Peri-implantation Period Of Pregnancy Research Articles

Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, invivo (high developmental potential (IV)), invitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to invivo versus invitro and invivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between invitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between invivo and cloned conceptuses, while only 6 miRNAs were different between invivo and invitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium invivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

Pig conceptuses utilize extracellular vesicles for IFNG-mediated paracrine communication with the endometrium.

Interferon-gamma (IFNG) is a pro-inflammatory cytokine secreted by the porcine conceptus (embryo and extra-embryonic membranes) during the peri-implantation period of pregnancy. IFNG modifies the endometrial inflammatory immune response and is required for the implantation and survival of the conceptus. It is not known how IFNG from the conceptus trophectoderm is transported across the endometrial luminal epithelium (LE). In the present study, immunofluorescence analyses detected immunoreactive IFNG protein in both the trophectoderm and endometrial LE on Day 15 of pregnancy, while our previous research localized IFNG mRNA only to conceptus trophectoderm. Using minced endometrial explants to disrupt the barrier posed by the intact endometrial LE, treatment with recombinant IFNG induced the expression of genes that were not induced when IFNG was infused into the uterine lumen in vivo by McLendon et al. (Biology of Reproduction. 2020;103(5):1018-1029). We hypothesized that during pregnancy extracellular vesicles (EVs) serve as intercellular signaling vehicles to transport conceptus-derived IFNG across the intact endometrial LE and into the stromal compartment of the uterus. Western blotting detected the presence of IFNG in EVs isolated from the uterine fluid of pregnant gilts, but not nonpregnant gilts. Real-time PCR demonstrated increased expression of IFNG-stimulated genes in EV-treated endometrial explants and EV-mediated IFNG transport was confirmed in whole uterine sections cultured with EVs from day 15 of pregnancy. These results suggest that EVs are involved in IFNG transport across the endometrial LE to enable paracrine communication between the conceptus and cells within the endometrial stroma.

Expression of circulating oar-miR-485-5p and oar-miR-493-5p during the estrous cycle and early pregnancy in ovine plasma.

In the current study, we aimed to assess the expression levels of two circulating microRNAs (miRNA) (oar-miR-485-5p and oar-miR-493-5p) in the ovine plasma during the peri-implantation. After mating, we collected the plasma samples from a total of 8 ewes on day 22 of pregnancy (P22; n = 4) and day 22 of the estrous cycle (C22; n=4). We used mature miRNA sequences for oar-miR-485-5p and oar-miR-493-5p out of one hundred fifty, which were retrieved from our microarray results of previous study. We showed that the miRNA expression of oar-miR-485-5p and oar-miR-493-5p were upregulated in P22 (P<0.05) when compared to C22. Those two miRNAs targeted 311 target genes in the peri-implantation period of pregnancy. Furthermore, we revealed 151 GO/pathway terms in biological process (BP) and 25 GO/pathway terms in molecular function (MF), while we demonstrated 13 GO/pathway terms in cellular component (CC). We revealed three hub genes as interleukin 2 (IL2), interleukin 18 (IL18), and C-X-C Motif Chemokine Ligand 10 (CXCL10). In conclusion, both miR-485-5p and oar-miR-493-5p have the potential to be a biomarker to understand peri-implantation of the ovine pregnancy in the aspect of pregnancy-reflected changes in maternal plasma.

43 Metabolomic Analysis of Uterine Luminal Fluid During the Peri-Implantation Period of Pregnancy in Pigs

Abstract During the peri-implantation period of pregnancy in pigs, the rapid elongation of conceptuses (embryonic/fetus and its extraembryonic membranes) is highly dependent on the composition of histotroph secreted from uterine luminal (LE) and glandular (GE) epithelial and stroma cells, as well as selective transport of nutrients. However, little is known about the metabolites in the uterine luminal fluid (ULF) associated with the uterine-conceptus communication during early pregnancy in pigs. Thus, this study was conducted to profile the metabolome in porcine ULF between days 10 and 16 of estrus cycle (C) and pregnancy (P). Gilts were observed for estrus and/or bred via artificial insemination at 12 and 24 h after onset of estrus (day 0). On days 10, 12, 14, or 16 of the estrous cycle and pregnancy (n = 6 gilts per day and status), uteri were flushed with 20 mL sterile PBS (pH 7.2) after gilts were subjected to a midventral laparotomy. Pregnancy was confirmed by the presence of one or more morphologically normal conceptuses. Recoverable uterine flushings (i.e., ULF) were then subjected to metabolomic analysis by LC-MS. Overall, 222 metabolites were detected in the ULF of which 102 were altered by status and 63 were altered by days (P &amp;lt; 0.05; fold change&amp;gt;1.5). Comparing to cyclic day 10 (10C), pregnancy stimulated increases in citric acid, lysine, arginine and other 19 metabolites in the ULF at pregnant day 10 (10P). At 12P when porcine conceptus initiates its morphological changes, ornithine, α-D-glucose, phenylalanine and other 13 metabolites were up-regulated as compared with 12C. At 14P when conceptus initiates implantation, 15 metabolites changed in ULF. At 16P, 123 metabolites were altered in ULF as compared with 16C. Interestingly, of 15 altered metabolites in ULF, 12 were downregulated including asparagine, nicotinamide riboside, and citrulline at 14P. Whereas 102 of 123 altered metabolites were upregulated including phosphorylcholine, 6-phosphate fructose and 6-phosphate glucose in ULF at 16P. To determine the absolute amount of amino acids in the ULF, we performed the targeted metabolomic analyses for 19 amino acids. As a result, arginine, asparagine, glutamic acid, glutamine, histidine, leucine/isoleucine, lysine, phenylalanine, proline, and valine were increased between 10P and 16P. They were less expressed in the ULF of estrus cycle as compared with pregnancy, and remained unchanged between 10C and 16C. Pathway analysis based on KEGG database indicating that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis have the highest degree of enrichment. These novel findings provide the foundation for future investigation of metabolomic and functional studies on both endometrium and conceptus required for elongation of porcine conceptus during peri-implantation period of pregnancy. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture

42 Adrenomedullin Stimulates Proliferation, Migration and Adhesion of Porcine Trophectoderm Cells Via CALCRL-AKT-TSC2-MTORC1 Cell Signaling Pathway

Abstract Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr1) isolated from day -12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via AKT-TSC2-MTOR cell signaling pathway in pTr1 cells. The pTr1 cells were cultured in DMEM/F12 medium with 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, 0.1 mM each for nutritionally nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 4 μg/mL insulin. Opti-MEM supplied with 2.5% (vol/vol) charcoal-stripped FBS was used for siRNA-mediated knockdown targeting non-treated control (siNTC) or specific genes including ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL). Cells were starved in FBS- and insulin-free medium for 24 hours before treatment. For proliferation assay, cell numbers were determined by staining with Janus-Green B after 48 h incubation. For migration assay, cells were treated with ADM after straight scratch in 6-well plates, and area of cell migration was calculated after 12 h treatment. For adhesion assay, cells were trypsinized in T-25 flasks and allowed for seeding in 96-well plates with density of 2×105 cells/0.2 mL/well, and the numbers of attached cells were determined after 12 h incubation. Western blot analyses were used to determine the expressions of target proteins at total and phosphorylated level. Porcine ADM at 10-7 M stimulated (P &amp;lt; 0.05) pTr1 cell proliferation, migration and adhesion by 1.4%-, 1.5%- and 1.2%-folds, respectively. These ADM-induced effects were abrogated (P &amp;lt; 0.05) by siADM and siCALCRL, as well as by rapamycin, the inhibitor of mechanistic target of rapamycin (MTOR). Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph act on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.

46 Adrenomedullin: A Novel Peptide Hormone for Uterine Receptivity and Conceptus Elongation in Pigs?

Abstract Adrenomedullin (ADM) is an evolutionarily conserved multi-functional peptide hormone in mammalian species. The implantation sites of conceptuses (embryo/fetus and its extra-embryonic membranes) in pregnant Adm+/− female mice are abnormally spaced leading to fetal crowding, intrauterine growth restriction (IUGR) and increases in pregnancy loss. As litter-bearing species, pigs exhibit a high incidence of early embryonic death (30-40%) and naturally occurring IUGR (15-25%), and significant numbers of stillborn piglets (3-9%). Therefore, it is imperative that the roles of ADM in regulation of uterine receptivity, as well as growth and development of conceptus in pigs be established. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components in uteri from cyclic and pregnant gilts, as well as conceptuses during the peri-implantation period of pregnancy. ADM receptor components include: calcitonin receptor-like receptor (CALCRL; G protein-coupled receptor bound by ADM), receptor activity modifying protein (RAMP2; dimerized with CALCRL to form ADM1 receptor) and RAMP3 (dimerized with CALCRL to form ADM2 receptor with lower specificity to ADM) and atypical chemokine receptor 3 (ACKR3; a decoy receptor that serves as a cell-autonomous molecular rheostat to dampen ADM signaling). Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P &amp;lt; 0.05), pregnant status (P &amp;lt; 0.01) and/or day x status (P &amp;lt; 0.05). Within porcine conceptuses, expression of CALCRL, RAMP2 and ACKR3 increased between days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line isolated from elongating porcine conceptuses recovered on day 2 post-breeding, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P &amp;lt; 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein (p-4EBP1) by 6.1- and 4.9-fold (P &amp;lt; 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation and placentation in pigs. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.

Uterine histotroph and conceptus development: III. Adrenomedullin stimulates proliferation, migration and adhesion of porcine trophectoderm cells via AKT-TSC2-MTOR cell signaling pathway.

Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10-7M stimulated (P < 0.05) pTr2 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of MTOR. Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph acts on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses.

Extracellular vesicles: Focus on peri-implantation period of pregnancy in pigs.

The establishment of cell-to-cell communication between the endometrium and the developing embryo is the most important step in successful mammalian pregnancy. Close interaction between the uterine luminal epithelium and trophoblast cells requires triggering timely molecular dialog for successful maternal recognition of pregnancy, embryo implantation, and placenta development. Quite recently, extracellular vesicles (EVs) carrying unique molecular cargo emerged as evolutionarily conserved mediators of cell-to-cell communication during early pregnancy. To date, the presence of EVs at the embryo-maternal interface has been demonstrated in numerous mammals, including domestic livestock, such as pigs. However, few studies have focused on revealing the mechanism of EV-mediated crosstalk between developing early embryos and receptive endometrium. Over the past years, it has appeared that understanding the role of EVs in mammalian reproduction can substantially improve our understanding of the biological challenges of successful reproductive performance. This review describes current knowledge of EVs, specifically in relation to the peri-implantation period in pigs, characterized by common features of embryo implantation and high embryonic mortality in mammals.

The ovine conceptus utilizes extracellular serine, glucose, and fructose to generate formate via the one carbon metabolism pathway.

Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4mM D-glucose + 2mM [U-13C]serine; 2) 6mM glycine + 4mM D-glucose + 2mM [U-13C]serine; 3) 4mM D-fructose + 2mM [U-13C]serine; 4) 6mM glycine + 4mM D-fructose + 2mM [U-13C]serine; 5) 4mM D-glucose + 4mM D-fructose + 2mM [U-13C]serine; or 6) 6mM glycine + 4mM D-glucose + 4mM D-fructose + 2mM [U-13C]serine. After 2h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells.

Effects of progesterone and interferon tau on ovine endometrial phosphate, calcium, and vitamin D signaling

Given recent reports of expression of postnatal mineral transport regulators at the maternal-conceptus interface during the peri-implantation period, this study tested the hypothesis that progesterone (P4) and interferon tau (IFNT) regulate phosphate, calcium, and vitamin D signaling in the ovine endometrium. Mature Rambouillet ewes (n= 24) were surgically fitted with intrauterine catheters on day 7 of the estrous cycle. Ewes received daily intramuscular injections of 50mg of P4 in corn oil vehicle and 75mg of progesterone receptor antagonist (RU486) in corn oil from days 8 to 15, and twice-daily intrauterine injections of either control proteins (CX) or IFNT (25μg/uterine horn/day) from days 11 to 15 resulting in four treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT. On day 16, ewes were hysterectomized. RU486 + P4 + CX treated ewes had lower concentrations of 25 (OH) D in plasma than P4 + CX treated ewes (P< 0.05). Endometria from ewes treated with IFNT had greater expression of FGF23 (P< 0.01), S100A9 (P< 0.05), and S100A12 (P= 0.05) mRNAs and lower expression of ADAM10 mRNA (P< 0.01) than of ewes treated with CX proteins. Expression of FGF23 mRNA was greater in endometria of ewes that received RU486 + P4 + IFNT than in ewes that received RU486 + P4 + CX (hormone × protein interaction, P< 0.05). The expression of S100G mRNA was greater in endometria of ewes that received P4 + IFNT compared to ewes that received RU486 + P4 + IFNT (P< 0.05; hormone × protein interaction, P< 0.01). These data implicate P4 and IFNT in the regulation of phosphate, calcium, and vitamin D signaling during the peri-implantation period of pregnancy and provide a platform for continued mechanistic investigations.

Progesterone and interferon tau regulate expression of polyamine enzymes during the ovine peri-implantation period†.

Progesterone (P4) and interferon tau (IFNT) are important for establishment and maintenance of pregnancy in ruminants. Agmatine and polyamines (putrescine, spermidine, and spermine) have important roles in the survival, growth, and development of mammalian conceptuses. This study tested the hypothesis that P4 and/or IFNT stimulate the expression of genes and proteins involved in the metabolism and transport of polyamines in the ovine endometrium. Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on Day 7 of the estrous cycle. They received daily intramuscular injections of 50mg P4 in corn oil vehicle and/or 75-mg progesterone receptor antagonist (RU486) in corn oil vehicle from Days 8-15, and twice daily intrauterine injections (25μg/uterine horn/day) of either control serum proteins (CX) or IFNT from Days 11-15, resulting in four treatment groups: (i) P4 + CX; (ii) P4 + IFNT; (iii) RU486 + P4 + CX; or (iv) RU486 + P4 + IFNT. On Day 16, ewes were hysterectomized. The total amounts of arginine, citrulline, ornithine, agmatine, and putrescine in uterine flushings were affected (P < 0.05) by P4 and/or IFNT. P4 increased endometrial expression of SLC22A2 (P < 0.01) and SLC22A3 (P < 0.05) mRNAs. IFNT affected endometrial expression of MAT2B (P < 0.001), SAT1 (P < 0.01), and SMOX (P < 0.05) mRNAs, independent of P4. IFNT increased the abundance of SRM protein in uterine luminal (LE), superficial glandular (sGE), and glandular epithelia (GE), as well as MAT2B protein in uterine LE and sGE. These results indicate that P4 and IFNT act synergistically to regulate the expression of key genes required for cell-specific metabolism and transport of polyamines in the ovine endometrium during the peri-implantation period of pregnancy.

Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells

BackgroundA number of studies have examined mRNA expression profiles of bovine endometrium at estrus and around the peri-implantation period of pregnancy. However, to date, these studies have been performed on the whole endometrium which is a complex tissue. Consequently, the knowledge of cell-specific gene expression, when analysis performed with whole endometrium, is still weak and obviously limits the relevance of the results of gene expression studies. Thus, the aim of this study was to characterize specific transcriptome of the three main cell-types of the bovine endometrium at day-15 of the estrus cycle.ResultsIn the RNA-Seq analysis, the number of expressed genes detected over 10 transcripts per million was 6622, 7814 and 8242 for LE, GE and ST respectively. ST expressed exclusively 1236 genes while only 551 transcripts were specific to the GE and 330 specific to LE. For ST, over-represented biological processes included many regulation processes and response to stimulus, cell communication and cell adhesion, extracellular matrix organization as well as developmental process. For GE, cilium organization, cilium movement, protein localization to cilium and microtubule-based process were the only four main biological processes enriched. For LE, over-represented biological processes were enzyme linked receptor protein signaling pathway, cell-substrate adhesion and circulatory system process.ConclusionThe data show that each endometrial cell-type has a distinct molecular signature and provide a significantly improved overview on the biological process supported by specific cell-types. The most interesting result is that stromal cells express more genes than the two epithelial types and are associated with a greater number of pathways and ontology terms.

Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses.

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

Adrenomedullin and Its Receptor Components in the Porcine Uterus and Peri-implantation Conceptuses

Abstract Adrenomedullin (ADM) is an evolutionarily conserved peptide hormone that regulates implantation, embryo spacing and placentation in humans and rodents. However, the potential roles of ADM in domestic animals, particularly pigs (as litter-bearing species), are not known. This study investigated expression patterns of ADM and its receptor components that include calcitonin receptor-like receptor (CALCRL), and receptor activity modifying protein (RAMP2) in porcine uteri and conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy when 30–40% of embryonic death loss occurs. Gilts were observed for estrus and/or bred via artificial insemination at 12 and 24 h after onset of estrus (Day 0). On D 10, 11, 12, 13, 14, 15, or 16 of the estrous cycle and pregnancy (n=6 gilts per day and status), uteri were flushed with 20 ml sterile PBS (pH 7.2) after gilts were subjected to a midventral laparotomy. Pregnancy was confirmed by the presence of one or more morphologically normal conceptuses. Real-time quantitative RT-PCR analyses revealed that ADM mRNA in the endometrium was elevated for pregnant than for cyclic gilts between D 10 and 16 of pregnancy (day x status, P &amp;lt; 0.05). In cyclic gilts, endometrial ADM mRNA increased 9.0-fold (P &amp;lt; 0.05) between D 10 and 16; whereas it increased 58.1-fold (P &amp;lt; 0.01) in pregnant gilts between D 10 and 16. As ADM receptors, expression of mRNAs for CALCRL and RAMP2 also increased (P &amp;lt; 0.05) 13.4- and 5.5-fold, respectively, in the porcine conceptuses between D 10 and 16 of pregnancy. Further, ELISA analyses showed that total recoverable ADM in the uterine flushings was greater (status, P &amp;lt; 0.0001) for pregnant than for cyclic gilts in which ADM increased 173.8-fold (P &amp;lt; 0.0001) between D 10 and 16 of pregnancy. These results indicate that ADM may play functional roles in survival, growth and development of the porcine conceptus during the peri-implantation period of pregnancy.

374 Early embryonic loss is an important mechanism for maximizing litter size in the pig

Abstract Litter size of commercial dam lines of pigs has seen a significant increase over the last 20 years. Although the current large litter size of gilts and sows can be attributed to improved genetic selection and nutrition, much of the increase in total number born has occurred through the continued increase in ovulation rate over the last four decades. Ovulation rate has increased from 14 in the 80’s to today’s average of 20–25 in commercial dam lines. However, high ovulation rate not only causes increased competition between embryos (uterine crowding), but the number of potential piglets born can out strip the number available teats. In addition, fetal crowding can reduce fetal growth and comprise health and survivability of lighter weight piglets at birth. Since there is usually minimal embryo loss observed up to blastocyst formation and hatching, the majority (20 to 30%) of embryonic loss occurs during Days 10 to 30 of gestation, which encompasses a critical period of conceptus development and differentiation, pregnancy recognition signaling, and onset of implantation. This period of early embryo loss is actually essential for establishing adequate uterine space to support fetal survival for a large litter. The period of rapid conceptus elongation on Day 12 of pregnancy provides a key “selection point” for regulating potential litter size in the pig. Establishing the fundamental roles of conceptus- and uterine-secreted factors is an essential step to develop strategies to increase placental and fetal growth to improve overall health and survivability of piglets before and after birth. During the peri-implantation period of pregnancy, porcine conceptuses produce interleukin-1B2, estrogens, prostaglandins and interferons. CRISPR/Cas9 genomic engineering technology has provided a direct method to evaluate the role of key pig conceptus genes. Loss-of-function studies have increased our understanding of the multiplicity of uterine/conceptus factors that are involved with maintenance of pregnancy.

Loss of ITGB3 in ovine conceptuses decreases conceptus expression of NOS3 and SPP1: implications for the developing placental vasculature†.

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the β3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo.

Effect of induced rumination on cortisol habituation to repeated acute stress

The peri-implantation period of pregnancy is critical for conceptus development, implantation, and signaling for establishment of pregnancy. This study evaluated the effects of bisphenol A (BPA) on proliferation, adhesion, and migration of porcine trophectoderm (pTr2) cells, expression of transporters of arginine and synthesis of amino acids. All concentrations of BPA decreased proliferation and adhesion of pTr2 cells after 96 h compared to the control group. Lower concentrations of BPA (1 × 10−9, 1 × 10-8, 10-7M) increased (P < 0.05), but higher concentrations of BPA (1 × 10-5, 1 × 10-4 M) decreased migration of pTr2 cells. BPA increased expression of SLC7A1 mRNA at lower concentrations (1 × 10−9 to 1 × 10-6M) and SL7A6, another cationic acid transporter, at higher concentrations (1 × 10-5, 1 × 10-4 M). BPA also down-regulated the expression of IGF1 and IGF1 receptor at concentrations of 1 × 10-7 to 1 × 10-4 M compared to the control group. The expression of mRNAs for aquaporins (AQP) 3 and 4 were reduced at all concentrations of BPA, but at lower concentrations of BPA, (1 × 10−9 to 1 × 10-8M) expression of AQP9 mRNA increased and the expression of AQP11 was not affected by BPA (P > 0.05). There was an inhibitory effect of BPA on the release of synthesis of asparagine, threonine, taurine, tryptophan, and ornithine into the culture medium by pTr2 cells. Collectively, BPA adversely affected the expression of transporters for cationic amino acids like arginine, as well as AQPs, IGF1, and IGF1R associated with proliferation, migration, and adhesion of pTr2 cells. Those adverse effects would likely increase pregnancy losses during the peri-implantation period of pregnancy.

Effects of Bisphenol A on expression of genes related to amino acid transporters, insulin- like growth factor, aquaporin and amino acid release by porcine trophectoderm cells

The peri-implantation period of pregnancy is critical for conceptus development, implantation, and signaling for establishment of pregnancy. This study evaluated the effects of bisphenol A (BPA) on proliferation, adhesion, and migration of porcine trophectoderm (pTr2) cells, expression of transporters of arginine and synthesis of amino acids. All concentrations of BPA decreased proliferation and adhesion of pTr2 cells after 96 h compared to the control group. Lower concentrations of BPA (1 × 10−9, 1 × 10-8, 10-7M) increased (P < 0.05), but higher concentrations of BPA (1 × 10-5, 1 × 10-4 M) decreased migration of pTr2 cells. BPA increased expression of SLC7A1 mRNA at lower concentrations (1 × 10−9 to 1 × 10-6M) and SL7A6, another cationic acid transporter, at higher concentrations (1 × 10-5, 1 × 10-4 M). BPA also down-regulated the expression of IGF1 and IGF1 receptor at concentrations of 1 × 10-7 to 1 × 10-4 M compared to the control group. The expression of mRNAs for aquaporins (AQP) 3 and 4 were reduced at all concentrations of BPA, but at lower concentrations of BPA, (1 × 10−9 to 1 × 10-8M) expression of AQP9 mRNA increased and the expression of AQP11 was not affected by BPA (P > 0.05). There was an inhibitory effect of BPA on the release of synthesis of asparagine, threonine, taurine, tryptophan, and ornithine into the culture medium by pTr2 cells. Collectively, BPA adversely affected the expression of transporters for cationic amino acids like arginine, as well as AQPs, IGF1, and IGF1R associated with proliferation, migration, and adhesion of pTr2 cells. Those adverse effects would likely increase pregnancy losses during the peri-implantation period of pregnancy.

Interferon tau: Influences on growth and development of the conceptus

Interferon tau (IFNT), the pregnancy recognition signal secreted from trophectoderm cells of ruminant conceptuses abrogates the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for production of progesterone (P4). Importantly, IFNT, in concert with P4, also induces expression of genes in uterine luminal (LE) and superficial glandular (sGE) epithelia for transport and/or secretion of histotroph into the uterine lumen to support growth and development of the conceptus. For example, IFNT and P4 induce transporters responsible foer transport of glucose and arginine into the uterine lumen during the peri-implantation period of pregnancy. Arginine activates the mechanistic target of rapamycin (MTOR) nutrient sensing cell signaling pathway to stimulate proliferation, migration, differentiation and translation of mRNAs essential for growth and development of the conceptus. Glucose not utilized by the conceptus is converted to fructose and those two hexose sugars are metabolized via aerobic glycolysis to produce metabolites used in the hexosamine biosynthesis pathway, pathways for one-carbon metabolism, and pentose phosphate pathway for synthesis of ribose sugars and NADPH. Arginine is metabolized to nitric oxide (NO) that stimulates angiogenesis in uterine and placental tissues, and to polyamines required for many cellular functions critical for growth and development of the conceptus. In summary, IFNT and P4 regulate expression of genes for transport of select nutrients into the pregnant uterus during the peri-implantation period of pregnancy. Those nutrients are then metabolized via multiple metabolic pathways to not only provide ATP, but also substrates for synthesis of nucleotides, amino acids, co-factors required for growth, development, and survival of conceptuses during the peri-implantation period of pregnancy.

COPS5 negatively regulates goat endometrial function via the ERN1 and mTOR‐autophagy pathways during early pregnancy

In ruminant, adequate endometrial function is a major factor affecting implantation and economic efficiency. However, the precise mechanisms regulating goat endometrial function during the peri-implantation period of pregnancy are still unclear. Here, we investigated the functional role and signal transduction of the fifth component of the constitutive photomorphogenic-9 signalosome (COPS5) in the regulation of endometrial function in endometrial epithelial cells (EECs). Our results showed that hormones decreased COPS5 expression, and COPS5-mediated regulation of endometrial function. We also found that knockdown of COPS5 hindered EECs proliferation by the G1-phase cell cycle arrest. Hormones affected the activity of COPS5 through hormones receptors, while feedback from the expression of COPS5 regulated the transcription of the receptor. Moreover, knockdown of endoplasmic reticulum (ER) to nucleus signaling 1 (ERN1) via si-ERN1 partly inhibited endometrial function in shCOPS5 EECs. In addition, blocking the mTOR pathway by rapamycin promoted endometrial function in si-ERN1-transfected shCOPS5 EECs. Overall, these results suggest that COPS5 negatively regulates goat endometrial function via the ERN1 and mTOR-autophagy pathways and provide new insights into the mechanistic pathways of COPS5 during female reproductive development.