CCL11, CCL14, CCL16, and CXCL10) in endometrial function in pregnant cows. The mRNA expression of IFNT-stimulated genes, including CCL2 and CCL8 , is higher in the endometrial tissues on day 16 of pregnant cows compared to non-pregnant cows [ 61 ]. In addition, there is a considerable increase in the populations of monocytes, macrophages, and dendritic cells in the endometrium in response to pregnancy [ 61 ]. Both CCL2 and CCL8 act through CCR2, which is found primarily on macrophages, monocytes, NK cells [ 18 , 23 ], and endometrial epithelial cells [ 48 ], suggesting that CCL2 and CCL8 may affect endometrial function during the early gestation period. Indeed, CCL2 decreases COX2 expression and CCL8 decreases the expression of both COX2 and OT receptors in cultured bovine endometrial tissues [ 48 ]. Because OT stimulates endometrial PG production by activating COX2 in endometrial cells [ 62 ], CCL2 and CCL8 may subsequently contribute to the inhibition of PGF2α production via CCR1 or CCR2, thereby establishing pregnancy in cows ( Fig. 1 Fig. 1. Hypothetical model of the inhibition of luteolysis and maternal recognition of pregnancy by IFNT and chemokines in cows. Although this model does not consider the effects of steroids or growth factors, IFNT, CCL2, CCL8, CCL14, and CXCL10 may block COX2 or OT receptor expression in bovine endometrial cells, leading to a reduction in OT-induced PGF2α output from the cells. Furthermore, IFNT and CCL16 may stimulate antiviral activity by upregulating ISG15 and MX1 expression in the endometrium as well as immune cells at the time of maternal recognition. IFNT may stimulate both CCL8 and CXCL10 production from immune cells and the endometrium and inhibit CCL14 production from the endometrium. The effects of CCL3, CCL5, and CCL11 on bovine endometrial function are still unclear, although their receptors (CCR1 and CCR3) are expressed in endometrial epithelial cells. The CCL19 actions via its receptor (CCR7) in the endometrium and immune cells still need to be clarified. Red and blue arrows show the stimulatory and inhibitory actions of each substance, respectively. This schematic figure was modified and adapted from the original figure in [ 48 ]. ). Hypothetical model of the inhibition of luteolysis and maternal recognition of pregnancy by IFNT and chemokines in cows. Although this model does not consider the effects of steroids or growth factors, IFNT, CCL2, CCL8, CCL14, and CXCL10 may block COX2 or OT receptor expression in bovine endometrial cells, leading to a reduction in OT-induced PGF2α output from the cells. Furthermore, IFNT and CCL16 may stimulate antiviral activity by upregulating ISG15 and MX1 expression in the endometrium as well as immune cells at the time of maternal recognition. IFNT may stimulate both CCL8 and CXCL10 production from immune cells and the endometrium and inhibit CCL14 production from the endometrium. The effects of CCL3, CCL5, and CCL11 on bovine endometrial function are still unclear, although their receptors (CCR1 and CCR3) are expressed in endometrial epithelial cells. The CCL19 actions via its receptor (CCR7) in the endometrium and immune cells still need to be clarified. Red and blue arrows show the stimulatory and inhibitory actions of each substance, respectively. This schematic figure was modified and adapted from the original figure in [ 48 ]. CCL11 mRNA levels considerably increased in the endometrium on day 18 of pregnancy ( Table 3 ). Because the CCL11 receptor (CCR3) is expressed in bovine [ 48 ] and human [ 63 ] endometrial epithelial cells, we expected this chemokine to act as a paracrine factor in the endometrium after day 18 of pregnancy. However, CCL11 expression is not affected by IFNT, and CCL11 does not affect the expression of any genes in cultured endometrial tissues [ 48 ]. CCL11 induces angiogenic responses in human endothelial cells [ 44 ], and CCL11 acts concomitant with estradiol-17β (E2) to recruit eosinophils to the uterine stroma during the estrous cycle in mice [ 64 ]. Additionally, CCL11 regulates extravillous trophoblast migration, invasion, and adhesion, highlighting the potential regulatory role of other chemokines during uterine decidual spiral arteriole remodeling in the first trimester of pregnancy in humans [ 45 ]. CCL11 is expressed not only in the endometrium but also in trophoblasts during the peri-implantation period in pigs [ 65 ]. Because the CCR3 protein is expressed in bovine fetal trophoblasts (day 18) and endometrial epithelial cells [ 48 ], CCL11 may act on the trophoblast conceptus via CCR3 rather than on endometrial cells in pregnant cows. CCL14 expression increases in the endometrium on day 15 of pregnant cows ( Table 3 ). In humans, CCL14 is produced maximally by the endometrium at the time of embryo implantation and during early pregnancy, predominantly by decidualized stroma and epithelial cells [ 66 ]. It stimulates trophoblast migration by activating its receptor (CCR1) [ 67 ]. Because CCR1 is expressed in fetal trophoblasts and endometrial epithelial cells in cows [ 48 ], CCL14 may promote trophoblast migration in cows. Moreover, CCL14 reduces OT receptor and COX2 expression in cultured endometrial tissues [ 48 ], suggesting that CCL14 decreases PG production in the bovine uterus ( Fig. 1 ). Although further studies regarding the physiological impact of CCL14 in regulating endometrial function are required, CCL14 may be an important factor in maternal-fetal communication in cows. CCL16 mRNA expression is higher in the endometrium on day 15 in pregnant cows than in non-pregnant cows ( Table 3 ). Moreover, CCL16 stimulates the expression of ISG15 and MX1 in cultured bovine endometrial tissues [ 48 ]. CCL16 increases the antigen presentation of macrophages, enhances T-cell cytotoxicity, and stimulates the production of interleukin (IL)-1β, tumor necrosis factor-α (TNF), and IL-12 in mice [ 68 ]. CCL16 and its receptors (CCR1 and CCR2) have been identified in the preterm human placenta [ 69 ] and bovine endometrium [ 48 ]. CCL16 induces human endothelial cell motility, which is pivotal for vessel formation, by stimulating the release of pro-inflammatory and proangiogenic chemokines [ 70 , 71 ]. Moreover, the expression of both CCL8 and CXCL10 in cultured bovine PBLs is stimulated by CCL16 and IFNT [ 36 ]. Therefore, CCL16 may influence angiogenesis and antiviral activity during maternal recognition in cows ( Fig. 1 ). The expression of CXCL10 in the endometrium is higher in pregnant cows than in non-pregnant cows [ 48 , 72 ]. Furthermore, CXCL10 mRNA expression is stimulated by IFNT, and CXCR3, which is the receptor of CXCL10, is expressed in the bovine endometrium and fetal trophoblasts on day 18 of pregnancy [ 48 ]. CXCL10 induces the recruitment of numerous leukocytes, lymphocytes, and/or monocytes to the ovine uterus and stimulates trophoblasts to attach to the endometrium [ 73 ]. Moreover, CXCL10 induces caprine trophoblast adhesion [ 74 ] and chemotaxis in human trophoblast cell lines [ 75 ]. CXCL10 inhibits OT receptor expression in cultured endometrial tissues [ 48 ]. These findings suggest that CXCL10 may affect not only PG production from the endometrium but also conceptus elongation at an early stage of pregnancy in cows, as in other animal species ( Fig. 1 ).

Numerous physiological changes occur in the CL and the endometrium during pregnancy. The expression of various chemokines is also altered in the CL and uterus, suggesting that chemokines play important roles in these organs during pregnancy as well as during the estrous cycle. Maternal recognition is regulated by a complex network mediated by chemokines and many factors in the bovine endometrium, conceptus, and immune cells. In contrast, our study, which examined changes in gene expression in the bovine CL during maternal recognition by microarray analysis, showed no significant changes in chemokine gene expression, unlike in the endometrium [ 76 ]. In the future, it will be necessary to examine the effects of chemokines on the reproductive physiology of pregnant cows, including their effects on the CL and uterus, as these are not yet understood.