Margaret M. Joyce

Prolactin Receptor and Uterine Milk Protein Expression in the Ovine Endometrium During the Estrous Cycle and Pregnancy

Abstract 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.

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 β-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 th...

Interferon Regulatory Factor-Two Restricts Expression of Interferon-Stimulated Genes to the Endometrial Stroma and Glandular Epithelium of the Ovine Uterus

Abstract Interferon tau (IFNτ) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFNτ 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 IFNτ 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 IFNτ 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 IFNτ 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 IFNτ induction of ISGs to the S and GE. In the S and GE, IFNτ 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

Abstract 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-α), 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-α, and PR gene expression were detected in both nascent and developing GE. Higher levels of PCNA and ER-α 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-17β 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-α, PR, and PRL-R gene expression; 3) PRL-R expression is a unique marker of GE differentiation and proliferation; and 4) serum estradiol-17β 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-α activation to stimulate and maintain endometrial gland genesis and branching morphogenesis.

Interferon-Tau and Progesterone Regulate Ubiquitin Cross-Reactive Protein Expression in the Ovine Uterus

Abstract Ubiquitin cross-reactive protein (UCRP) is a functional ubiquitin homolog synthesized by the ruminant endometrium in response to conceptus-derived interferon-tau (IFNτ). Progesterone is required for IFNτ to exert antiluteolytic actions on the endometrium. Therefore, this study was designed to determine whether progesterone is requisite for IFNτ 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 IFNτ (roIFNτ, Days 11–24); or 4) P and ZK and roIFNτ. All ewes were hysterectomized on Day 25. In P-treated ewes, roIFNτ increased endometrial UCRP mRNA and protein levels. However, administration of ZK to ewes ablated roIFNτ induction of UCRP. Recombinant ovine IFNτ 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 IFNτ induction of type I IFN-responsive genes, such as UCRP, in the ruminant uterus.

Steroid Regulation of Cell Specific Secreted Phosphoprotein 1 (Osteopontin) Expression in the Pregnant Porcine Uterus

Abstract 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.

Osteopontin Expression in Uterine Stroma Indicates a Decidualization-Like Differentiation During Ovine Pregnancy

Abstract 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 (αSMA) 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 αSMA 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, αSMA 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.

Osteopontin Is Synthesized by Uterine Glands and a 45-kDa Cleavage Fragment Is Localized at the Uterine-Placental Interface Throughout Ovine Pregnancy

Abstract 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.

Uterine MHC Class I Molecules and β2-Microglobulin Are Regulated by Progesterone and Conceptus Interferons during Pig Pregnancy

MHC class I molecules and β2-microglobulin (β2m) are membrane glycoproteins that present peptide Ags to TCRs, and bind to inhibitory and activating receptors on NK cells and other leukocytes. They are involved in the discrimination of self from non-self. Modification of these molecules in the placenta benefits pregnancy, but little is known about their genes in the uterus. We examined the classical class I swine leukocyte Ags (SLA) genes SLA-1, SLA-2, and SLA-3, the nonclassical SLA-6, SLA-7, and SLA-8 genes, and the β2m gene in pig uterus during pregnancy. Uterine SLA and β2m increased in luminal epithelium between days 5 and 9, then decreased between days 15 and 20. By day 15 of pregnancy, SLA and β2m increased in stroma and remained detectable through day 40. To determine effects of estrogens, which are secreted by conceptuses to prevent corpus luteum regression, nonpregnant pigs were treated with estradiol benzoate, which did not affect the SLA or β2m genes. In contrast, progesterone, which is secreted by corpora lutea, increased SLA and β2m in luminal epithelium, whereas a progesterone receptor antagonist (ZK137,316) ablated this up-regulation. To determine effects of conceptus secretory proteins (CSP) containing IFN-δ and IFN-γ, nonpregnant pigs were implanted with mini-osmotic pumps that delivered CSP to uterine horns. CSP increased SLA and β2m in stroma. Cell-type specific regulation of SLA and β2m genes by progesterone and IFNs suggests that placental secretions control expression of immune regulatory molecules on uterine cells to provide an immunologically favorable environment for survival of the fetal-placental semiallograft.

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.