Fibroblast growth factor-10: a stromal mediator of epithelial function in the ovine uterus

Fibroblast growth factor-10 (FGF-10) is a stromal-derived paracrine growth factor considered to be important during embryogenesis; however, its expression by cells in the female reproductive tract has not been investigated. Therefore, an ovine FGF-10 cDNA was cloned from an ovine endometrial cDNA library to investigate expression and potential paracrine characteristics of FGF-10 in the ovine uterus. The ovine FGF-10 cDNA encodes a protein of 213 amino acids and possesses an unusually long 5' untranslated region (UTR). In situ hybridization demonstrated that ovine FGF-10 mRNA was expressed by endometrial stromal cells and by mesenchymal cells of the chorioallantoic placenta. The mRNA for FGF-7, a homologue of FGF-10, was localized in the tunica muscularis of blood vessels in endometrium and myometrium. In contrast, FGF receptor 2IIIb, the high-affinity receptor for both FGF-10 and FGF-7, was expressed exclusively in luminal epithelium, glandular epithelium, and placental trophectoderm. The in vivo spatial expression pattern suggests that FGF-10 is a novel endometrial stromal cell-derived mediator of uterine epithelial and conceptus trophectodermal functions. The nonoverlapping spatial patterns of expression for FGF-10 and FGF-7 in ovine uterus and conceptus suggest independent roles in uterine function and conceptus development.     

Keratinocyte growth factor: expression by endometrial epithelia of the porcine uterus

Keratinocyte growth factor/fibroblast growth factor-7 (KGF/FGF-7) is an established paracrine mediator of hormone-regulated epithelial growth and differentiation. In all organs studied, KGF is uniquely expressed in cells of mesenchymal origin. To determine whether KGF and its receptor, keratinocyte growth factor receptor (KGFR) or fibroblast growth factor receptor-2IIIb, were expressed in the porcine uterus as a potential paracrine system mediating progesterone action, we cloned KGF and KGFR partial cDNAs from the porcine endometrium. KGF and KGFR expression was detected in endometrium by Northern blot hybridization. Interestingly, in situ hybridization results demonstrated that KGF was expressed by endometrial epithelia and was particularly abundant between Days 12 and 15 of the estrous cycle and pregnancy. KGF secretion into the lumen of the porcine uterus was also detected on Day 12 of the estrous cycle and pregnancy. KGFR was expressed in both endometrial epithelia and conceptus trophectoderm. These novel findings suggest that KGF may act on the uterine endometrial epithelium in an autocrine manner and on the conceptus trophectoderm in a paracrine manner in the pig, which is the only species possessing a true epitheliochorial type of placentation.     

Expression and action of hepatocyte growth factor in bovine endometrial stromal and epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2alpha secretion in epithelial, but not stromal cells. RT-PCR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion.     

Swine leukocyte antigen-DQ expression and its regulation by interferon-gamma at the maternal-fetal interface in pigs

Successful pregnancy requires an appropriate intrauterine immune response to the conceptus, which is a semiallograft within the uterus. We reported that swine leukocyte antigen-DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene, is expressed in the uterine endometrium at the time of conceptus implantation in pigs. Because MHC molecules play critical roles in the immune system, SLA-DQ was hypothesized to be involved in immune regulation during pregnancy. Therefore, we examined expression of SLA-DQ in uterine endometrial tissues obtained during the estrous cycle and pregnancy. SLA-DQA and SLA-DQB mRNAs were detected as 1.3-kb and 1.2-kb bands, respectively. Real-time RT-PCR analysis indicated that SLA-DQA and SLA-DQB mRNA expression was affected by day and pregnancy status, with the highest expression on Day 15 of pregnancy. SLA-DQ was localized primarily to subepithelial stromal cells and endothelial cells of the uterus. Using endometrial explant cultures from Day 12 of the estrous cycle, we determined that expression of SLA-DQA and SLA-DQB mRNAs increased in response to interferon-gamma (IFNG), which is produced by pig conceptus trophectoderm between Days 14 and 18 of pregnancy. The abundance of SLA-DQ protein was less in endometria from gilts with conceptuses resulting from somatic cell nuclear transfer compared with endometria from gilts with conceptuses resulting from natural mating. These results support our hypothesis that SLA-DQ is expressed in response to IFNG from the conceptus, and likely regulates immune response at the maternal-fetal interface to support the maintenance of pregnancy in pigs.     

Mechanisms regulating norgestomet inhibition of endometrial gland morphogenesis in the neonatal ovine uterus

In many species, endometrial gland adenogenesis occurs neonatally in an ovary- and steroid-independent manner. Chronic exposure of the developing neonatal ovine uterus to norgestomet (NOR) from birth permanently ablates endometrial gland morphogenesis or adenogenesis, creating an adult ovine uterine gland knockout (UGKO) phenotype. This study was conducted to determine the mechanism(s) whereby NOR inhibits adenogenesis in the neonatal ewe. Ewe lambs received no implant or a NOR implant at birth and on postnatal day (PND) 14, and they were necropsied on PND28. Histological analyses of the tracts indicated NOR exposure specifically inhibited endometrial adenogenesis, but no histoarchitectural differences were observed in the oviduct, cervix, or vagina. No effect of NOR treatment was detected on proliferating cell nuclear antigen (PCNA) expression in the endometrial luminal epithelium (LE), stroma, or myometrium. In control (CX) ewes, estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) mRNA and protein were expressed strongly in nascent and proliferating glandular epithelium (GE) but were undetected in epithelium of NOR uteri. Expression of c-met and fibroblast growth factor receptor 2IIIb (FGFR2IIIb) mRNA was detected in the LE and GE of CX uteri. In NOR uteri, c-met was expressed in the LE similar to CX uteri, but FGFR2IIIb mRNA levels were lower than in the LE of CX uteri. Uterine hepatocyte growth factor (HGF), the ligand for c-met, and FGFR2IIIb mRNA expression was substantially lower in NOR ewes, but expression of FGF-7 and FGF-10 mRNAs, ligands for FGFR2IIIb, was unaffected. These results indicate that NOR disrupts endometrial adenogenesis by ablating epithelial ER-alpha expression and altering expression of paracrine growth factors and/or receptors involved in epitheliomesenchymal interactions. Likewise, these mechanisms are proposed to be important regulators of normal uterine gland morphogenesis in the neonate.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Prostaglandin H synthase Type 2 is differentially expressed in endometrium based on pregnancy status in pony mares and responds to oxytocin and conceptus secretions in explant culture

The equine embryo must signal its presence to the uterus for pregnancy to continue to term. Mobility of the conceptus throughout the uterus is crucial for its survival, and this action presumably permits the conceptus to transmit its antiluteolytic signal to the endometrium. Studies were completed to establish whether this unidentified antiluteolytic signal targets prostaglandin G/H synthase 2 (PGHS2), a rate limiting enzyme in converting arachidonic acid to prostaglandins (PGs). In the first study, quantitative RT-PCR was used to determine the relative abundance of PGHS2 mRNA in endometrium derived from estrous cyclic and pregnant mares on day 14 post-ovulation. PGHS2 mRNA abundance was substantially greater in endometrium from estrous cyclic mares. Additional studies were completed to better understand PGHS2 in equine endometrium. An estrogen and progesterone treatment regimen in ovariectomized mares was developed as a test model for detecting endometrial PGHS2 mRNA. Also, exposing endometrial explants to conceptus secretions (conditioned culture medium) decreased PGHS2 mRNA abundance whereas exposing explants to oxytocin increased PGHS2 mRNA abundance. Exposure to conceptus secretions also decreased PGF2α concentrations in explant-conditioned medium whereas oxytocin supplementation increased PGF2α concentrations in medium. These data support the hypothesis that PGHS2 is a target for the antiluteolytic signal produced by equine conceptuses during early pregnancy. Also, the endometrial explant culture system used for these studies can serve as a model for identifying and characterizing the maternal recognition of pregnancy factor in equids.     

Analysis of interferon‐γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal‐conceptus interface and the role of interferon‐γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs

It has long been known that pig conceptuses produce interferon‐γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal‐conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin‐1β did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy‐ and cell‐type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.     

Paracrine regulation of endometrial function: interaction between progesterone and corticotropin-releasing factor (CRF) and activin A

Under the influence of ovarian steroid hormones, endometrial cells aer able to produce a wide variety of growth factors and peptide hormones that area believed to promote: (1) physiological growth and differentiation during the endometrial cycle; (2) decidualization, an essential preparative event for establishment of pregnancy; and (3) pathological growth and differentiation in endometriosis and cancer. Among the local factors produced by the human endometrium, corticotropin-releasing factor (CRF) and activin A have been evaluated in terms of localization and effects. CRF is a neuropeptide expressed by the epithelial and stromal cells of the human endometrium in increasing amounts from the endometrial proliferative to the secretory phase. CRF expression also increases in the pregnant endometrium, from early in the pregnancy until term. CRF-type 1 receptor mRNA is only expressed by stromal cells. Progesterone induces CRF gene expression and release from decidualized cells and CRF decidualizes cultured stromal endometrial cells. Urocortin, a CRF-related peptide, has been identified in endometrial epithelial and stromal cells, and its function is still under investigation. Activin A is a growth factor expressed in increasing amounts throughout endometrial phases by both epithelial and stromal cells. This growth factor is secreted into the uterine cavity with higher levels in the secretory phase. Maternal decidua expresses activin A mRNA in increasing amounts from early pregnancy until term. Human endometrium also expresses activin-A receptors and follistatin, its binding protein. Activin A decidualizes cultured human endometrial stromal cells (an effect reversed by follistatin) and modulates embryonic trophoblast differentiation and adhesion. Activin A is expressed in endometriosis and endometrial adenocarcinoma.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

Galectin 15 (LGALS15): a gene uniquely expressed in the uteri of sheep and goats that functions in trophoblast attachment

Galectins are a family of secreted animal lectins with biological roles in cell adhesion and migration. In sheep, galectin 15 (LGALS15) is expressed specifically in the endometrial luminal (LE) and superficial glandular (sGE) epithelia of the uterus in concert with blastocyst elongation during the peri-implantation period. The present study examined LGALS15 expression in the uterus of cattle, goats, and pigs. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels, and all contained a conserved carbohydrate recognition domain and RGD recognition sequence for integrin binding. Endometrial LGALS15 mRNA levels increased after Day 11 of both the estrous cycle and pregnancy, and were considerably increased after Day 15 of pregnancy in goats. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Immunoreactive LGALS15 protein was present in endometrial epithelia and conceptus trophectoderm of goat uteri and detected within intracellular crystal structures in trophectoderm and LE. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypothesis that LGALS15 is uniquely expressed in Caprinae endometria and functions as an attachment factor important for peri-implantation blastocyst elongation.     

Conceptus-mediated integrity of endometrial epithelial cells and maintenance of relaxin synthesis in pregnant rabbits: effects of unilateral oviduct ligation

A previous study indicated rabbit endometrial relaxin synthesis is stimulated by blastocyst (Lee VH, Fields PA, Biol Reprod 1990; 40:737-745). To evaluate this hypothesis, unilateral oviduct ligations were placed (A) at the oviduct isthmus on Day 1 post-copulation and (B), in a separate group of rabbits, at the infundibulum before copulation. Blastocysts migrate into and implant in the uterine horn contralateral to the ligated oviduct only (conceptus-bearing uterus). The uterine horn ipsilateral to the ligated oviduct will be referred to as the non-conceptus-bearing uterus. Uteri and ovaries were removed on Days 4-28 of pregnancy and were evaluated for relaxin using guinea pig anti-porcine relaxin serum and avidin-biotin light microscopy immunohistochemistry. Results were identical for both models. Blastocysts first attach to the antimesometrial uterine surface by Day 7 post-copulation. Implantation on the mesometrial surface occurs on Days 8-11. Relaxin was observed in antimesometrial endometrial glands of both conceptus and non-conceptus-bearing uteri on Days 4-7 of pregnancy. Beyond Day 7, relaxin was observed in antimesometrial and mesometrial endometrial glandular and luminal epithelial cells at implantation sites of the conceptus-bearing uterus only. Relaxin was not found between implantation sites. Endometrial epithelial cells of the non-conceptus-bearing uterus were regressing by Day 9. These data indicate a conceptus-mediated maintenance of endometrial epithelial cells. Furthermore, the data suggest a paracrine maintenance of epithelial cell integrity and relaxin synthesis since these parameters are preserved only in the conceptus-bearing uterus. Cell-cell communication between conceptus and endometrium appears to be specific since endometrium between implantation sites does not contain relaxin. Uterine tissue from pseudopregnant rabbits (Days 1-16) was evaluated. Relaxin was observed in the antimesometrial glands on Day 7 only. Like the endometrium in the ligation model, endometrial epithelial cells of the pseudopregnant rabbit uterus were regressing by Day 9. These results indicate that pregnancy is not required for, but may enhance, relaxin synthesis. In addition, endometrial epithelial cells regress in the absence of pregnancy. Regression of endometrial epithelial cells on Day 9 suggests that maternal recognition of pregnancy occurs during the preimplantation period (Days 4-8).