Premature estrogen exposure alters endometrial gene expression to disrupt pregnancy in the pig.

Establishment and maintenance of pregnancy in the pig involve intricate communication between the developing conceptuses and maternal endometrium. Conceptus synthesis and release of estrogen during trophoblastic elongation are essential factors involved with establishing conceptus-uterine communication. The present study identified endometrial changes in gene expression associated with implantation failure and complete pregnancy loss after premature exposure of pregnant gilts to exogenous estrogen. Gilts were treated with either 5 mg estradiol cypionate (EC) or corn oil on d-9 and -10 gestation, which was associated with complete conceptus degeneration by d-17 gestation. Microarray analysis of gene expression revealed that a total of eight, 32, and five genes were up-regulated in the EC endometrium, whereas one, 39, and 16 genes were down-regulated, on d 10, 13, and 15, respectively. Four endometrial genes altered by EC, aldose reductase (AKR1B1), secreted phosphoprotein 1 (SPP1), CD24 antigen (CD24), and neuromedin B (NMB), were evaluated using quantitative RT-PCR and in situ hybridization. In situ hybridization localized gene expression for NMB, CD24, AKR1B1, and SPP1 in the luminal epithelium, and confirmed the expression patterns from RT-PCR analysis. The aberrant expression patterns of endometrial AKR1B1, SPP1, CD24, and NMB 3-4 d after premature estrogen exposure to pregnant gilts may be involved with conceptus attachment failure to the uterine surface epithelium and induction of endometrial responses that disrupt the establishment of a viable pregnancy.

Secreted phosphoprotein 1 (osteopontin) is expressed by stromal macrophages in cyclic and pregnant endometrium of mice, but is induced by estrogen in luminal epithelium during conceptus attachment for implantation.

Secreted phosphoprotein 1 (SPP1, osteopontin) is the most highly upregulated extracellular matrix/adhesion molecule/cytokine in the receptive phase human uterus, and Spp1 null mice manifest decreased pregnancy rates during mid-gestation as compared with wild-type counterparts. We hypothesize that Spp1 is required for proliferation, migration, survival, adhesion, and remodeling of cells at the conceptus-maternal interface. Our objective was to define the temporal/spatial distribution and steroid regulation of Spp1 in mouse uterus during estrous cycle and early gestation. In situ hybridization localized Spp1 to luminal epithelium (LE) and immune cells. LE expression was prominent at proestrus, decreased by estrus, and was nearly undetectable at diestrus. During pregnancy, Spp1 mRNA was not detected in LE until day 4.5 (day 1 = vaginal plug). Spp1-expressing immune cells were scattered within the endometrial stroma throughout the estrous cycle and early pregnancy. Immunoreactive Spp1 was prominent at the apical LE surface by day 4.5 of pregnancy and Spp1 protein was also co-localized with subsets of CD45-positive (leukocytes) and F4/80-positive (macrophages) cells. In ovariectomized mice, estrogen, but not progesterone, induced Spp1 mRNA, whereas estrogen plus progesterone did not induce Spp1 in LE. These results establish that estrogen regulates Spp1 in mouse LE and are the first to identify macrophages that produce Spp1 within the peri-implantation endometrium of any species. We suggest that Spp1 at the apical surface of LE provides a mechanism to bridge conceptus to LE during implantation, and that Spp1-positive macrophages within the stroma may be involved in uterine remodeling for conceptus invasion.

The temporal expression of osteopontin (SPP-1) in the rodent model of alcoholic steatohepatitis: a potential biomarker.

Previous studies from our laboratory have shown that osteopontin (OPN) mediates neutrophil infiltration into the liver in a rodent model of alcoholic steatohepatitis (ASH). The objective of this study was to investigate the temporal and spatial pattern of hepatic OPN mRNA and protein expression during the progression of alcoholic liver disease. OPN mRNA and protein expression were evaluated using real time PCR, in situ hybridization, Western blot and immunohistochemistry respectively. ASH was induced in male Sprague-Dawley rats by feeding EtOH-containing Lieber-DeCarli diet for 6 weeks, followed by a single injection of lipopolysaccharide (LPS, 10 mg/kg, ip). Rats were sacrificed 2-, 12-and 24-hour post LPS injection. A progressive induction of OPN mRNA was observed that preceded hepatic neutrophil infiltration and the increase in OPN mRNA correlated with increases in OPN protein expression. OPN mRNA was localized primarily to the biliary epithelium. The data indicates that OPN is transcribed and translated within the biliary epithelium. These findings suggest a potential role of OPN as an early biomarker in predicting inflammatory liver diseases such as ASH.

Expression of porcine endometrial prostaglandin synthase during the estrous cycle and early pregnancy, and following endocrine disruption of pregnancy.

Porcine trophoblast attachment to the uterine surface is associated with increased conceptus and endometrial production of prostaglandins. Conceptus secretion of estrogen on Day 12 of gestation is important for establishment of pregnancy; however, early (Days 9 and 10) exposure to exogenous estrogens results in embryonic mortality. Present studies established the temporal and spatial pattern of endometrial PTGS1 (prostaglandin-endoperoxide synthase 1) and PTGS2 expression during the estrous cycle and early pregnancy and determined the effect of early estrogen treatment on endometrial PTGS expression in pregnant gilts. Endometrial PTGS1 mRNA expression increased 2- to 3-fold after Day 10 of the estrous cycle and pregnancy, whereas PTGS2 mRNA expression increased 76-fold between Days 5 and 15 of the estrous cycle and pregnancy. Increased expression of the PTGS2 transcript was detected in the lumenal epithelium after Day 10 in both cyclic and pregnant gilts. There was a 10- and 20-fold increase in endometrial PTGS2 protein expression between Days 5 and 18 of the estrous cycle and pregnancy respectively. Administration of estrogen on Days 9 and 10 of gestation increased endometrial PTGS2 mRNA and protein on Day 10, but decreased PTGS2 mRNA and protein in lumenal epithelium (LE) on Day 12 of gestation compared to vehicle-treated gilts. The present study demonstrates that an increase in uterine epithelial PTGS2 expression occurs after Day 10 of the estrous cycle and early pregnancy in the pig. The conceptus-independent increase in the uterine LE indicates that a novel pathway exists for endometrial induction PTGS2 expression before conceptus elongation and attachment to the uterine surface. Epithelial expression of PTGS2 may serve as one of the signals for placental attachment and embryo survival in the pig. Early administration of estrogen on Days 9 and 10 of pregnancy alters endometrial PTGS2 mRNA and protein expression, which may, at least in part, represent a mechanism by which endocrine disruption of pregnancy causes total embryonic loss during implantation in the pig.

Steroid regulation of cell specific secreted phosphoprotein 1 (osteopontin) expression in the pregnant porcine uterus.

Secreted phosphoprotein 1 (SPP1, commonly referred to as osteopontin and formerly known as bone sialoprotein 1, early T-lymphocyte activation 1) is an extracellular matrix/adhesion molecule that is upregulated in the pregnant uterus of all mammals examined to date. This study focused on the pig, which has true epitheliochorial placentation and exhibits induction of SPP1 mRNA in luminal epithelium (LE) just before conceptus attachment and in glandular epithelium (GE) after Day 30 of pregnancy. The objective of this study was to determine steroid regulation of SPP1 mRNA and protein in porcine uterine epithelium. To examine the effect of estrogen, cyclic gilts were treated daily (Days 11-14) with 5 mg estradiol benzoate (i.m.) and hysterectomized on Day 15. To evaluate the long-term effect of pseudopregnancy, cyclic gilts were given daily injections (Days 11-15) with steroid as above and hysterectomized on Day 90. In situ hybridization showed high expression of SPP1 mRNA only in LE contiguous with apposing conceptus tissue on Day 15 of pregnancy. In contrast, estrogen injection resulted in moderate but uniform SPP1 mRNA in all LE of Day 15 nonpregnant gilts, with expression maintained through Day 90 of pseudopregnancy. SPP1 mRNA also localized to the GE of Day 90 pseudopregnant gilts, similar to expression in late gestation. Consistent with in situ hybridization results, SPP1 protein localized to the apical surface of LE in all estrogen-treated gilts and in the GE on Day 90 of pseudopregnancy. We conclude that, in pregnant pigs, SPP1 is induced by conceptus estrogen in uterine LE and is regulated in GE in a manner coincident with CL/placental progesterone production.

Interferon stimulated gene 15 conjugates to endometrial cytosolic proteins and is expressed at the uterine-placental interface throughout pregnancy in sheep.

Interferon-stimulated gene 15 (ISG15) is a ubiquitin homolog expressed in uteri of ruminants in response to interferon (IFN)-tau and is also induced during pregnancy in the uteri of mice, pigs, humans, and baboons. This study examined expression of ISG15 and its conjugation to target proteins in the ovine uterus beyond the period of IFNtau secretion by the conceptus. Although steady-state levels of ISG15 mRNA decreased after d 25 of pregnancy, ISG15 persisted in endometrium through d 120. In situ hybridization and immunocytochemistry localized ISG15 across the entire uterine wall through d 25, after which expression was restricted to endometrial stroma along the maternal-placental interface. Western blots revealed ISG15 and ISG15-conjugated proteins in endometrium. Treatment of ovariectomized sheep with progesterone and IFNtau increased both free and conjugated ISG15. These results are the first to show in vivo regulation of ISG15 function (i.e. conjugation to target proteins) by a type I IFN in the uterus of any species and that ISG15 is expressed at contacts between the placenta and uterus when trophectoderm no longer produces IFNtau. Interestingly, mRNA for the type II IFNgamma was present in the endometrial stromal compartment on d 15-50, which may stimulate the synthesis of ISG15 through later pregnancy. We hypothesize that ISG15 is not merely a consequence of an antiviral state induced by trophoblast IFNtau but represents a critical component of the microenvironment at the uterine-placental interface during the progressive events of conceptus development, implantation, and placentation in sheep and perhaps other mammalian species.