Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus.

During early pregnancy in ruminants, progesterone (P4) from the corpus luteum and interferon tau (IFNT) from the conceptus act on the endometrium to regulate genes important for uterine receptivity and conceptus growth. The use of the uterine gland knockout (UGKO) ewe has demonstrated the critical role of epithelial secretions in regulation of conceptus survival and growth. A custom ovine cDNA array was used to identify alterations in gene expression of endometria from Day 14 cyclic, pregnant, and UGKO ewes (study 1) and from cyclic ewes treated with P4 or P4 with ZK 136,317 antiprogestin and control proteins or IFNT (study 2). In study 1, expression of 47 genes was more than 2-fold different between Day 14 pregnant and cyclic endometria, whereas 23 genes was different between Day 14 cyclic and UGKO endometria. In study 2, 70 genes were different due to P4 alone, 74 genes were affected by IFNT in a P4-dependent manner, and 180 genes were regulated by IFNT in a P4-independent manner. In each study, an approximately equal number of genes were found to be activated or repressed in each group. Endometrial genes increased by pregnancy and P4 and/or IFNT include B2M, CTSL, CXCL10, G1P3, GRP, IFI27, IFIT1, IFITM3, LGALS15, MX1, POSTN, RSAD2, and STAT5A. Transcripts decreased by pregnancy and P4 and/or IFNT include COL3A1, LUM, PTMA, PUM1, RPL9, SPARC, and VIM. Identification and analysis of these hormonally responsive genes will help define endometrial pathways critical for uterine support of peri-implantation conceptus survival, growth, and implantation.

Sheep uterine gland knockout (UGKO) model.

Endometrial gland development is a postnatal event in the ovine uterus that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin after birth. The uterus of neonatally progestinized ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype. Progestin ablation of endometrial gland development is specific, because it does not affect development of extra-uterine reproductive tract structures or the hypothalamic-pituitary-ovarian axis. The UGKO ewe is a useful model for study of uterine development and the role of endometrial glands in uterine function during the estrous cycle and pregnancy. UGKO ewes exhibit altered estrous cycles due to the inability of the uterus to produce luteolytic pulses of prostaglandin F2alpha. UGKO ewes are infertile, and blastocysts hatch normally but fail to survive or elongate during early pregnancy. This pregnancy defect is primarily due to the absence of endometrial glands and their secretions rather than alterations in expression of either anti-adhesive or adhesive molecules on the endometrial epithelium. Genomics and proteomics are being used to identify specific components of histotroph that are absent or diminished in the UGKO ewe and will serve as markers of endometrial function and uterine receptivity.

Galectin-15 in ovine uteroplacental tissues.

Galectin-15 is the newest member of a secreted beta-galactoside-binding lectin family. The galectin-15 gene is expressed specifically by the endometrial luminal epithelium (LE) and superficial ductal glandular epithelium (sGE) of the ovine uterus. The proposed extracellular role of secreted galec7tin-15 is to regulate implantation and placentation by functioning as a heterophilic cell adhesion molecule between the conceptus trophectoderm and endometrial LE, while that of intracellular galectin-15 is to regulate cell survival, differentiation and function. The present study determined galectin-15 expression in uteroplacental tissues during gestation and in the postpartum uterus. In the uterine lumen, secreted galectin-15 was found as multimers, particularly on days 14 and 16 of pregnancy. In the endometrial epithelium and conceptus trophectoderm, intracellular galectin-15 protein was found associated with crystalline structures. Between days 20 and 120 of pregnancy, galectin-15 mRNA was expressed specifically by the LE and sGE of the intercaruncular endometrium of ewes. Immunoreactive galectin-15 protein was most abundant in the trophectoderm with lower levels in the endometrial LE and sGE. Galectin-15 protein was detected in allantoic fluid, but not in amniotic fluid. After parturition, galectin-15 mRNA declined in the endometrium from postpartum day (PPD) 1 to 28 and exhibited a variegated expression pattern in the LE and sGE. These results indicate that galectin-15 is synthesized and secreted throughout gestation by the endometrial LE/sGE and is absorbed by the placenta and forms crystals within the trophectoderm, whereas the remainder is cleared into the allantois after being transported into the fetal circulation via the placental areolae. Based on the biological properties of other galectin family members, galectin-15 is hypothesized to have biological roles in conceptus-endometrial interactions, uterine immune and inflammatory responses, and placental morphogenesis and function.

Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm.

Secretions of the uterus support survival and growth of the conceptus (embryo/fetus and associated membranes) during pregnancy. Galectin-15, also known as OVGAL11 and a previously uncharacterized member of the galectin family of secreted beta-galactoside lectins containing a conserved carbohydrate recognition domain and a separate putative integrin binding domain, was discovered in the uterus of sheep. In endometria of cyclic and pregnant sheep, galectin-15 mRNA was expressed specifically in the endometrial luminal epithelium but not in the conceptus. In pregnant sheep, galectin-15 mRNA expression appeared in the epithelia between days 10 and 12 and increased between days 12 and 16. Progesterone induced and IFN-tau stimulated galectin-15 mRNA in the endometrial epithelium. Galectin-15 protein was concentrated near and on the apical surface of the endometrial luminal epithelia and localized within discrete cytoplasmic crystalline structures of conceptus trophectoderm (Tr). In the uterine lumen, secreted galectin-15 protein increased between days 14 and 16 of pregnancy. Galectin-15 protein was functional in binding lactose and mannose sugars and immunologically identical to the unnamed Mr 14,000 (14K) protein from the ovine uterus that forms crystalline inclusion bodies in endometrial epithelia and conceptus Tr. Based on the functional studies of other galectins, galectin-15 is hypothesized to function extracellularly to regulate Tr migration and adhesion to the endometrial epithelium and intracellularly to regulate Tr cell survival, growth, and differentiation. Galectins may be useful as cellular and molecular markers for endometrial function and receptivity, to enhance conceptus survival and development, and to evaluate and enhance fertility.

Gene expression profiling of neonatal mouse uterine development.

Postnatal uterine development involves differentiation and development of the endometrial glandular epithelium from the luminal epithelium as well as development of the mesenchyme into the endometrial stroma and myometrium. This period of development is critical because exposure of neonates to endocrine disruptors compromises reproductive cycles and pregnancy in the adult. However, the hormonal, cellular, and molecular mechanisms regulating postnatal uterine development remain largely unknown. In order to identify candidate genes and gene networks that regulate postnatal uterine development, uteri were collected from CD-1 outbred mice on postnatal days (PND) 3, 6, 9, 12, and 15, and gene expression profiling was conducted using Affymetrix mouse genome U74Av2 GeneChips in study 1. Of the approximately 12,000 genes analyzed, 9002 genes were expressed in the uterus and expression of 3012 genes increased or decreased 2-fold during uterine development. In study 2, the uterine epithelium was enzymatically separated from the stroma/myometrium on PNDs 3, 6, and 9, and gene expression profiling was conducted using CodeLink UniSet Mouse I Expression Bioarrays. Results from these two studies support the hypothesis that postnatal uterine development is a complex process involving overlapping positive and negative changes in uterine epithelial and stromal/myometrial gene expression. Candidate genes regulating uterine development encode secreted factors (Wnt5a, Wnt7a), transcription factors (Hoxa10, Hoxa11, Hoxd10, MSX-1), enzymes (matrix metalloproteinases, cathepsin, carbonic anhydrase), growth factors (IGF-II, IGF binding proteins), and components of the extracellular matrix (osteopontin) to name a few. The candidate genes and gene networks identified by transcriptional profiling provide an important foundation to discern and understand mechanisms regulating postnatal uterine morphogenesis.

The activin-follistatin system in the neonatal ovine uterus.

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding and tubulogenesis followed by coiling and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (Postnatal Day [PND] 0) and PND 56. Activins, which are members of the transforming growth factor beta superfamily, and follistatin, an inhibitor of activins, regulate epithelial branching morphogenesis in other organs. The objective of the present study was to determine effects of postnatal age on expression of follistatin, inhibin alpha subunit, betaA subunit, betaB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin alpha subunit mRNA or protein was detected in the neonatal uterus. Expression of betaA and betaB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.

Estrogen and antiestrogen effects on neonatal ovine uterine development.

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. These critical events coincide with expression of estrogen receptor alpha (ERalpha) by nascent endometrial glands and stroma. To test the working hypothesis that estrogen and uterine ERalpha regulate uterine growth and endometrial gland morphogenesis in the neonatal ewe, ewes were treated daily from birth (PND 0) to PND 55 with 1) saline and corn oil as a vehicle control (CX), 2) estradiol-17 beta (E2) valerate (EV), an ERalpha agonist, 3) EM-800, an ERalpha antagonist, or 4) CGS 20267, a nonsteroidal aromatase inhibitor. On PND 14, ewes were hemihysterectomized, and the ipsilateral oviduct and ovary were removed. The remaining uterine horn, oviduct, and ovary were removed on PND 56. Treatment with CGS 20267 decreased plasma E2 levels, whereas EM-800 had no effect compared with CX ewes. Uterine horn weight and length were not affected by EM-800 or CGS 20267 but were decreased in EV ewes on PND 56. On PND 14 and PND 56, treatment with EV decreased endometrial thickness but increased myometrial thickness. The numbers of ductal gland invaginations and endometrial glands were not affected by CGS but were lower in EM-800 ewes on PND 56. Exposure to EV completely inhibited endometrial gland development and induced luminal epithelial hypertrophy but did not alter uterine cell proliferation. Exposure to EV substantially decreased expression of ERalpha, insulin-like growth factor (IGF) I, and IGF-II in the endometrium. Results indicate that circulating E2 does not regulate endometrial gland differentiation or development. Although ERalpha does not regulate initial differentiation of the endometrial glandular epithelium, results indicate that ERalpha does regulate, in part, coiling and branching morphogenesis of endometrial glands in the neonatal ewe. Ablation of endometrial gland genesis by EV indicates that postnatal uterine development is extremely sensitive to the detrimental effects of inappropriate steroid exposure.

Osteopontin is synthesized by uterine glands and a 45-kDa cleavage fragment is localized at the uterine-placental interface throughout ovine pregnancy.

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus.

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.

Osteopontin expression in uterine stroma indicates a decidualization-like differentiation during ovine pregnancy.

Osteopontin (OPN) is a component of the extracellular matrix that interacts with cell surface receptors, including integrins, to mediate cell adhesion, migration, differentiation, survival, and immune function. In pregnant mice and primates, OPN has been detected in decidualized stroma and is considered to be a gene marker for decidualization. Decidualization involves transformation of spindle-like fibroblasts into polygonal epithelial-like cells that are hypothesized to limit conceptus trophoblast invasion through the uterine wall during invasive implantation. Decidualization is not considered characteristic of species with noninvasive implantation, such as domestic animals. However, the extent of trophoblast invasion between sheep and pigs differs, with sheep exhibiting erosion of the uterine luminal epithelium (LE) and fusion of trophectoderm with LE to form syncytia, and pigs maintaining an intact LE throughout pregnancy. Therefore, the present study measured changes in the decidualization marker genes OPN, desmin, and alpha smooth muscle actin (alphaSMA) in ovine and porcine uterine stroma throughout pregnancy. The morphology of endometrial stromal cells in pregnant ewes changes following conceptus attachment, with cells increasing in size and becoming polyhedral in shape by Day 35 of pregnancy. Expression of OPN mRNA and protein, as well as desmin and alphaSMA proteins, was observed in this same uterine stromal compartment. In contrast, no morphological changes in uterine stroma nor induction of OPN mRNA and protein, or desmin protein, were detected during porcine pregnancy. Interestingly, alphaSMA protein was absent on Day 20, but prominent in uterine stroma of pregnant pigs on Day 45. Collectively, these results indicate that the uterine stroma of sheep undergoes a program of differentiation similar to decidualization in invasive implanting species, whereas porcine stroma exhibits differentiation that is more limited than that in sheep, rodents, or primates. Results suggest that uterine stromal decidualization is common to species with different types of placentation, but the extent is variable and correlates with the depth of trophoblast invasion during implantation.

Prolactin regulation of neonatal ovine uterine gland morphogenesis.

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding, proliferation, and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (postnatal day or PND 0) and PND 56. This critical developmental event is coincident with increases in serum PRL and expression of long and short PRL receptors specifically in the nascent and proliferating GE. In study one, ewes were treated with a placebo pellet as a control (CX) or a bromocryptine mesylate pellet from PNDs 0-56. On PND 56, the endometrium of bromocryptine mesylate ewes contained fewer glands, particularly in the stratum spongiosum that contained numerous coiled and branched glands in CX uteri. In study two, ewes were treated with saline as a CX or recombinant ovine PRL from PNDs 0-56. Treatment with PRL increased gland number and density on PND 14 and PND 56. In study three, expression of signal transducers and activators of transcription (STAT) 1, 3, and 5 proteins was detected in the developing glands from PNDs 7-56. In study four, Western blot analyses indicated that PRL increased levels of phosphorylated STATs 1 and 5, but not STAT 3, and phosphorylated ERK 1 and 2 MAPKs and c-Jun N-terminal kinase/stress-activated protein kinase proteins in explanted PND 28 ovine uteri. Collectively, results indicate that PRL regulates endometrial adenogenesis in the neonatal ovine uterus.

Receptor usage and fetal expression of ovine endogenous betaretroviruses: implications for coevolution of endogenous and exogenous retroviruses.

Betaretroviruses of sheep include two exogenous viruses, Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), and a group of endogenous viruses known as enJSRVs. The exogenous JSRV and ENTV are the etiological agents of ovine pulmonary adenocarcinoma (OPA) and enzootic nasal tumor (ENT), respectively. Sheep affected by OPA or ENT do not show an appreciable antibody response to JSRV or ENTV. Consequently, it is conceivable that enJSRV expression in the fetal lamb tolerizes sheep to the related exogenous viruses. In this study, possible mechanisms of interference between the sheep exogenous and endogenous betaretroviruses were investigated. In situ hybridization detected enJSRV RNAs in lymphoid cells associated with the lamina propria of the small intestine and in the thymus of sheep fetuses. Low-level expression of enJSRVs was also detected in the lungs. In addition, expression of enJSRVs was found to block entry of the exogenous JSRV, presumably via mechanisms of receptor interference. Indeed, enJSRVs, like JSRV and ENTV, were found to utilize hyaluronidase-2 as a cellular receptor.