Establishment Of Pregnancy In Ruminants Research Articles

LncRNA STAT3-AS regulates endometrial receptivity via the STAT3 signaling pathway

Endometrial receptivity is critical for the successful establishment of pregnancy in ruminants. Interferon tau (IFNT) plays a key role in promoting embryo attachment by activating the Janus kinase/signal transducer and activator of transcription pathway, which induces the expression of a series of interferon-stimulated genes (ISGs). In our previous study, sequencing analysis of goat endometrial epithelial cells (gEECs) treated with 20 ng/mL IFNT revealed a differentially expressed long non-coding RNA located on the STAT3 antisense chain, which we designated STAT3-AS. The aim of this study was to investigate the role and mechanism of STAT3-AS in establishing endometrial receptivity in goats. The results showed that STAT3-AS was expressed in both the nucleus and cytoplasm of gEECs, and its expression increased significantly in the uterus on day 15 of pregnancy. STAT3-AS expression was upregulated in gEECs treated with IFNT alone or in combination with progesterone and estradiol. Knockdown of STAT3-AS using specific short interfering RNA significantly inhibited the expression of classical ISGs such as interferon-stimulated gene 15 and 2′,5′-oligodenylate synthetase 2, as well as uterine endometrial receptivity-related genes including homeobox gene A11, integrin beta 3, and vascular endothelial growth factor. Moreover, gEEC proliferation and the STAT3 pathway were suppressed in the absence of STAT3-AS. However, pretreatment with the STAT3 activator RO8191 restored the effect of silencing STAT3-AS on endometrial receptivity. Overall, these results suggest that STAT3-AS is an important regulator of endometrial receptivity in goats and that it regulates endometrial receptivity through the STAT3 pathway.

Interferon-τ -induced ISG15-AS regulates endometrial receptivity during early goat pregnancy

Endometrial receptivity is a critical process for the successful establishment of pregnancy in ruminants. However, the biological role of long non-coding RNAs (lncRNAs) in the development of endometrial receptivity is poorly understood. In this study, we performed RNA-seq analysis of immortalised goat endometrial epithelial cells (gEECs) treated with interferon-τ (IFNT). Transcriptome profiles showed that 8069 high-confidence putative lncRNAs, including 6498 intronic lncRNA transcripts, 1078 lincRNAs and 493 antisense lncRNAs were identified in gEECs with or without IFNT treatment. Functional clustering analysis was performed by using cis and trans lncRNAs prediction. GO and KEGG analyses revealed that differentially expressed lncRNAs may regulate tissue remodelling and immune responses. Subsequently, six of the 21 differentially expressed antisense lncRNAs were validated using qRT-PCR. Through functional screening and co-expression analysis of lncRNAs in gEECs, we identified that ISG15-AS was mainly expressed in the luminal and glandular epithelium on days 5 and 15 and was strongly upregulated on day 18 of pregnancy in vivo. Similarly, ISG15-AS was abundant in the nucleus and cytoplasm, and was significantly upregulated after treatment with IFNT in gEECs. In addition, ISG15 is an IFNT-responsive gene, that displayed an evident increase in vivo and in vitro. Moreover, sense ISG15 was significantly upregulated following ISG15-AS silencing. The key genes related to ISGylation and endometrial receptivity in gEECs dramatically increased after ISG15-AS inhibition. Collectively, our results indicate that a novel antisense lncRNA, ISG15-AS, may be important in regulating endometrial receptivity through ISGylation.

Galectin-1 induces gene and protein expression related to maternal-conceptus immune tolerance in bovine endometrium†.

Conceptus secretory factors include galectins, a family of carbohydrate binding proteins that elicit cell adhesion and immune suppression by interacting with intracellular and extracellular glycans. In rodents, galectin-1 (LGALS1) promotes maternal-fetal immune tolerance in the decidua through expansion of tolerogenic cluster of differentiation 11c (CD11c) positive dendritic cells, increased anti-inflammatory interleukin (IL)-10, and activation of forkhead box P3 (FOXP3) positive regulatory T cells (Treg). This study characterized galectin expression in early ruminant conceptuses and endometrium. We also tested the effect of recombinant bovine LGALS1 (rbLGALS1) and progesterone (P4) on endometrial expression of genes and protein related to maternal-conceptus immune tolerance in cattle. Elongating bovine and ovine conceptuses expressed several galectins, particularly, LGALS1, LGALS3, and LGALS8. Within bovine endometrium, expression of LGALS3, LGALS7, and LGALS9 was greater on Day 16 of pregnancy compared to the estrous cycle. Within ovine endometrium, LGALS7 was greater during pregnancy compared to the estrous cycle and endometrium of pregnant sheep tended to have greater LGALS9 and LGALS15. Expression of endometrial LGALS4 was less during pregnancy in sheep. Treating bovine endometrium with rbLGALS1 increased endometrial expression of CD11c, IL-10, and FOXP3, within 24h. Specifically, within caruncular endometrium, both rbLGALS1 and P4 increased FOXP3, suggesting that both ligands may promote Treg expansion. Using IHC, FOXP3+ cells with a leukocyte phenotype were localized to the bovine uterine stratum compactum near the uterine surface and increased in response to rbLGALS1. We hypothesize that galectins have important functions during establishment of pregnancy in ruminants and bovine conceptus LGALS1 and luteal P4 confer mechanisms of maternal-conceptus immune tolerance in cattle.

Analysis of novel embryonic factors of cattle and effects on endometrial cells in vitro

Interferon tau (IFNT) is thought to have essential functions in maternal recognition and establishment of pregnancy in ruminants. There, however, is a lack of research on embryonic factors that affect pregnancy other than IFNT. The present study was conducted to determine what are other embryo-derived factors involved in pregnancy recognition and to identify effects on endometrial cells using an in vitro culture system. With use of LC–MS/MS procedures to evaluate the supernatant of elongating embryos of cattle in culture, there was detection of 78 secretary proteins including five cytokines and two growth factors. Then there was analysis for up-regulated genes using ingenuity pathway procedure, IFNT and MIF were identified as upstream regulators of 37 and five genes, respectively. The mRNA transcript of MIF receptors was identified in endometrial cells, however, not in embryos. Among genes induced by MIF, CCL2, IL7 and IL23A transcripts were identified in endometrial cells. When endometrial cells were treated with interferon alpha (IFNA) and MIF, the CCL2 transcript was in a larger abundance of endometrial epithelial and polymorphonuclear neutrophil cells, and there was a larger abundance of there mRNA transcripts as a result of MIF treatment of peripheral blood mononuclear cells. In conclusion, MIF secreted by elongating embryos of cattle synergistically regulates relative abundances of specific mRNA transcripts of endometrial cells when there is treatment with IFNA, indicating further there are several factors other than IFNT that have effects on gene expression in the endometrium during early stages of gestation in cattle.

Fetal-maternal interactions during the establishment of pregnancy in ruminants

This review integrates established information with new insights into molecular and physiological mechanisms responsible for events leading to pregnancy recognition, endometrial receptivity, and implantation with emphasis on sheep. After formation of the corpus luteum, progesterone acts on the endometrium and stimulates blastocyst growth and elongation to form a filamentous conceptus (embryo/fetus and associated extraembryonic membranes). Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that endometrial epithelial secretions are essential for peri-implantation blastocyst survival and growth. The elongating sheep conceptus secretes interferon tau (IFNT) that acts on the endometrium to inhibit development of the luteolytic mechanism by inhibiting transcription of the estrogen receptor alpha (ESR1) gene in the luminal (LE) and superficial ductal glandular (sGE) epithelia, which prevents estrogen-induction of oxytocin receptors (OXTR) and production of luteolytic prostaglandin F2-alpha pulses. Progesterone downregulates its receptors (PGR) in LE and then GE, correlating with a reduction of anti-adhesive MUC1 (mucin glycoprotein one) and induction of secreted LGALS15 (galectin 15) and SPP1 (secreted phosphoprotein one), that are proposed to regulate trophectoderm growth and adhesion. IFNT acts on the LE to induce WNT7A (wingless-type MMTV integration site family member 7A) and to stimulate LGALS15, CTSL (cathepsin L), and CST3 (cystatin C), which may regulate conceptus development and implantation. During the peri-implantation period, trophoblast giant binucleate cells (BNC) begin to differentiate from mononuclear trophectoderm cells, migrate and then fuse with the uterine LE as well as each other to form multinucleated syncytial plaques. Trophoblast giant BNC secrete chorionic somatomammotropin (CSH1 or placental lactogen) that acts on the endometrial glands to stimulate their morphogenesis and differentiated function. The interactive, coordinated and stage-specific effects of ovarian and placental hormones regulate endometrial events necessary for fetal-maternal interactions and successful establishment of pregnancy.

Insights into conceptus elongation and establishment of pregnancy in ruminants.

This review integrates established and new information on the factors and pathways regulating conceptus-endometrial interactions, conceptus elongation and establishment of pregnancy in sheep and cattle. Establishment of pregnancy in domestic ruminants begins at the conceptus stage (embryo or fetus and associated extra-embryonic membranes) and includes pregnancy recognition signalling, implantation and the onset of placentation. Survival and growth of the preimplantation blastocyst and elongating conceptus require embryotrophic factors (amino acids, carbohydrates, proteins, lipids and other substances) provided by the uterus. The coordinated and interactive actions of ovarian progesterone and conceptus-derived factors (interferon-τ and prostaglandins) regulate expression of elongation- and implantation-related genes in the endometrial epithelia that alter the uterine luminal milieu and affect trophectoderm proliferation, migration, attachment, differentiation and function. A comparison of sheep and cattle finds both conserved and non-conserved embryotrophic factors in the uterus; however, the overall biological pathways governing conceptus elongation and establishment of pregnancy are likely conserved. Given that most pregnancy losses in ruminants occur during the first month of pregnancy, increased knowledge is necessary to understand why and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency.

Novel concepts on the role of prostaglandins on luteal maintenance and maternal recognition and establishment of pregnancy in ruminants

In ruminants, the corpus luteum (CL) of early pregnancy is resistant to luteolysis. Prostaglandin (PG)E2 is considered a luteoprotective mediator. Early studies indicate that during maternal recognition of pregnancy (MRP) in ruminants, a factor(s) from the conceptus or gravid uterus reaches the ovary locally through the utero-ovarian plexus (UOP) and protects the CL from luteolysis. The local nature of the embryonic antiluteolytic or luteoprotective effect precludes any direct effect of a protein transported or acting between the gravid uterus and CL in ruminants. During MRP, interferon tau (IFNT) secreted by the trophoblast of the conceptus inhibits endometrial pulsatile release of PGF2α and increases endometrial PGE2. Our recent studies indicate that (1) luteal PG biosynthesis is selectively directed toward PGF2α at the time of luteolysis and toward PGE2 at the time of establishment of pregnancy (ESP); (2) the ability of the CL of early pregnancy to resist luteolysis is likely due to increased intraluteal biosynthesis and signaling of PGE2; and (3) endometrial PGE2 is transported from the uterus to the CL through the UOP vascular route during ESP in sheep. Intrauterine co-administration of IFNT and prostaglandin E2 synthase 1 (PGES-1) inhibitor reestablishes endometrial PGF2α pulses and regresses the CL. In contrast, intrauterine co-administration of IFNT and PGES-1 inhibitor along with intraovarian administration of PGE2 rescues the CL. Together, the accumulating information provides compelling evidence that PGE2 produced by the CL in response to endometrial PGE2 induced by pregnancy may counteract the luteolytic effect of PGF2α as an additional luteoprotective mechanism during MRP or ESP in ruminants. Targeting PGE2 biosynthesis and signaling selectively in the endometrium or CL may provide luteoprotective therapy to improve reproductive efficiency in ruminants.

The role of progesterone and conceptus-derived factors in uterine biology during early pregnancy in ruminants

This review integrates established and new information on the role of progesterone, interferon tau (IFNT), and prostaglandins in uterine biology of ruminants. Establishment of pregnancy in ruminants encompasses growth of the posthatching blastocyst, elongation of the conceptus (embryo and extraembryonic membranes), and suppression of the endometrial luteolytic mechanism to maintain progesterone production by the ovary. Conceptus elongation involves exponential increases in length of the trophectoderm for pregnancy recognition signaling, implantation, and establishment of pregnancy. Pregnancy recognition signaling is accomplished by IFNT from the trophectoderm that has a paracrine antiluteolytic effect to inhibit upregulation of oxytocin receptors in the endometrial epithelia, thereby inhibiting production of luteolytic PGF2α pulses by the uterus. Survival and growth of the preimplantation blastocyst and elongating conceptus clearly requires embryotrophic factors (AA, carbohydrates, proteins, lipids, and other substances) in the uterine lumen. Individual, interactive, and coordinated actions of progesterone, IFNT, and prostaglandins regulate expression of elongation- and implantation-related genes in the endometrial epithelia that, in turn alter the uterine luminal histotroph and govern conceptus survival and growth. An increased knowledge of progesterone biology and conceptus-endometrial interactions is necessary to understand and elucidate the causes of pregnancy loss and provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency in ruminants.

Implantation and Establishment of Pregnancy in Ruminants.

The establishment of pregnancy in ruminants occurs during the peri-implantation period and involves the suppression of the endometrial luteolytic mechanism to maintain progesterone production by the corpus luteum (CL). Reciprocal interactions between the elongating conceptus (embryo/fetus and associated extraembryonic membranes) and endometrium culminate in implantation. Antiluteolytic effects of the conceptus are due to the production of interferon tau (IFNT) by the trophoblast that has a paracrine effect to inhibit the upregulation of oxytocin receptors in the endometrial epithelia, thereby disrupting uterine release of luteolytic prostaglandin F2 alpha (PGF) pulses. Additionally, IFNT is released into the uterine vein and has endocrine actions to induce ISGs in peripheral tissues. For example, IFNT may induce luteal resistance to PGF, thereby ensuring survival of the CL and maintenance of pregnancy. Survival of the blastocyst and elongation of the conceptus requires embryotrophic factors from the epithelia of the uterus, and those embryotrophic factors are regulated by ovarian progesterone as well as conceptus-derived factors including IFNT and prostaglandins. This review provides new concepts on mechanisms of the establishment of pregnancy and implantation in ruminants with emphasis on conceptus-maternal signaling associated with elongation of the blastocyst and endometrial responses to the presence of a conceptus.

Intrauterine coadministration of ERK1/2 inhibitor U0126 inhibits interferon TAU action in the endometrium and restores luteolytic PGF2alpha pulses in sheep.

In ruminants, prostaglandin F2 alpha (PGF2alpha) is synthesized and released in a pulsatile pattern from the endometrial luminal epithelial (LE) cells during the process of luteolysis. Interferon tau (IFNT) is a Type 1 IFN secreted by the trophoblast cells of the developing conceptus. IFNT acts locally on endometrial LE cells to inhibit pulsatile releases of PGF2alpha and thus establish an endocrine environment for recognition of pregnancy. Cell signaling pathways through which IFNT stimulates expression of multiple genes or proteins in endometrial LE are largely unknown. Results of the present investigation indicate that intrauterine administration of IFNT inhibits pulsatile release of PGF2alpha, while coadministration IFNT and ERK 1/2 inhibitor U0126 restores luteolytic PGF2alpha pulses in sheep. IFNT increases phosphorylation of ERK1/2 proteins and increases its interaction with PGT proteins in endometrial LE. Blockade of ERK1/2 pathways inhibits IFNT action, decreases pERK1/2 and PGT protein interactions, and re-establishes the spatial expression of the oxytocin receptor protein completely and the estrogen receptor protein partially without modulating the expression of interferon regulatory factor-2 (IRF-2) protein in endometrial LE. IFNT does not decrease expression of COX-2, PGDH, or PGT protein in endometrial LE. Our results provide important new insights into IFNT signaling and the molecular endocrine control of PGF2alpha release at the time of establishment of pregnancy in ruminants. This novel IFNT-ERK1/2 signaling module needs to be explored in future studies to understand molecular and cellular mechanisms of IFNT action in endometrial LE in ruminants.

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE 2) plays pleiotropic roles at fetal–maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12–16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE 2 signaling in endometrial functions and establishment of pregnancy in ruminants.

Intrauterine Infusion of Interferon Tau Selectively Directs Intraluteal Prostaglandin Biosynthesis Towards PGE2 and Activates EP2 and EP4-Mediated Signaling in the Corpus Luteum at the Time of Establishment of Pregnancy in Ruminants.

Intrauterine Infusion of Interferon Tau Selectively Directs Intraluteal Prostaglandin Biosynthesis Towards PGE2 and Activates EP2 and EP4-Mediated Signaling in the Corpus Luteum at the Time of Establishment of Pregnancy in Ruminants.

Progesterone Regulation of Endometrial Gene Expression in the Early Pregnant Ovine Uterus.

Progesterone Regulation of Endometrial Gene Expression in the Early Pregnant Ovine Uterus. (August 2011) Megan A. Minten, B.S., North Dakota State University Co-Chairs of Advisory Committee: Dr. Thomas E. Spencer Dr. Fuller W. Bazer Establishment of pregnancy in ruminants requires blastocyst development to form an elongated filamentous conceptus that produces interferon tau (IFNT), the pregnancy recognition signal, and initiate implantation. Blastocyst growth and development is dependent upon secretions from the uterine endometrium. An early increase in post-ovulatory circulating levels of progesterone (P4) stimulates blastocyst growth and conceptus elongation in ruminants. Microarray analysis was used to identify candidate P4-regulated genes and regulatory networks in the endometrium that govern peri-implantation blastocyst/conceptus growth and development. The first study was conducted to validate effects of P4 and/or pregnancy on expression of candidate genes identified by microarray analysis. The genes included: ANGPTL3, CHGA, CLEC4E, CXCL14, EFNA1, EFNB1, FABP3, IFNG, IL6, LGALS3, PTH, RBP4, SLIT2, SLIT3, and VWF. Early P4 treatment up-regulated CXCL14 gene expression in Day 9 ovine endometrium compared to control endometrium, and FABP3, IFNG, IL6 and LGALS3 in Day 12 early P4-treated ovine endometrium. Expression of ANGPTL3, CHGA, CXCL14, EFNA1, EFNB1, LGALS3 and RBP4 was affected by day of pregnancy. Treatment of ewes with P4+RU486 (P4 receptor antagonist) reduced expression of ANGPTL3, CHGA, EFNA1, EFNB1, FABP3, IFNG, IL6, LGALS3, RBP4, and SLIT2, SLIT3 and VWF in comparison to Day 12 P4treated endometrium.

Interferon Tau Regulates PGF2α Release from the Ovine Endometrial Epithelial Cells via Activation of Novel JAK/EGFR/ERK/EGR-1 Pathways

In ruminants, pulsatile release of prostaglandin F2α (PGF(2α)) from the endometrium is transported to the ovary and induces luteolysis thereby allowing new estrous cycle. Interferon tau (IFNT), a type 1 IFN secreted by the trophoblast cells of the developing conceptus, acts on endometrial luminal epithelial (LE) cells and inhibits pulsatile release of PGF(2α) and establishes pregnancy. One of the unknown mechanisms is that endometrial pulsatile release of PGF(2α) is inhibited whereas basal release of PGF(2α) is increased in pregnant compared with nonpregnant sheep. We have recently found that pulsatile release of PGF(2α) from the endometrium is regulated by prostaglandin transporter (PGT)-mediated mechanisms. We hypothesize that modulation in the endometrial pulsatile vs. basal release of PGF(2α) likely requires PGT-mediated selective transport, and IFNT interacts with PGT protein and modulates pulsatile vs. basal release of PGF(2α). The new findings of the present study are: 1) IFNT activates novel JAK-SRC kinase-EGFR-RAS-RAF-ERK1/2-early growth response (EGR)-1 signaling module in LE cells; 2) IFNT increases interactions between PGT and ERK1/2 or EGR-1 proteins and alters phosphorylation of PGT protein; 3) IFNT precludes action of protein kinase C and Ca(2+) on PGT function; and 4) IFNT inhibits 80% PGT-mediated but not 20% simple diffusion-mediated release of PGF(2α) from the endometrial LE cells through this novel signaling module. The results of the present study provide important new insights on IFNT signaling and molecular control of PGT-mediated release of PGF(2α) and unravel the underlying mechanisms responsible for the increased basal release of PGF(2α) at the time of establishment of pregnancy in ruminants.

HSD11B1, HSD11B2, PTGS2, and NR3C1 Expression in the Peri-Implantation Ovine Uterus: Effects of Pregnancy, Progesterone, and Interferon Tau1

Establishment of pregnancy in ruminants requires conceptus elongation and production of interferon tau (IFNT), the pregnancy recognition signal that maintains the corpus luteum and progesterone (P4) secretion. The enzymes hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) and HSD11B2 catalyze the interconversion of inactive cortisone and active cortisol, which is a biologically active glucorticoid and ligand for the receptor subfamily 3, group C, member 1 (glucocorticoid receptor) (NR3C1). The activity of HSD11B1 is stimulated by P4, prostaglandins, and cortisol. These studies determined the effects of pregnancy, P4, and IFNT on HSD11B1, HSD11B2, prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) (PTGS2), and nuclear NR3C1 in the ovine uterus. Endometrial HSD11B1 mRNA levels were more abundant between Days 12 and 16 of pregnancy than the estrous cycle, and HSD11B1 and PTGS2 expression in the endometrial luminal and superficial glandular epithelia was coincident with conceptus elongation. HSD11B1 mRNA was very low in the conceptus, whereas HSD11B2 mRNA was abundant in the conceptus but not in the uterus. Treatment of ewes with P4 induced, and intrauterine infusions of IFNT modestly stimulated, HSD11B1 expression in the endometrial luminal and superficial glandular epithelia. In all of the studies, HSD11B1 and PTGS2 expression was coincident in the endometrial epithelia, and NR3C1 was present in all endometrial cell types. Collectively, these results support hypotheses that endometrial epithelial HSD11B1 expression is induced by P4 as well as stimulated by IFNT and PTGS2-derived prostaglandins and that HSD11B1-regenerated cortisol acts via NR3C1 to regulate ovine endometrial functions during early pregnancy.

Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation

Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern preimplantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of preimplantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and preimplantation blastocyst growth and development in sheep.

Fetal-maternal interactions during the establishment of pregnancy in ruminants

This review integrates established information with new insights into molecular and physiological mechanisms responsible for events leading to pregnancy recognition, endometrial receptivity, and implantation with emphasis on sheep. After formation of the corpus luteum, progesterone acts on the endometrium and stimulates blastocyst growth and elongation to form a filamentous conceptus (embryo/fetus and associated extraembryonic membranes). Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that endometrial epithelial secretions are essential for peri-implantation blastocyst survival and growth. The elongating sheep conceptus secretes interferon tau (IFNT) that acts on the endometrium to inhibit development of the luteolytic mechanism by inhibiting transcription of the estrogen receptor alpha (ESR1) gene in the luminal (LE) and superficial ductal glandular (sGE) epithelia, which prevents estrogen-induction of oxytocin receptors (OXTR) and production of luteolytic prostaglandin F2-alpha pulses. Progesterone downregulates its receptors (PGR) in LE and then GE, correlating with a reduction of anti-adhesive MUC1 (mucin glycoprotein one) and induction of secreted LGALS15 (galectin 15) and SPP1 (secreted phosphoprotein one), that are proposed to regulate trophectoderm growth and adhesion. IFNT acts on the LE to induce WNT7A (wingless-type MMTV integration site family member 7A) and to stimulate LGALS15, CTSL (cathepsin L), and CST3 (cystatin C), which may regulate conceptus development and implantation. During the peri-implantation period, trophoblast giant binucleate cells (BNC) begin to differentiate from mononuclear trophectoderm cells, migrate and then fuse with the uterine LE as well as each other to form multinucleated syncytial plaques. Trophoblast giant BNC secrete chorionic somatomammotropin (CSH1 or placental lactogen) that acts on the endometrial glands to stimulate their morphogenesis and differentiated function. The interactive, coordinated and stage-specific effects of ovarian and placental hormones regulate endometrial events necessary for fetal-maternal interactions and successful establishment of pregnancy.

Estrogen Regulates Transcription of the Ovine Oxytocin Receptor Gene through GC-Rich SP1 Promoter Elements

Establishment of pregnancy in ruminants results from paracrine signaling by interferon tau (IFNT) from the conceptus to uterine endometrial luminal epithelia (LE) that prevents release of luteolytic prostaglandin F(2alpha) pulses. In cyclic and pregnant ewes, progesterone down-regulates progesterone receptor (PGR) gene expression in LE. In cyclic ewes, loss of PGR allows for increases in estrogen receptor alpha (ESR1) and then oxytocin receptor (OXTR) gene expression followed by oxytocin-induced prostaglandin F(2alpha) pulses. In pregnant ewes, IFNT inhibits transcription of the ESR1 gene, which presumably inhibits OXTR gene transcription. Alternatively, IFNT may directly inhibit OXTR gene transcription. The 5' promoter/enhancer region of the ovine OXTR gene was cloned and found to contain predicted binding sites for activator protein 1, SP1, and PGR, but not for ESR1. Deletion analysis showed that the basal promoter activity was dependent on the region from -144 to -4 bp that contained only SP1 sites. IFNT did not affect activity of the OXTR promoter. In cells transfected with ESR1, E2, and ICI 182,780 increased promoter activity due to GC-rich SP1 binding sites at positions -104 and -64. Mutation analyses showed that the proximal SP1 sites mediated ESR1 action as well as basal activity of the promoter. In response to progesterone, progesterone receptor B also increased OXTR promoter activity. SP1 protein was constitutively expressed and abundant in the LE of the ovine uterus. These results support the hypothesis that the antiluteolytic effects of IFNT are mediated by direct inhibition or silencing of ESR1 gene transcription, thereby precluding ESR1/SP1 from stimulating OXTR gene transcription.

A Novel Integrated View of the Role of Prostaglandins for Recognition and Establishment of Pregnancy in Ruminants

Prostaglandins are important regulators of fertility in most mammals. Prostaglandins F 2 α (PGF 2 α) and E 2 (PGE 2 ) appear as the most active PGs in reproduction and often mediate opposite reactions at the time of establishment and termination of pregnancy. These prostaglandins and possibly also prostacyclin (PGI 2 ) are responsible for the regulation of luteolysis, implantation, and parturition. In cows, endometrial PGF 2 α is the luteolytic hormone whereas PGE, may favour maternal recognition of pregnancy. During the bovine oestrous cycle, days 16-17 are considered as the for either maternal recognition of pregnancy (MRP), in the presence of a viable embryo or luteolysis and return to a new oestrous cycle. In ruminants, trophoblastic interferon tau (IFNt) is known as the pregnancy recognition signal. We have found over the years that the mechanism of MRP is more complex than initially anticipated and involves regulation of PG action at the level of biosynthesis, catabolism, transport and receptors. For instance, in the bovine endometrium where the production of PGF 2 α is abundant and tightly modulated, we have found that the PGF synthase responsible for its production is not the enzyme previously identified in lung or liver but rather an aldose reductase with a new function, AKR1B5. This enzyme and others like PGH synthase 1 and 2 and PGE synthases are also tightly modulated to control the relative concentration of PGF 2 α and PGE 2 . Moreover, inspite of their lipid structure, PGs diffuse pooriy through plasma membranes because they are charged negatively. We have identified a novel prostaglandin transporter bPGT that is expressed in the genital tract in a spatio-temporal manner in order to allow the transfer of PGs produced at critical periods of the oestrous cycle or during pregnancy. Finally, the action of individual PGs is regulated at the level of their receptors. A good understanding of PG action in the regulation of reproductive function requires that we consider all these factors. We intend to present here an integrated view of PG action on the function of the female genital tract at the critical period of recognition of pregnancy.

Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants

AbstractThe effects of high and low dietary dietary intake on reproduction in female cattle and sheep will be considered at the level of the pituitary gland, ovary and uterus. In sheep, increased dietary intake for a relatively short time will increase ovulation rate, by increasing gonadotropin secretion. Dietary intake can affect steroids such as progesterone and also intra-follicular concentrations of some growth factors such as IGF-1 and IGF-2. The effects of altered energy intake on gonadotropins and steroids in cattle are not as repeatable as those in sheep but follicular growth rates can be altered. High nutrition has a negative effect on oocyte quality, with animals onad-libitumhigh energy diets particularly at risk. Overfeeding can decrease embryo quality in both sheep and cattle and it appears that this results from changes primarily at the level of the follicle or oocyte. Restricted nutrition for a short time will enhance pregnancy rates in cattle; most of this benefit appears to occur if food is restricted before insemination. Thus feeding levels before mating are particularly important to subsequent reproductive success. High dietary crude protein may decrease pregnancy rate in lactating cows. In ewes and heifers supplementation with urea failed to have any effect on pregnancy rates when good quality embryos were transferred to recipient animals exposed to high dietary crude protein. In donor ewes there were adverse effects on early embryo development following urea treatment, suggesting that the mechanism affecting the reproductive process was primarily operating at the level of the oocyte. Collectively, these data identify the overall deleterious effects of high dietary intake and excess crude protein on fertility and highlight the importance of dietary intake before ovulation on the likelihood of establishing a viable pregnancy.