Temporal and spatial associations of oestrogen receptor alpha and progesterone receptor in the endometrium of cyclic and early pregnant mares.

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.

Caprine uterine and placental osteopontin expression is distinct among epitheliochorial implanting species.

Osteopontin (OPN) is the most highly up-regulated extracellular matrix/adhesion molecule in the uterus of humans and domestic animals as it becomes receptive to implantation. Studies in sheep and pigs have shown that OPN is a component of ovine and porcine histotroph characterized by a complex temporal and spatial pattern of uterine and conceptus expression involving immune, epithelial, and stromal cells. It is proposed that these expression events are orchestrated to contribute to conceptus attachment and placentation. However, differences in OPN expression between sheep and pigs have been detected that relate to differences in placentation. Therefore, this study examined OPN expression in the caprine uterus and conceptus to gain insight into mechanisms underlying OPN function(s) during pregnancy through comparative analysis of differences in placentation between pigs, sheep, and goats. Goats were hysterectomized (n = 5/day) on Days 5, 11, 13, 15, 17 or 19 of the estrous cycle, and Days 5, 11, 13, 15, 17, 19 or 25 of pregnancy. Slot-blot hybridization showed increases in endometrial OPN mRNA beginning on Day 17 of the estrous cycle and Day 19 of pregnancy. In situ hybridization localized OPN mRNA to endometrial glandular epithelium (GE), Day 25 myometrium, and cells scattered within the placenta hypothesized to be immune. Immunofluorescence microscopy detected OPN protein on the apical surface of endometrial lumenal epithelium (LE), in GE, and on conceptus (Tr). Western blot analysis detected primarily the native 70-kDa OPN protein in endometrial extracts from the estrous cycle and pregnancy, as well as in uterine flushings from pregnant goats. Co-induction of OPN and alpha-smooth muscle actin, but not desmin proteins, was observed in uterine stroma by Day 25 of pregnancy. OPN in cyclic GE, Day 25 myometrium, and desmin-negative endometrial stroma is unique and reflects subtle differences among superficial implanting species that correlate with the depth of Tr invasion.

Expression of the uterine Mx protein in cyclic and pregnant cows, gilts, and mares.

Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts--animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.

The Interferon Stimulated Genes (ISG) 17 and Mx have different temporal and spatial expression in the ovine uterus suggesting more complex regulation of the Mx gene.

Interferon stimulated gene 17 (ISG17) and Mx are up-regulated in the ruminant uterus in response to interferon-tau (IFNtau) during early pregnancy. Recent evidence strongly indicates that expression of ISGs occur only in stroma (ST) and glandular epithelium (GE) during this time as a result of transcriptional repression by interferon regulatory factor two (IRF-2) expression in the LE. The present report tested this hypothesis by examining mRNA and protein expression of ISG17 and Mx in serial uterine cross-sections obtained from cyclic and early pregnant ewes. In situ and immunocytochemical analysis revealed that ISG17 mRNA and protein were low to undetectable, whereas Mx mRNA was expressed in the lumenal (LE) and superficial GE at all days of the estrous cycle examined. Both ISG17 and Mx mRNA increased in the stratum compactum ST between Days 11 and 13, and expression extended into the deep GE and stratum spongiosum ST on Days 15 through 17 in pregnant ewes. Interestingly the Mx gene continued to be strongly expressed in LE and superficial GE through Day 17 of pregnancy, whereas ISG17 remained low to undetectable in these cells. Collectively, this study highlights the complexity of the uterine environment by unequivocally illustrating differential temporal and spatial expression of the IFN-responsive genes ISG17 and Mx.

Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion.

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function.

Differential expression of ribosomal L31, Zis, gas-5 and mitochondrial mRNAs following oxidant induction of proliferative vascular smooth muscle cell phenotypes.

Treatment of cultured vascular smooth muscle cells (vSMCs) with benzo(a)pyrene (BaP), a prooxidant present in the particulate phase of tobacco smoke, induces highly proliferative (i.e. atherogenic) phenotypes. Critical early target genes in vSMCs have been identified, but patterns of gene expression following repeated cycles of carcinogen treatment in vivo have yet to be evaluated. In the present study, male Sprague-Dawley rats (175-200 g) were given weekly injections of BaP (10 mg/kg) for 8 weeks to induce atherogenic phenotypes. At the end of this atherogenic regimen, vSMCs were established in serial culture and monitored for patterns of proliferative activity and gene expression. vSMCs isolated from BaP-treated animals (hence forth referred to as BaP cells) exhibited constitutively increased growth rates, and marked enhancement of proliferation in response to serum mitogens. Differential display polymerase chain reaction (DD-PCR) and Northern blot analyses revealed that mRNAs for ribosomal protein L31 and Zis genes were suppressed, while gas-5 and mitochondrial mRNAs were overexpressed in BaP cells relative to control mRNA populations. In situ hybridization experiments in vascular tissue confirmed these alterations in vivo. This is the first report linking expression of these genes to proliferative dysregulation during the course of experimentally-induced atherogenesis.

Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus.

Interferon tau (IFNtau) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFNtau on IFN-stimulated gene (ISG) expression are mediated by ISG factor 3 (ISGF3), which is composed of signal transducer and activator of transcription (Stat) 1, Stat 2, and IFN regulatory factor-9 (IRF-9), and by gamma-activated factor (GAF), which is a Stat 1 homodimer. Induction of ISGs, such as ISG17 and 2',5'-oligoadenylate synthetase, by IFNtau during pregnancy is limited to the endometrial stroma (S) and glandular epithelium (GE) of the ovine uterus. The IRF-2, a potent transcriptional repressor of ISG expression, is expressed in the luminal epithelium (LE). This study determined effects of the estrous cycle, pregnancy, and IFNtau on expression of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in the ovine endometrium. In cyclic ewes, Stat 1, Stat 2, IRF-1, and IRF-9 mRNA and protein were detected at low levels in the S and GE. During pregnancy, expression of these genes increased only in the S and GE. Expression of IRF-2 was detected only in the LE and superficial GE (sGE) of both cyclic and pregnant ewes. In cyclic ewes, intrauterine administration of IFNtau stimulated Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Ovine IRF-2 repressed transcriptional activity driven by IFN-stimulated response elements that bind ISGF3, but not by gamma-activation sequences that bind GAF. These results suggest that IRF-2 in the LE and sGE restricts IFNtau induction of ISGs to the S and GE. In the S and GE, IFNtau hyperactivation of ISG expression likely involves formation and actions of the transcription factors ISGF3 and, perhaps, IRF-1.

Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin.

Effects of age on uterine histoarchitecture, cell proliferation, and hormone receptor expression were determined for neonatal ewe lambs from birth (Postnatal Day [PND] 0) to PND 56. Uteri were histologically evaluated and proliferating cell nuclear antigen (PCNA), estrogen receptor alpha (ER-alpha), progesterone receptor (PR), and prolactin receptor (PRL-R) expression were characterized by in situ hybridization (ISH), immunohistochemistry, or both. The most striking feature of neonatal uterine development was the genesis and development of glands in the intercaruncular areas of endometrium. After birth, endometrial glandular epithelium (GE) budded and differentiated into the underlying stroma from the luminal epithelium (LE) between PNDs 1 and 7. Between PNDs 14 and 56, extensive coiling and branching morphogenesis of nascent endometrial glands occurred. By PND 56, the uterine wall appeared to be histoarchitecturally mature. At birth, nuclear PCNA protein was strongly detected in LE. Between PNDs 7 and 56, high levels of PCNA, ER-alpha, and PR gene expression were detected in both nascent and developing GE. Higher levels of PCNA and ER-alpha expression were detected in GE at the tips of developing glands as well as in the surrounding stroma. Progesterone was below detectable limits in serum. Serum estradiol-17beta levels were high on PND 1, increased from PNDs 14 to 28, and declined from PND 42 to PND 56. Serum PRL levels increased from PNDs 1 to 14 and declined thereafter. Using ISH and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, expression of mRNAs for short and long forms of the ovine PRL-R were first detected in nascent GE on PND 7 and increased between PNDs 7 and 56 in proliferating and differentiating GE. These results indicate that 1) uterine gland genesis is initiated between PNDs 1 and 7 after birth and is essentially completed by PND 56; 2) neonatal uterine morphogenesis involves temporal and spatial alterations in cell proliferation and ER-alpha, PR, and PRL-R gene expression; 3) PRL-R expression is a unique marker of GE differentiation and proliferation; and 4) serum estradiol-17beta and PRL levels increase during the onset of GE tubular branching morphogenesis. Results support the hypothesis that neonatal ovine uterine development involves epithelial PRL-R and ER-alpha activation to stimulate and maintain endometrial gland genesis and branching morphogenesis.

Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy.

Lactogenic hormones regulate epithelial proliferation, differentiation, and function in a variety of epitheliomesenchymal organs. During pregnancy, the ovine uterus is a potential site for endocrine and paracrine actions of lactogenic hormones in the form of pituitary prolactin (PRL) and placental lactogen (PL). These studies determined temporal and spatial alterations in PRL receptor (PRL-R) and expression of uterine milk proteins (UTMP), a marker of endometrial secretory activity, in the ovine endometrium during the estrous cycle and pregnancy. Slot-blot hybridization analysis indicated that steady-state levels of endometrial PRL-R mRNA increased during pregnancy. In situ hybridization and immunohistochemical analyses indicated that PRL-R mRNA and protein were exclusively expressed in the endometrial glandular epithelium (GE). No PRL-R mRNA expression was detected in luminal epithelium, stroma, myometrium, or conceptus trophectoderm. Reverse transcription-polymerase chain reaction analyses determined that the endometrial GE expressed both long and short alternative splice forms of the ovine PRL-R gene. Slot-blot hybridization analysis indicated that steady-state levels of intercaruncular endometrial UTMP mRNA increased about 3-fold between Days 20 and 60, increased another 3-fold between Days 60 and 80, and then declined slightly to Day 120. In pregnant ewes, UTMP mRNA expression was restricted to the endometrial GE in the stratum spongiosum (sGE), increased substantially between Days 15 and 17, and, between Days 17 to 50 of gestation, was markedly higher in upper than lower sGE. After Day 50, hyperplasia of the sGE was accompanied by increased UTMP mRNA expression by all sGE. Collectively, results indicate that 1) endometrial sGE is a primary target for actions of lactogenic hormones and 2) UTMP mRNA expression is correlated with PL production by the trophectoderm and state of sGE differentiation during pregnancy. It is proposed that activation of PRL-R signal transduction pathways by PRL and PL plays a major role in endometrial GE remodeling and differentiated function during pregnancy in support of conceptus growth and development.

Interferon-tau and progesterone regulate ubiquitin cross-reactive protein expression in the ovine uterus.

Ubiquitin cross-reactive protein (UCRP) is a functional ubiquitin homolog synthesized by the ruminant endometrium in response to conceptus-derived interferon-tau (IFNtau). Progesterone is required for IFNtau to exert antiluteolytic actions on the endometrium. Therefore, this study was designed to determine whether progesterone is requisite for IFNtau induction of UCRP expression within the ovine uterus. Cyclic ewes were ovariectomized and fitted with intrauterine (i.u.) catheters on Day 5 and treated daily with steroids (i.m.) and protein (i.u.) as follows: 1) progesterone (P, Days 5-24) and control serum proteins (CX, Days 11-24); 2) P and ZK 137.316 (ZK; progesterone receptor antagonist, Days 11-24) and CX proteins; 3) P and recombinant ovine IFNtau (roIFNtau, Days 11-24); or 4) P and ZK and roIFNtau. All ewes were hysterectomized on Day 25. In P-treated ewes, roIFNtau increased endometrial UCRP mRNA and protein levels. However, administration of ZK to ewes ablated roIFNtau induction of UCRP. Recombinant ovine IFNtau induced expression of UCRP mRNA in progestinized endometrial luminal (LE) and glandular (GE) epithelium as well as in both stratum compactum and spongiosum layers of the stroma (ST). Progesterone receptor protein was located in endometrial ST, but not in LE and GE from these ewes. Results support the hypothesis that progesterone is required for IFNtau induction of type I IFN-responsive genes, such as UCRP, in the ruminant uterus.

Discovery and characterization of endometrial epithelial messenger ribonucleic acids using the ovine uterine gland knockout model.

Prolonged exposure of the developing neonatal ovine uterus to a progestin from birth prevents uterine gland development and creates an adult endometrial phenotype characterized by the absence of glandular epithelium, the uterine gland knockout (UGKO) phenotype. This study used endometrium from normal and UGKO sheep to identify messenger RNAs (mRNAs) expressed differentially in the endometrial epithelium using the molecular techniques of mRNA differential display PCR (DD-PCR) and suppression subtractive complementary DNA (cDNA) hybridization (SSH). Sequence analyses of DD- and SSH-identified and cloned cDNAs indicated similarity of some to known mRNAs, including beta-lactoglobulin, alkaline phosphatase, type B and D endogenous sheep retroviruses, gp330/megalin, matrix Gla protein, and others. Other cDNAs were not similar to any known sequences and are considered novel, although some of these match human expressed sequence tags. In situ hybridization analyses of uteri from cyclic and pregnant ewes indicated that all DD-PCR- and SSH-identified mRNAs were expressed in either the endometrial lumenal and/or glandular epithelium, although some were also expressed in other uterine cell types. Northern and in situ hybridization analyses revealed that patterns of mRNA expression for most clones were affected by the day of the estrous cycle and pregnancy in a manner consistent with regulation by progesterone. Studies demonstrate the utility of the ovine UGKO model as a tool with which to identify known and novel uterine epithelial-specific genes. Cloned cDNAs identified here are expressed sequence tags useful for comparative and physical genetic mapping and may be used to reveal new factors and pathways regulating endometrial function.

Identification and characterization of glycosylation-dependent cell adhesion molecule 1-like protein expression in the ovine uterus.

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is an endothelial glycoprotein secreted in lymph nodes that serves as a ligand for leukocyte cell surface selectin and mediates lymphocyte extravasation. In the present studies, rabbit anti-rat GlyCAM-1 IgG was used in immunochemical analyses of GlyCAM-1-like protein in the ovine uterus. In cyclic ewes, GlyCAM-1 expression increased in the endometrial luminal epithelium (LE) and shallow glandular epithelium (cGE) between Days 1 and 5 and then decreased between Days 11 and 15. In pregnant ewes, GlyCAM-1 in the LE and cGE was low on Days 11 and 13, increased on Day 15, and was abundant on Days 17 and 19. Immunoreactive GlyCAM-1 was also detected in the conceptus trophectoderm on Days 13-19. Staining for GlyCAM-1 in the smooth muscle of the vasculature and myometrium was constitutive, and no staining was detected in the stroma. An immunoreactive protein of approximately 45 kDa was identified in endometrial extracts and uterine flushings from cyclic and pregnant ewes. In pregnant ewes, the relative amount of immunoreactive GlyCAM-1 in uterine flushings was low on Days 11 and 13 but high on Days 15 and 17. Results suggest that a GlyCAM-1-like protein may be a secretory product of the endometrial epithelium and/or conceptus trophectoderm. Patterns of distribution observed for immunoreactive GlyCAM-1-like protein in the endometrial epithelium, combined with proposed functions for lymphoid GlyCAM-1, suggest that this mucin glycoprotein may be involved in conceptus-maternal interactions during the periimplantation period of pregnancy in sheep.

Effects of exogenous recombinant ovine interferon tau on circulating concentrations of progesterone, cortisol, luteinizing hormone, and antiviral activity; interestrous interval; rectal temperature; and uterine response to oxytocin in cyclic ewes.

Interferon tau (IFN tau) is the conceptus-produced antiluteolytic signal in ruminants. Three experiments examined the effects of s.c. administration of recombinant ovine (ro)IFN tau on interestrous interval (IEI), oxytocin (OT)-induced uterine prostaglandin F2alpha metabolite (PGFM) production, rectal temperature (RT), respiration rate (RR), and plasma concentrations of progesterone, cortisol, LH, and antiviral activity (AVA) in plasma and uterine flushings. In experiment I, 20 ewes were treated s.c. with either 0, 1, 2, or 4 mg/day roIFN tau (0.7 x 10(8) U/mg; 5 ewes/dosage) from Days 11 to 15 of the estrous cycle (estrus = Day 0) and were challenged with OT (30 IU) on Day 15. Jugular blood samples were collected at -10, 0, 10, 20, 30, 40, 50, and 60 min relative to the OT challenge and assayed for PGFM. Recombinant oIFN tau increased IEI (16.7, 18.7, and 22.6 +/- 0.6 days for 0, 2, and 4 mg roIFN tau, respectively, p < 0.01). Recombinant oIFN tau did not affect peak PGFM response to OT (2309 +/- 172 pg/ml; p > 0.1). However, the 4 mg/day dosage delayed the time to peak PGFM (32.4 vs. 47.5 +/- 3.4 min; p < 0.01, 0 vs. 4 mg) and resulted in approximately 200% higher concentrations of PGFM at 60 min post-OT (0 vs. 4 mg/day, p < 0.07). Experiment II was similar to experiment I, except that only the 0- and 4-mg/day dosages of roIFN tau were administered. Ewes were hysterectomized on Day 16, and assay of uterine flushes detected no AVA from ewes treated with either 0 or 4 mg/day roIFN tau. In experiment III, 20 ewes were treated s.c. with either 0, 2, 4, or 6 mg roIFN tau on Day 12. Blood samples, RT, and RR were obtained at frequent intervals for 24 h, and plasma was assayed for progesterone, cortisol, LH, and AVA. Plasma AVA, which increased in a dose-dependent manner, was detectable within 60 min and remained elevated at 24 h compared to control values. RT (elevated 0.5-1.0 degrees C), RR, and cortisol increased in response to all dosages of roIFN tau, with peak values occurring 150-180 min postinjection. For all dosages of roIFN tau, plasma progesterone declined from 120 to 360 min posttreatment and then returned to pretreatment values by 24 h (p < 0.01) as compared to controls. Overall, exogenous roIFN tau altered uterine PGFM response to OT from a pulse to a gradual and sustained elevation and extended IEI with only a transient decline in progesterone and mild hyperthermia, effects that are not expected to compromise pregnancy.

Identification of a cell-specific transcription activation domain within the human Ah receptor nuclear translocator.

In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals, the Ah receptor nuclear translocator (Arnt) forms a heterodimeric complex with the ligand-bound Ah receptor, leading to recognition of dioxin-responsive elements within the enhancer of the CYP1A1 gene and transcription activation by an unknown mechanism. To understand the role of Arnt in transcription activation by the Ah receptor-Arnt heterodimer, we performed a deletion analysis of Arnt to locate domains that are directly involved in transcription activation. We showed that the C-terminal 34 amino acids of Arnt encode a transcription activation domain (TAD) that functions independently of other sequences in the Ah receptor complex when attached to the heterologous Gal4 DNA binding domain. Deletion of the C-terminal acidic-rich 14 amino acids completely abolishes activity. Sequences important in Arnt TAD function were independent of the glutamine-rich region which is an important structural feature in the TAD of other transcription factors. The strength of the Arnt TAD when compared with the strong TAD from the herpes simplex virus VP16 protein was cell-type specific. Both the Arnt and VP16 TAD were equally strong in COS-1 cells, but the Arnt TAD had weak activity in an Arnt-deficient mouse hepatoma cell line and was not needed for restoration of CYP1A1 activation. These results imply that for CYP1A1 activation the Ah receptor provides the dominant activation function for the heterodimer in hepatoma cells. The potential of the Arnt TAD to contribute to activation by the Ah receptor complex is likely determined by availability or activity of cell-specific factors with which the TAD interacts.