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.

Impact of surface receptors TLR2, CR3, and FcγRIII on Rhodococcus equi phagocytosis and intracellular survival in macrophages.

The virulence-associated protein A (VapA) produced by virulent Rhodococcus equi allows it to replicate in macrophages and cause pneumonia in foals. It is unknown how VapA interacts with mammalian cell receptors, but intracellular replication of avirulent R. equi lacking vapA can be restored by supplementation with recombinant VapA (rVapA). Our objectives were to determine whether the absence of the surface receptors Toll-like receptor 2 (TLR2), complement receptor 3 (CR3), or Fc gamma receptor III (FcγRIII) impacts R. equi phagocytosis and intracellular replication in macrophages, and whether rVapA restoration of virulence in R. equi is dependent upon these receptors. Wild-type (WT) murine macrophages with TLR2, CR3, or FcγRIII blocked or knocked out (KO) were infected with virulent or avirulent R. equi, with or without rVapA supplementation. Quantitative bacterial culture and immunofluorescence imaging were performed. Phagocytosis of R. equi was not affected by blockade or KO of TLR2 or CR3. Intracellular replication of virulent R. equi was not affected by TLR2, CR3, or FcγRIII blockade or KO; however, avirulent R. equi replicated in TLR2-/- and CR3-/- macrophages but not in WT and FcγRIII-/-. rVapA supplementation did not affect avirulent R. equi phagocytosis but promoted intracellular replication in WT and all KO cells. By demonstrating that TLR2 and CR3 limit replication of avirulent but not virulent R. equi and that VapA-mediated virulence is independent of TLR2, CR3, or FcγRIII, our study provides novel insights into the role of these specific surface receptors in determining the entry and intracellular fate of R. equi.

Understanding placentation in ruminants: a review focusing on cows and sheep.

Mammals differ regarding their placentae, but in all species placental trophoblasts interact intimately with the uterine endometrium to mediate the transfer of nutrients from the mother to the embryo/fetus through the closely juxtaposed microcirculatory systems of the uterus and placenta. Placentation in ruminants is intermediate between the non-invasive type, as observed in the epitheliochorial placenta of pigs, and the invasive type, as observed in the haemochorial placentae of mice and humans. In ruminants, placental trophoblast cells invade uterine endometrial tissue, but invasion is believed to be limited to the endometrial luminal epithelium (LE). In the LE there are varying degrees of syncytialisation among species, with syncytialisation being more extensive in sheep than cows. The hallmarks of placentation in ruminants include: (1) an extended period in which conceptuses (embryos and associated placental membranes) elongate and must be supported by secretions (histotroph) from the uterus; (2) a cascade involving an array of adhesion molecules that includes integrin-mediated attachment of the conceptus trophoblast to the endometrial LE for implantation; (3) syncytialisation of the developing early placenta, a process for which there is currently limited understanding; and (4) development of placentomes that define the cotyledonary placentae of cows and sheep, and provide haemotrophic support of fetal development.

Integrins and their potential roles in mammalian pregnancy.

Integrins are a highly complex family of receptors that, when expressed on the surface of cells, can mediate reciprocal cell-to-cell and cell-to-extracellular matrix (ECM) interactions leading to assembly of integrin adhesion complexes (IACs) that initiate many signaling functions both at the membrane and deeper within the cytoplasm to coordinate processes including cell adhesion, migration, proliferation, survival, differentiation, and metabolism. All metazoan organisms possess integrins, and it is generally agreed that integrins were associated with the evolution of multicellularity, being essential for the association of cells with their neighbors and surroundings, during embryonic development and many aspects of cellular and molecular biology. Integrins have important roles in many aspects of embryonic development, normal physiology, and disease processes with a multitude of functions discovered and elucidated for integrins that directly influence many areas of biology and medicine, including mammalian pregnancy, in particular implantation of the blastocyst to the uterine wall, subsequent placentation and conceptus (embryo/fetus and associated placental membranes) development. This review provides a succinct overview of integrin structure, ligand binding, and signaling followed with a concise overview of embryonic development, implantation, and early placentation in pigs, sheep, humans, and mice as an example for rodents. A brief timeline of the initial localization of integrin subunits to the uterine luminal epithelium (LE) and conceptus trophoblast is then presented, followed by sequential summaries of integrin expression and function during gestation in pigs, sheep, humans, and rodents. As appropriate for this journal, summaries of integrin expression and function during gestation in pigs and sheep are in depth, whereas summaries for humans and rodents are brief. Because similar models to those illustrated in Fig. 1, 2, 3, 4, 5 and 6 are present throughout the scientific literature, the illustrations in this manuscript are drafted as Viking imagery for entertainment purposes.

Equine bronchial epithelial cells are susceptible to cell entry with a SARS-CoV-2 pseudovirus but reveal low replication efficiency.

OBJECTIVE: To examine the susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus relative to human bronchial epithelial cells (HBECs). SAMPLE: Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control. METHODS: Angiotensin-converting enzyme 2 (ACE2) expression by EBECs was demonstrated using immunofluorescence, western immunoblot, and flow cytometry. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were transduced with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media for 96 hours. After 96 hours, eGFP expression in EBECs was assessed by fluorescence microscopy of cell cultures and quantitative PCR. RESULTS: ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean ΔCt values from quantitative PCR were significantly (P < .0001) higher in EBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. CLINICAL RELEVANCE: Equine respiratory tract cells were susceptible to cell entry with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infective to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted.

Hexavalent Chromium Disrupts Oocyte Development in Rats by Elevating Oxidative Stress, DNA Double-Strand Breaks, Microtubule Disruption, and Aberrant Segregation of Chromosomes.

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22-29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9-12 fold) damage, and increased DNA double-strand breaks (~5-10 fold) and DNA repair protein RAD51 (~3-6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes.

Creatine metabolism at the uterine-placental interface throughout gestation in sheep†.

The placenta requires high levels of adenosine triphosphate to maintain a metabolically active state throughout gestation. The creatine-creatine kinase-phosphocreatine system is known to buffer adenosine triphosphate levels; however, the role(s) creatine-creatine kinase-phosphocreatine system plays in uterine and placental metabolism throughout gestation is poorly understood. In this study, Suffolk ewes were ovariohysterectomized on Days 30, 50, 70, 90, 110 and 125 of gestation (n = 3-5 ewes/per day, except n = 2 on Day 50) and uterine and placental tissues subjected to analyses to measure metabolites, mRNAs, and proteins related to the creatine-creatine kinase-phosphocreatine system. Day of gestation affected concentrations and total amounts of guanidinoacetate and creatine in maternal plasma, amniotic fluid and allantoic fluid (P < 0.05). Expression of mRNAs for arginine:glycine amidinotransferase, guanidinoacetate methyltransferase, creatine kinase B, and solute carrier 16A12 in endometria and for arginine:glycine amidinotransferase and creatine kinase B in placentomes changed significantly across days of gestation (P < 0.05). The arginine:glycine amidinotransferase protein was more abundant in uterine luminal epithelium on Days 90 and 125 compared to Days 30 and 50 (P < 0.01). The chorionic epithelium of placentomes expressed guanidinoacetate methyltransferase and solute carrier 6A13 throughout gestation. Creatine transporter (solute carrier 6A8) was expressed by the uterine luminal epithelium and trophectoderm of placentomes throughout gestation. Creatine kinase (creatine kinase B and CKMT1) proteins were localized primarily to the uterine luminal epithelium and to the placental chorionic epithelium of placentomes throughout gestation. Collectively, these results demonstrate cell-specific and temporal regulation of components of the creatine-creatine kinase-phosphocreatine system that likely influence energy homeostasis for fetal-placental development.

Dietary supplementation with L-citrulline improves placental angiogenesis and embryonic survival in gilts.

This study was conducted with gilts as an animal model to test the hypothesis that dietary supplementation with L-citrulline (Cit) improves placental angiogenesis and embryonic survival. Between Days 14 and 25 of gestation, each gilt was fed a corn- and soybean-meal-based diet (2 kg/day) supplemented with 0.4% Cit or an isonitrogenous amount of L-alanine (Control). On Day 25 of gestation, gilts were hysterectomized to obtain conceptuses. Amniotic and allantoic fluids and placentae were analyzed for NOx [stable oxidation products of nitric oxide (NO)], polyamines, and amino acids (AAs). Placentae were also analyzed for syntheses of NO and polyamines; concentrations of AAs and related metabolites; and the expression of angiogenic factors and aquaporins (AQPs). Compared to the control group, Cit supplementation increased (P < 0.01) the number of viable fetuses by 2.0 per litter, the number and diameter of placental blood vessels (21% and 24%, respectively), placental weight (15%), and total allantoic and amniotic fluid volumes (20% and 47%, respectively). Cit supplementation also increased (P < 0.01) enzymatic activities of GTP-cyclohydrolase-1 (32%) and ornithine decarboxylase (27%) in placentae; syntheses of NO (29%) and polyamines (26%); concentrations of NOx (19%), tetrahydrobiopterin (28%), polyamines (22%), cAMP (26%), and cGMP (24%) in placentae; total amounts of NOx (22-40%), polyamines (23-40%), AAs (16-255%), glucose (22-44%), and fructose (22-43%) in allantoic and amniotic fluids. Furthermore, Cit supplementation increased (P < 0.05) placental mRNA levels for angiogenic factors (eNOS [84%], GTP-CH1 [55%], PGF [61%], VEGFA120 [26%], and VEGFR2 [137%], as well as AQPs - AQP1 [105%], AQP3 [53%], AQP5 [77%], AQP8 [57%], and AQP9 [31%]). Collectively, dietary Cit supplementation enhanced placental NO and polyamine syntheses as well as angiogenesis to improve conceptus development and survival.

Temporospatial expression of osteopontin in both left and right uterine horns during the peri-implantation period of dromedary camel.

Pregnancy in camels is established and maintained predominantly in the left uterine horn (98% frequency), whereas pregnancies occurring in the right horn result in early embryonic death. Aside from other reasons such as asynchrony of conceptus signaling and uterine receptivity, this phenomenon contributes to low reproductive efficiency in camels. The current research focuses on the expression of osteopontin (OPN), an extracellular matrix protein and adhesion molecule involved in implantation in mammals. Based on the differences in the pregnancy rate between the left and right horns, the temporal and spatial OPN expression was analyzed during the peri-implantation period on Days 8, 10, and 12. Results showed that OPN expression on Day 10 significantly increased by 14.5 fold in the left and 8.4-fold in the right uterine horn. By Day 12, OPN expression increased to 39.4 fold in the left and increased 7-fold in the right horn compared with non-mated females. Only the full length, 70-kDa OPN, was detected and upregulated with advancing pregnancy, with higher intensity in the left uterine horns than in the right. Spatially, OPN was predominantly localized on the apical uterine luminal and glandular epithelium in all camels. Moreover, OPN was detected in the stratum compactum stroma of pregnant camels. In conclusion, OPN mRNA and protein were detected and upregulated during the peri-implantation period, with higher OPN expression detected in the left uterine horn than in the right. OPN may be regulated by the presence of the embryo in the left uterine horn due to its role in embryo adhesion, implantation and placentation.

Creatine metabolism at the uterine-conceptus interface during early gestation in sheep†.

Ruminant conceptuses that elongate and attach to the uterine luminal epithelium (LE) to establish pregnancy require a large amount of adenosine triphosphate (ATP). The creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system re-generates ATP in dividing and migrating cells such as the conceptus trophectoderm cells. However, little is known about metabolism of Cr within uterine and conceptus tissues in livestock species during early gestation. In this study, Suffolk ewes were ovariohysterectomized on Days 9, 12, 15, 16, 17, 18, 20, or 21 of pregnancy (n = 2-5 animals/per day) to investigate metabolites, mRNAs, and proteins of the Cr-CK-PCr system at uterine-conceptus interface. Amounts of Cr and guanidinoacetate (GA) in uterine flushings increased between Days 12 and 17 of pregnancy. Endometrial expression of mRNAs for GA formation (AGAT), Cr synthesis (GAMT), and Cr/PCr utilization (CKB) was greater on Days 17 and 21 than on Days 9 and 12 of pregnancy. Immunoreactive AGAT was detected in uteri only on Day 21 but not in uteri or conceptuses at earlier days of pregnancy. GAMT, SLC6A8, and CKs were expressed in uterine luminal and glandular epithelia. Immunoreactive CKs (CKB, CKM, and CKMT1) appeared greater on Day 9 than Day 17 of pregnancy. Immunoreactive GAMT and CKs appeared greater in trophectoderm of conceptuses on Day 20 than on Day 15 of pregnancy, whereas the opposite was observed for that of SLC6A8. This study provides insights into cell-, tissue-, and time-specific metabolism of Cr at the uterine-conceptus interface suggesting a role for the Cr-CK-PCr system in ovine conceptus development and implantation.

Inhibition of SHMT2 mRNA translation increases embryonic mortality in sheep†.

The one-carbon metabolism (OCM) pathway provides purines and thymidine for synthesis of nucleic acids required for cell division, and S-adenosyl methionine for polyamine and creatine syntheses and the epigenetic regulation of gene expression. This study aimed to determine if serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in the OCM pathway, is critical for ovine trophectoderm (oTr) cell function and conceptus development by inhibiting translation of SHMT2 mRNA using a morpholino antisense oligonucleotide (MAO). In vitro treatment of oTr cells with MAO-SHMT2 decreased expression of SHMT2 protein, which was accompanied by reduced proliferation (P = 0.053) and migration (P < 0.05) of those cells. Intrauterine injection of MAO-SHMT2 in ewes on Day 11 post-breeding tended to decrease the overall pregnancy rate (on Days 16 and 18) compared with MAO-control (3/10 vs. 7/10, P = 0.07). The three viable conceptuses (n = 2 on Day 16 and n = 1 on Day 18) recovered from MAO-SHMT2 ewes had only partial inhibition of SHMT2 mRNA translation. Conceptuses from the three pregnant MAO-SHMT2 ewes had similar levels of expression of mRNAs and proteins involved in OCM as compared with conceptuses from MAO-control ewes. These results indicate that knockdown of SHMT2 protein reduces proliferation and migration of oTr cells (in vitro) to decrease elongation of blastocysts from spherical to elongated forms. These in vitro effects suggest that increased embryonic deaths in ewes treated with MAO-SHMT2 are the result of decreased SHMT2-mediated trophectoderm cell proliferation and migration supporting a role for the OCM pathway in survival and development of ovine conceptuses.

Elongating porcine conceptuses can utilize glutaminolysis as an anaplerotic pathway to maintain the TCA cycle†.

During the peri-implantation period of pregnancy, the trophectoderm of pig conceptuses utilize glucose via multiple biosynthetic pathways to support elongation and implantation, resulting in limited availability of pyruvate for metabolism via the TCA cycle. Therefore, we hypothesized that porcine trophectoderm cells replenish tricarboxylic acid (TCA) cycle intermediates via a process known as anaplerosis and that trophectoderm cells convert glutamine to α-ketoglutarate, a TCA cycle intermediate, through glutaminolysis. Results demonstrate: (1) that expression of glutaminase (GLS) increases in trophectoderm and glutamine synthetase (GLUL) increases in extra-embryonic endoderm of conceptuses, suggesting that extra-embryonic endoderm synthesizes glutamine, and trophectoderm converts glutamine into glutamate; and (2) that expression of glutamate dehydrogenase 1 (GLUD1) decreases and expression of aminotransferases including PSAT1 increase in trophectoderm, suggesting that glutaminolysis occurs in the trophectoderm through the GLS-aminotransferase pathway during the peri-implantation period. We then incubated porcine conceptuses with 13C-glutamine in the presence or absence of glucose in the culture media and then monitored the movement of glutamine-derived carbons through metabolic intermediates within glutaminolysis and the TCA cycle. The 13C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of 13C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux.

Temporal and spatial expression of aquaporins 1, 5, 8, and 9: Potential transport of water across the endometrium and chorioallantois of pigs.

INTRODUCTION: The uterus and placenta transport water during pregnancy recognition signaling, conceptus implantation, and placental development/placentation. This is likely influenced by aquaporins (AQPs) in the reproductive tract. This study determined mRNA and cell-type specific expression of AQP 1, 5, 8, and 9 proteins in the porcine uterus and placenta. METHODS: Porcine uteri and Chorioallantois were subjected to real-time PCR and immunofluorescence microscopy. RESULTS: AQP1 mRNA was maximal by Day 25 in endometrium and remained stable thereafter. AQP1 mRNA did not change in chorioallantois. AQP1 protein localized to erythrocytes and endothelium of the endometrium and allantois, and to smooth muscle of the myometrium. AQP5 protein localized to apical and lateral surfaces of the chorionic epithelia of areolae, but mRNA did not change in chorioallantois. AQP8 mRNA was high in the endometrium from Days 15 through 60 of gestation, and protein localized to multiple cell types within the endometrium and chorioallantois. AQP9 mRNA was highest in the endometrium on Days 10, 12 and 25, but did not change in the chorioallantois. AQP9 protein localized to the apical surface of endometrial luminal epithelial cells during early pregnancy, with a shift towards the basal surface later. AQP9 protein was observed in the allantoic epithelium. DISCUSSION: Results reveal pigs can potentially use AQP1, AQP5, AQP8, and AQP9 to transport water from the endometrial bloodstream to the allantoic bloodstream or allantoic fluid. The reverse is also possible and may explain the mechanism for changing volumes of allantoic fluid and hydration of allantoic connective tissues during pregnancy.

Microarray analysis reveals an important role for dietary L-arginine in regulating global gene expression in porcine placentae during early gestation.

BACKGROUND: Increasing the dietary provision of L-arginine to pregnant swine beginning at Day 14 of gestation enhances embryonic survival, but the underlying mechanisms are largely unknown. OBJECTIVE: This study determined the effects of dietary supplementation with 0.8% L-arginine to gilts between Days 14 and 25 of gestation on the global expression of genes in their placentae. METHODS: Between Days 14 and 24 of gestation, gilts were fed 2 kg of a corn- and soybean meal-based diet (containing 12.0% crude protein and 0.70% Arg) supplemented with 0.8% L-arginine or without L-arginine (0.0%; with 1.64% L-alanine as the isonitrogenous control). On Day 25 of gestation, 30 min after the consumption of their top dressing containing 8 g L-arginine or 16.4 g L-alanine, gilts underwent hysterectomy to obtain placentae, which were snap-frozen in liquid nitrogen. Total RNAs were extracted from the frozen tissues and used for microarray analysis based on the 44-K Agilent porcine gene platform. RESULTS: L-Arginine supplementation affected placental expression of 575 genes, with 146 genes being up-regulated and 429 genes being down-regulated. These differentially expressed genes play important roles in nutrient metabolism, polyamine production, protein synthesis, proteolysis, angiogenesis, immune development, anti-oxidative responses, and adhesion force between the chorioallantoic membrane and the endometrial epithelium, as well as functions of insulin, transforming growth factor beta, and Notch signaling pathways. CONCLUSION: Dietary supplementation with L-arginine plays an important role in regulating placental gene expression in gilts. Our findings help to elucidate mechanisms responsible for the beneficial effect of L-arginine in improving placental growth and embryonic/fetal survival in swine.

Dietary supplementation with L-arginine between days 14 and 25 of gestation enhances NO and polyamine syntheses and the expression of angiogenic proteins in porcine placentae.

Dietary supplementation with 0.4 or 0.8% L-arginine (Arg) to gilts between days 14 and 25 of gestation enhances embryonic survival and vascular development in placentae; however, the underlying mechanisms are largely unknown. This study tested the hypothesis that Arg supplementation stimulated placental expression of mRNAs and proteins that enhance angiogenesis, including endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), placental growth factor (PGF), GTP cyclohydrolase-I (GTP-CH1), ornithine decarboxylase (ODC1), and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2). Beginning on the day of breeding, gilts were fed daily 2 kg of a corn-soybean meal-based diet supplemented with 0.0 (control), 0.4, or 0.8% Arg. On day 25 of gestation, gilts were hysterectomized to obtain uteri and conceptuses for histochemical and biochemical analyses. eNOS and VEGFR1 proteins were localized to endothelial cells of maternal uterine blood vessels and to the uterine luminal epithelium, respectively. Compared with the control, dietary supplementation with 0.4 or 0.8% Arg increased (P < 0.05) the amounts of nitrite plus nitrate (NOx; oxidation products of NO) and polyamines in allantoic and amniotic fluids, concentrations of NOx, tetrahydrobiopterin (BH4, an essential cofactor for all NOS isoforms) and polyamines in placentae, as well as placental protein abundances of GTP-CH1 (the key enzyme for BH4 production) and ODC1 (the key enzyme for polyamine synthesis). Placental  mRNA levels for GTP-CH1, eNOS, PGF, VEGF, and VEGFR2 increased in response to both 0.4% and 0.8% Arg supplementation. Collectively, these results indicate that dietary Arg supplementation to gilts between days 14 and 25 of pregnancy promotes placental angiogenesis by increasing the expression of mRNAs and proteins for angiogenic factors as well as NO and polyamine syntheses.

Nutritional and Physiological Regulation of Water Transport in the Conceptus.

Water transport during pregnancy is essential for maintaining normal growth and development of conceptuses (embryo/fetus and associated membranes). Aquaporins (AQPs) are a family of small integral plasma membrane proteins that primarily transport water across the plasma membrane. At least 11 isoforms of AQPs (AQPs 1-9, 11, and 12) are differentially expressed in the mammalian placenta (amnion, allantois, and chorion), and organs (kidney, lung, brain, heart, and skin) of embryos/fetuses during prenatal development. Available evidence suggests that the presence of AQPs in the conceptus mediates water movement across the placenta to support the placentation, the homeostasis of amniotic and allantoic fluid volumes, as well as embryonic and fetal survival, growth and development. Abundances of AQPs in the conceptus can be modulated by nutritional status and physiological factors affecting the pregnant female. Here, we summarize the effects of maternal dietary factors (such as intakes of protein, arginine, lipids, all-trans retinoic acid, copper, zinc, and mercury) on the expression of AQPs in the conceptus. We also discuss the physiological changes in hormones (e.g., progesterone and estrogen), oxygen supply, nitric oxide, pH, and osmotic pressure associated with the regulation of fluid exchange between mother and fetus. These findings may help to improve the survival, growth, and development of embryo/fetus in livestock species and other mammals (including humans).

Dietary L-arginine supplementation during days 14-25 of gestation enhances aquaporin expression in the placentae and endometria of gestating gilts.

This study tested the hypothesis that dietary L-arginine (Arg) supplementation to pregnant gilts enhanced the expression of water channel proteins [aquaporins (AQPs)] in their placentae and endometria. Gilts were fed twice daily 1 kg of a corn and soybean meal-based diet supplemented with 0.0%, 0.4%, or 0.8% Arg between Days 14 and 25 of gestation. On Days 25 and 60 of gestation, gilts were hysterectomized to obtain placentae and endometria. On Day 25 of gestation, supplementation with 0.4% Arg increased (P < 0.05) the abundance of placental AQP9 protein, whereas supplementation with 0.8% Arg increased (P < 0.05) placental AQP1 and AQP9 proteins, compared with controls. On Day 60 of gestation, supplementation with 0.4% Arg increased (P < 0.05) endometrial AQP1 protein, whereas supplementation with 0.8% Arg increased (P < 0.05) endometrial AQP5 and AQP9 proteins. Supplementation with 0.8% Arg increased the endometrial expression of AQP1, AQP5, and AQP9 proteins located in the luminal epithelium and glandular epithelium of endometria, and placental transport of 3H2O. Collectively, these results indicate that dietary Arg supplementation stimulates the expression of selective AQPs in porcine placenta and endometria, thereby enhancing water transport from mother to fetus and expanding the chorioallantoic membranes during the period of placentation.

Integrin Adhesion Complex Organization in Sheep Myometrium Reflects Changing Mechanical Forces during Pregnancy and Postpartum.

Cells respond to extracellular mechanical forces through the assembly of integrin adhesion complexes (IACs) that provide a scaffold through which cells sense and transduce responses to those forces. IACs are composed of transmembrane integrin receptors that bind to extracellular matrix (ECM) proteins externally and connect with the actomyosin cytoskeleton internally. Myometrial smooth muscle cells respond to forces that arise due to increases in fetal growth/weight, placental fluid volumes, and blood flow. As a result, the uterus transforms into an organ that can forcefully expel the fetus and placental membranes during parturition. While earlier studies focused on IAC expression in the myometrial compartment of rodents and humans to explore pregnancy-associated responses, the present study examines IAC assembly in ovine myometrium where mechanical forces are expected to be amplified in a manner similar to humans. Results indicate that the ITGA5 and ITGB1 heterodimers associate with the ECM protein FN1 externally, and with VCL and TLN1 internally, to form IACs in myometrial cells during the first trimester of pregnancy. These IACs become increasingly ordered until parturition. This ordered structure is lost by one day postpartum; however, the abundance of the integrin proteins remains elevated for at least two weeks postpartum. Implications of the present study are that sheep are similar to humans regarding the assembly of IACs in the pregnant myometrium and suggest that IACs may form much earlier in human gestation than was previously implied by the rat model. Results highlight the continued value of the sheep model as a flagship gynecological model for understanding parturition in humans.

SPP1 expression in the mouse uterus and placenta: implications for implantation†.

Secreted phosphoprotein 1 (SPP1, also known as osteopontin) binds integrins to mediate cell-cell and cell-extracellular matrix communication to promote cell adhesion, migration, and differentiation. Considerable evidence links SPP1 to pregnancy in several species. Current evidence suggests that SPP1 is involved in implantation and placentation in mice, but in vivo localization of SPP1 and in vivo mechanistic studies to substantiate these roles are incomplete and contradictory. We localized Spp1 mRNA and protein in the endometrium and placenta of mice throughout gestation, and utilized delayed implantation of mouse blastocysts to link SPP1 expression to the implantation chamber. Spp1 mRNA and protein localized to the endometrial luminal (LE), but not glandular epithelia (GE) in interimplantation regions of the uterus throughout gestation. Spp1 mRNA and protein also localized to uterine naturel killer (uNK) cells of the decidua. Within the implantation chamber, Spp1 mRNA localized only to intermittent LE cells, and to the inner cell mass. SPP1 protein localized to intermittent trophoblast cells, and to the parietal endoderm. These results suggest that SPP1: (1) is secreted by the LE at interimplantation sites for closure of the uterine lumen to form the implantation chamber; (2) is secreted by LE adjacent to the attaching trophoblast cells for attachment and invasion of the blastocyst; and (3) is not a component of histotroph secreted from the GE, but is secreted from uNK cells in the decidua to increase angiogenesis within the decidua to augment hemotrophic support of embryonic/fetal development of the conceptus.

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 ß3 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.