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.

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.

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.

Implantation and Placentation in Ruminants.

In comparison to many other mammalian species, ruminant ungulates have a unique form of placentation. Ruminants initially display an epitheliochorial type of placentation; however, during the period of placental attachment, trophoblast giant binucleate cells (BNC) develop within the chorion to migrate and fuse with the uterine surface epithelium to form syncytial plaques. Binucleate cell migration and fusion continues throughout pregnancy but never appears to breach the basal lamina, beneath the uterine surface or luminal epithelium. Therefore, the semi-invasive type of placentation in ruminants is classified as synepitheliochorial. The endometrium of ruminant species also contains unique specialized aglandular structures termed "caruncles" in which the chorioallantois (cotyledons) interdigitates and forms highly vascularized fetal-maternal "placentomes." This chapter will discuss the current knowledge of early conceptus development during the peri-attachment period, establishment of pregnancy, conceptus attachment, and placentation in ruminant ungulates. The features of placentomes, BNCs, fetomaternal hybrid cells, and multinucleated syncytial plaques of the cotyledonary placenta of ruminant species will be reviewed to highlight the unique form of placentation compared to the placentae of other artiodactyls.

Pig conceptuses secrete interferon gamma to recruit T cells to the endometrium during the peri-implantation period†.

The emerging paradigm in the immunology of pregnancy is that implantation of conceptuses does not progress in an immunologically suppressed environment. Rather, the endometrium undergoes a controlled inflammatory response during implantation as trophectoderm of elongating and implanting pig conceptuses secrete the pro-inflammatory cytokine interferon gamma (IFNG). Results of this study with pigs revealed: (1) accumulation of immune cells and apoptosis of stromal cells within the endometrium at sites of implantation during the period of IFNG secretion by conceptuses; (2) accumulation of proliferating cell nuclear antigen (PCNA)-positive T cells within the endometrium at sites of implantation; (3) significant increases in expression of T cell co-signaling receptors including programmed cell death 1 (PDCD1), CD28, cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and inducible T cell co-stimulator (ICOS), as well as chemokines CXCL9, 10, and 11 within the endometrium at sites of implantation; (4) significant increases in T cell co-signaling receptors, PDCD1 and ICOS, and chemokine CXCL9 in the endometrium of cyclic gilts infused with IFNG; and (5) identification of CD4+ (22.59%) as the major T cell subpopulation, with minor subpopulations of CD8+ (1.38%), CD4+CD25+ (1.08%), and CD4+CD8+ (0.61%) T cells within the endometrium at sites of implantation. Our results provide new insights into the immunology of implantation to suggest that trophectoderm cells of pigs secrete IFNG to recruit various subpopulations of T cells to the endometrium to contribute to a controlled inflammatory environment that supports the active breakdown and restructuring of the endometrium in response to implantation of the conceptus.

Integrins and OPN localize to adhesion complexes during placentation in sheep.

Integrins and OPN are potential mediators of blastocyst attachment to the endometrium to initiate implantation. The goals were to examine the temporal/spatial pattern of expression of integrins at the endometrial-placental interface of sheep encompassing Days 9 through 80 of gestation and determine if OPN co-localizes with integrins. Results show the following: (1) αv, α4, ß1, ß3 and ß5 integrins at the apical surface of endometrial luminal epithelium (LE) from Days 11 through 16 of pregnancy that indicate a role for these integrins during implantation; (2) large, intermittent aggregates of αv, α4, α5, ß1 and ß5 integrins at the endometrial-placental interface from Days 20 through 55, suggesting adaptation to a localized tissue remodeling stage of placentation; and (3) integrin adhesion complexes (IACs) containing αv, α4, α5, ß1 and ß5 integrins precisely distribute at the apical surfaces of apposed endometrial LE and chorion along expanses of the interplacentomal endometrial-placental interface between Days 60 and 80 of gestation, suggesting engagement of these integrins with the ECM to stabilize adhesion between endometrial LE and chorion in response to the increasing mechanical stress on this interface by the increasing size of the fetus and volumes of fetal fluids. An advancement is the clear co-localization of OPN and integrins at the endometrial-placental interface throughout gestation in sheep. The comprehensive nature of these results provide evidence that integrins potentially interact with OPN to play key roles in the mechanisms required for implantation and placentation throughout pregnancy in sheep and have implications concerning implantation and placentation in other species.

OPN binds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature.

Angiogenesis is fundamental to the expansion of the placental vasculature during pregnancy. Integrins are associated with vascular formation; and osteopontin is a candidate ligand for integrins to promote angiogenesis. Endothelial progenitor cells (EPCs) are released from bone marrow into the blood and incorporate into newly vascularized tissue where they differentiate into mature endothelium. Results of studies in women suggest that EPCs may play an important role in maintaining placental vascular integrity during pregnancy, although little is known about how EPCs are recruited to these tissues. Our goal was to determine the αv integrin mediated effects of osteopontin on EPC adhesion and incorporation into angiogenic vascular networks. EPCs were isolated from 6 h old piglets. RT-PCR revealed that EPCs initially had a monocyte-like phenotype in culture that became more endothelial-like with cell passage. Immunofluorescence microscopy confirmed that the EPCs express platelet endothelial cell adhesion molecule, vascular endothelial cadherin, and von Willebrand factor. When EPCs were cultured on OPN-coated slides, the αv integrin subunit was observed in focal adhesions at the basal surface of EPCs. Silencing of αv integrin reduced EPC binding to OPN and focal adhesion assembly. In vitro siRNA knockdown in EPCs,demonstrated that OPN stimulates EPC incorporation into human umbilical vein endothelial cell (HUVEC) networks via αv-containing integrins. Finally, in situ hybridization and immunohistochemistry localized osteopontin near placental blood vessels. In summary, OPN binds the αv integrin subunit on EPCs to support EPC adhesion and increase EPC incorporation into angiogenic vascular networks.

Randomized trial of intermittent intraputamenal glial cell line-derived neurotrophic factor in Parkinson's disease.

We investigated the effects of glial cell line-derived neurotrophic factor (GDNF) in Parkinson's disease, using intermittent intraputamenal convection-enhanced delivery via a skull-mounted transcutaneous port as a novel administration paradigm to potentially afford putamen-wide therapeutic delivery. This was a single-centre, randomized, double-blind, placebo-controlled trial. Patients were 35-75 years old, had motor symptoms for 5 or more years, and presented with moderate disease severity in the OFF state [Hoehn and Yahr stage 2-3 and Unified Parkinson's Disease Rating Scale motor score (part III) (UPDRS-III) between 25 and 45] and motor fluctuations. Drug delivery devices were implanted and putamenal volume coverage was required to exceed a predefined threshold at a test infusion prior to randomization. Six pilot stage patients (randomization 2:1) and 35 primary stage patients (randomization 1:1) received bilateral intraputamenal infusions of GDNF (120 µg per putamen) or placebo every 4 weeks for 40 weeks. Efficacy analyses were based on the intention-to-treat principle and included all patients randomized. The primary outcome was the percentage change from baseline to Week 40 in the OFF state (UPDRS-III). The primary analysis was limited to primary stage patients, while further analyses included all patients from both study stages. The mean OFF state UPDRS motor score decreased by 17.3 ± 17.6% in the active group and 11.8 ± 15.8% in the placebo group (least squares mean difference: -4.9%, 95% CI: -16.9, 7.1, P = 0.41). Secondary endpoints did not show significant differences between the groups either. A post hoc analysis found nine (43%) patients in the active group but no placebo patients with a large clinically important motor improvement (≥10 points) in the OFF state (P = 0.0008). 18F-DOPA PET imaging demonstrated a significantly increased uptake throughout the putamen only in the active group, ranging from 25% (left anterior putamen; P = 0.0009) to 100% (both posterior putamina; P < 0.0001). GDNF appeared to be well tolerated and safe, and no drug-related serious adverse events were reported. The study did not meet its primary endpoint. 18F-DOPA imaging, however, suggested that intermittent convection-enhanced delivery of GDNF produced a putamen-wide tissue engagement effect, overcoming prior delivery limitations. Potential reasons for not proving clinical benefit at 40 weeks are discussed.

Immunohistochemical Examination of Trophoblast Syncytialization during Early Placentation in Sheep.

During the peri-implantation period, multinucleated syncytia are formed in the sheep placenta. For over 20 years the scientific consensus has been that during trophoblast syncytialization in sheep, binucleate trophoblast giant cells (BNCs) differentiate from mononuclear trophoblast cells, and individual BNCs fuse with individual luminal epithelial (LE) cells to form trinucleate cells. These trophoblast-LE syncytial plaques then grow through continued BNC migration and fusion. Therefore, LE cells are thought to be incorporated into syncytial plaques. However, these ideas were based on electron microscopy studies, without benefit of molecular markers for BNC and LE cells to support conclusions. The aim of this study was to observe interactions between BNCs and uterine LE cells using immunohistochemical localization for molecular markers for BNCs and uterine LE cells. We performed immunofluorescence staining, laser capture microdissection, and TUNEL staining on the uterine-placental tissues of sheep during early placentation. We observed: (1) syncytial cells containing more than two nuclei within the trophoblast cell layer; (2) depolarized LE cells that express caspase 3 and stain positively for TUNEL; (3) engulfment of caspase 3-positive LE cells by trophoblast giant cells (TGCs) and empty spaces within the LE layer at sites of implantation; (4) rapid enlargement of syncytial plaques; and (5) E-cadherin and TUNEL-positive cells within the uterine stroma underlying degenerating LE was coincident with accumulation of CD45-positive cells at these sites. These data suggest that during early placentation: (1) fusion between trophoblasts is not limited to the formation of BNCs, and the term 'trophoblast giant cell (TGC)' may be appropriate; (2) LE cells undergo apoptosis; (3) apoptotic LE cells are eliminated by TGCs; (4) fusion is not limited to the incorporation of new BNCs but involves the lateral fusion between growing syncytial plaques; and (5) TGCs carry apoptotic LE cells away from the uterine-placental interface for elimination by immune cells within the stroma. These data indicate that uterine LE cells are not incorporated into syncytial plaques, but are engulfed and eliminated, and that early placentation in sheep is more similar to early placentation in humans than is currently understood in that both develop mononucleated cytotrophoblast and multinucleated syncytiotrophoblast layers of entirely placental origin. The elimination of LE cells by sheep TGCs might provide insights into elimination and penetration of LE cells during human embryo implantation.

ITGAV (alpha v integrins) bind SPP1 (osteopontin) to support trophoblast cell adhesion.

Attachment of the conceptus trophoblast (Tr) to the uterine luminal epithelium (LE) is critical for successful implantation. This study determined whether alpha v (av) integrins (ITGAV) directly mediate porcine trophoblast cell adhesion to secreted phosphoprotein 1 (SPP1, also known as osteopontin (OPN)) and examined the temporal/spatial expression of ITGAV, beta 3 (b3, ITGB3) and beta 6 (b6, ITGB6) integrin subunits, and SPP1, at the uterine-placental interface of pigs. Knockdown of ITGAV in porcine Tr (pTr2) cells by siRNA reduced pTr2 attachment to SPP1. In situ hybridization confirmed the presence of ITGAV, ITGB3 and ITGB6 mRNAs in uterine LE and conceptus Tr between Days 9 and 60 of gestation, with no change in the magnitude of expression over the course of pregnancy. Exogenous E2 or P4 did not affect ITGAV, ITGB3 and ITGB6 mRNA expression in the uteri of ovariectomized gilts. Immunofluorescence identified ITGAV, ITGB3 and SPP1 proteins in large aggregates at the uterine LE-placental Tr/chorion interface on Day 25, but aggregates were no longer observed by Day 50 of gestation. These results are the first to directly demonstrate that pTr2 cells engage ITGAV-containing integrin receptors to adhere to SPP1 and suggest that mechanical forces generated by tethering elongating conceptuses to uterine LE leads to assembly of focal adhesions containing ITGAV and SPP1; however, as placentation progresses, subsequent folding/interdigitation at the uterine-placental interface disperses mechanical forces resulting in the loss of focal adhesions.

Placental Proteomics Reveal Insights into Fetal Alcohol Spectrum Disorders.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) describe many of the well-known neurodevelopmental deficits afflicting children exposed to alcohol in utero. The effects of alcohol on the maternal-fetal interface, especially the placenta, have been less explored. We herein hypothesized that chronic binge alcohol exposure during pregnancy significantly alters the placental protein profile in a rat FASD model. METHODS: Pregnant rats were orogastrically treated daily with alcohol (4.5 g/kg, gestational day [GD] 5 to 10; 6.0 g/kg, GD 11 to 19) or 50% maltose dextrin (isocalorically matched pair-fed controls). On GD 20, placentae were collected, flash-frozen, and stored until tissues were homogenized. Protein lysates were denatured, reduced, captured on a 10-kDa spin filter, and digested. Peptides were eluted, reconstituted, and analyzed by a Q Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer. RESULTS: Mass spectrometry (MS) analysis identified 2,285 placental proteins based on normalized spectral counts and 2,000 proteins by intensity-based absolute quantification. Forty-five placental proteins were significantly (p < 0.05) altered by gestational alcohol exposure by both quantification approaches. These included proteins directly related to alcohol metabolism; specific isoforms of alcohol dehydrogenase and aldehyde dehydrogenase were up-regulated in the alcohol group. Ingenuity analysis identified ethanol degradation as the most significantly altered canonical pathway in placenta, and fetal/organ development as most altered function, with increased risk for metabolic, neurological, and cardiovascular diseases. Physiological roles of the significantly altered proteins were related to early pregnancy adaptations, implantation, gestational diseases, fetal organ development, neurodevelopment, and immune functions. CONCLUSIONS: We conclude that the placenta is a valuable organ not only to understand FASD etiology but it may also serve as a diagnostic tool to identify novel biomarkers for detecting the outcome of fetal alcohol exposure. Placental MS analysis can offer sophisticated insights into identifying alcohol metabolism-related enzymes and regulators of fetal development.

Uterine Histotroph and Conceptus Development. II. Arginine and Secreted Phosphoprotein 1 Cooperatively Stimulate Migration and Adhesion of Ovine Trophectoderm Cells via Focal Adhesion-MTORC2 Mediated Cytoskeleton Reorganization.

In all mammalian species, critical events, including uterine receptivity and development of the conceptus (embryo/fetus and its associated extraembryonic membranes), must be intricately orchestrated and carefully timed during the window of implantation. Otherwise, failure of conceptuses to implant is inevitable, which accounts for 50%-75% of failures to establish pregnancy. Unlike human and rodent blastocysts, the blastocysts of pigs and ruminants undergo rapid transitions from spherical to tubular and filamentous conceptuses in response to histotroph during the peri-implantation period of pregnancy. Both arginine and secreted phosphoprotein 1 (SPP1; also known as osteopontin) are multifunctional molecules that increase significantly in ovine uterine histotroph during early pregnancy; however, little is known about their relationship and synergistic effects on conceptus development. Therefore, we conducted in vitro experiments using our established ovine trophectoderm cell line (oTr1) isolated from Day 15 ovine conceptuses to determine their migratory and adhesive responses to individual and combined effects of arginine and recombinant SPP1 (rSPP1) that contains an Arg-Gly-Asp (RGD) binding sequence. Migration and adhesion of oTr1 cells were significantly stimulated by rSPP1, whereas arginine alone only induced a significant increase in cell migration. However, the combination of arginine and rSPP1 had an additive effect on migration, and a synergistic effect on adhesion of oTr1 cells. Those cooperative effects of arginine and SPP1 were mediated by focal adhesion assembly-MTORC2-cytoskeletal reorganization and MAPK pathways. Collectively, results suggest that arginine and SPP1 in histotroph affect cellular events required for rapid elongation of ovine conceptuses during the peri-implantation period of pregnancy.

Fructose Synthesis and Transport at the Uterine-Placental Interface of Pigs: Cell-Specific Localization of SLC2A5, SLC2A8, and Components of the Polyol Pathway.

The fetal fluids and uterine flushings of pigs contain higher concentrations of fructose than glucose, but fructose is not detected in maternal blood. Fructose can be synthesized from glucose via enzymes of the polyol pathway, aldose reductase (AKR1B1) and sorbitol dehydrogenase (SORD), transported across cell membranes by solute carriers SLC2A5 and SLC2A8, and converted to fructose-1-phosphate by ketohexokinase (KHK). SLC2A8, SLC2A5, AKR1B1, SORD, and KHK mRNAs and proteins were analyzed using quantitative PCR and immunohistochemistry or in situ hybridization in endometria and placentae of cyclic and pregnant gilts, cyclic gilts injected with estrogen, and ovariectomized gilts injected with progesterone. Progesterone up-regulated SLC2A8 protein in uterine luminal (LE) and glandular epithelia during the peri-implantation period, and expression became exclusively placental, chorion and blood vessels, after Day 30. P4 up-regulated SLC2A5 mRNA in uterine LE and glandular epithelia after implantation, and the chorion expressed SLC2A5 between Days 30 and 85. AKR1B1 and SORD proteins localized to uterine LE during the peri-implantation period, but expression switched to chorion by Day 20 and was maintained through Day 85. Uterine expression of AKR1B1 mRNA was down-regulated by estrogen. KHK protein localized to trophectoderm/chorion throughout gestation. These results provide evidence that components for the conversion of glucose to fructose and for fructose transport are present at the uterine-placental interface of pigs. The shift in expression from LE to chorion during pregnancy suggests free-floating conceptuses are supported by fructose synthesized by the uterus, but after implantation, the chorion becomes self-sufficient for fructose synthesis and transport.

Uterine histotroph and conceptus development. I. cooperative effects of arginine and secreted phosphoprotein 1 on proliferation of ovine trophectoderm cells via activation of the PDK1-Akt/PKB-TSC2-MTORC1 signaling cascade.

The greatest limitation to reproductive performance in most mammals, including humans, is embryonic mortality, which, in general, claims 20%-40% of the embryos during the peri-implantation period of pregnancy. Both arginine and secreted phosphoprotein 1 (SPP1) are multifunctional molecules that increase significantly in ovine uterine histotroph during early pregnancy. However, little is known about the relationship and underlying mechanisms for synergistic effects of arginine and SPP1, if any, on conceptus (embryo/fetus and associated extraembryonic membranes) development. Therefore, we conducted in vitro experiments using our established ovine trophectoderm cell line (oTr1) isolated from Day 15 ovine conceptuses to determine their proliferative response to individual and synergistic effects of arginine and recombinant SPP1 (rSPP1) that contains an RGD binding sequence. At physiological concentrations, arginine (0.2 mM) stimulated oTr1 cell proliferation 1.7-fold (P < 0.05) at 48 h, whereas rSPP1 (10 ng/ml) had no such effect. However, an additive effect on oTr1 cell proliferation was induced by combination of arginine and SPP1 as compared to the control (2.1-fold increase; P < 0.01), arginine alone (1.3-fold increase; P < 0.05), and rSPP1 alone (1.5-fold increase; P < 0.01). This additive effect was mediated through cooperative activation of the PDK1-Akt/PKB-TSC2-MTORC1 cell signaling cascade. Collectively, results suggest that arginine and SPP1 in histotroph act cooperatively to enhance survival, growth, and development of ovine conceptuses.

The many faces of interferon tau.

Interferon tau (IFNT) was discovered as the pregnancy recognition signal in ruminants, but is now known to have a plethora of physiological functions in the mammalian uterus. The mammalian uterus includes, from the outer surface to the lumen, the serosa, myometrium and endometrium. The endometrium consists of the luminal, superficial glandular, and glandular epithelia, each with a unique phenotype, stromal cells, vascular elements, nerves and immune cells. The uterine epithelia secrete or selectively transport molecules into the uterine lumen that are collectively known as histotroph. Histotroph is required for growth and development of the conceptus (embryo and its associated extra-embryonic membranes) and includes nutrients such as amino acids and glucose, enzymes, growth factors, cytokines, lymphokines, transport proteins for vitamins and minerals and extracellular matrix molecules. Interferon tau and progesterone stimulate transport of amino acids in histotroph, particularly arginine. Arginine stimulates the mechanistic target of rapamycin pathway to induce proliferation, migration and protein synthesis by cells of the conceptus, and arginine is the substrate for synthesis of nitric oxide and polyamines required for growth and development of the conceptus. In ruminants, IFNT also acts in concert with progesterone from the corpus luteum to increase expression of genes for transport of nutrients into the uterine lumen, as well as proteases, protease inhibitors, growth factors for hematopoiesis and angiogenesis and other molecules critical for implantation and placentation. Collectively, the pleiotropic effects of IFNT contribute to survival, growth and development of the ruminant conceptus.

Uterine histotroph and conceptus development: select nutrients and secreted phosphoprotein 1 affect mechanistic target of rapamycin cell signaling in ewes.

Interferon tau (IFNT), the pregnancy recognition signal in ruminants, abrogates the uterine luteolytic mechanism to ensure maintenance of function for the corpora lutea to produce progesterone (P4). IFNT also suppresses expression of classical IFN-stimulated genes by uterine lumenal epithelium (LE) and superficial glandular (sGE) epithelium but, acting in concert with progesterone, affects expression of a multitude of genes critical to growth and development of the conceptus. The LE and sGE secrete proteins and transport nutrients into the uterine lumen necessary for conceptus development, pregnancy recognition signaling, and implantation. Secretions include arginine and secreted phosphoprotein 1 (SPP1). Arginine can be metabolized to nitric oxide and to polyamines or act directly to activate the mechanistic target of rapamycin cell signaling pathway to stimulate proliferation, migration, and mRNA translation in trophectoderm cells. SPP1 binds alphavbeta3 and alpha5beta1 integrins to induce focal adhesion assembly, adhesion, and migration of conceptus trophectoderm cells during implantation. Thus, arginine and SPP1 mediate growth, migration, cytoskeletal remodeling, and adhesion of trophectoderm essential for pregnancy recognition signaling and implantation. This minireview focuses on components of histotroph that affect conceptus development in the ewe.

Select nutrients in the ovine uterine lumen. VIII. Arginine stimulates proliferation of ovine trophectoderm cells through MTOR-RPS6K-RPS6 signaling cascade and synthesis of nitric oxide and polyamines.

During the peri-implantation period in sheep, L-arginine (L-Arg) in the uterine lumen is an essential substrate for the synthesis of nitric oxide (NO), by nitric oxide synthase (NOS), and polyamines, via arginase and ornithine decarboxylase, that are required for survival and development of ovine conceptuses (embryo and its extraembryonic membranes). L-Arginine can stimulate hypertrophy, hyperplasia, and differentiation of the ovine conceptus trophectoderm; however, the responsible signal transduction cascade has not been determined. Therefore, this study examined possible signaling pathways mediated by L-Arg, as well as the effects of two NO donors (S-nitroso-N-acetyl-DL-penicillamine and diethylenetriamine) and putrescine (precursor for spermidine and spermine) on oTr cell proliferation. Further, the inhibition of these effects by L-NAME (L-nitro-arginine methyl ester, an inhibitor of NOS) and nor-NOHA (N-omega-hydroxy-nor-arginine, an inhibitor of arginase) was assessed. L-Arginine treatment increased the abundance of phosphorylated MTOR, RPS6K, and EIF4EBP1 in oTr cells. Consistent with activation of these cell-signaling molecules, L-Arg increased protein synthesis and reduced protein degradation in oTr cells. Both NO and polyamines enhanced cell proliferation in a dose-dependent manner. The effects of L-Arg were partially inhibited by both L-NAME and nor-NOHA. These results indicate that L-Arg enhances production of polyamines and NO and activates the MTOR/FRAP1-RPS6K-RPS6 signaling pathway to stimulate proliferation and migration of oTr cells.

Select nutrients in the ovine uterine lumen. VII. Effects of arginine, leucine, glutamine, and glucose on trophectoderm cell signaling, proliferation, and migration.

Histotroph is required for survival and development of ovine conceptuses (embryo and extraembryonic membranes). Results from our laboratory indicate that arginine (Arg), leucine (Leu), glutamine (Gln), and glucose increase in the uterine lumen between Days 10 and 15 of pregnancy, coincident with increases in expression of amino acid and glucose transporters by uterine epithelia as well as trophectoderm and yolk sac of conceptuses and elongation of the conceptus trophectoderm. Therefore, we hypothesized that Arg, Leu, Gln, and glucose have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary ovine trophectoderm (oTr) cells isolated from Day 15 conceptuses were serum-starved for 24 h in a customized medium, deprived of select nutrients, and then treated with either Arg, Leu, Gln, or glucose. Western blot analyses of whole oTr cell extracts revealed that Arg, Leu, and glucose, but not Gln, increased phosphorylated AKT1 by 2.8-, 2.5-, and 1.8-fold, respectively, within 15 min, and the increase was maintained to 60 min. Arg, Leu, and glucose also stimulated increases in phosphorylated ribosomal protein S6K (pRPS6K) by 4.2-, 4.7-, and 2.3-fold, respectively, within 15 min, as well as increases in phosphorylated ribosomal protein S6 (pRPS6) between 0 and 30 min posttreatment, that were sustained to 60 min. When oTr cells were treated with Arg, pRPS6K protein increased in nuclei, but this was not observed in nuclei of oTr cells treated with Leu and glucose. Immunocytochemical analyses also revealed abundant amounts of pRPS6 protein in the cytoplasm of oTr cells treated with Arg, Leu, and glucose. Furthermore, Arg and Leu increased proliferation and migration of oTr cells. Collectively, these results indicate that Arg, Leu, and glucose, but not Gln, in histotroph coordinately activate AKT1-mechanistic target of rapamycin and RPS6K-RPS6 cell signaling pathways to stimulate hypertrophy, hyperplasia, and migration of oTr cells.

Select nutrients in the ovine uterine lumen. IX. Differential effects of arginine, leucine, glutamine, and glucose on interferon tau, ornithine decarboxylase, and nitric oxide synthase in the ovine conceptus.

Nutrients are primary requirements for development of conceptuses (embryo and extraembryonic membranes), including protein synthesis. We have shown that arginine (Arg), leucine (Leu), and glucose stimulate protein synthesis through phosphorylation of MTOR signaling molecules, thereby increasing proliferation of ovine trophectoderm cells. This study determined whether Arg, Leu, glutamine (Gln), and glucose influence gene expression and protein synthesis in explant cultures of ovine conceptuses recovered from ewes on Day 16 of pregnancy. Conceptuses were deprived of select nutrients and then cultured with either Arg, Leu, Gln, or glucose for 18 h, after which they were analyzed for abundance of MTOR, RPS6K, RPS6, EIF4EBP1 (also known as 4EBP1), IFNT, NOS2, NOS3, GCH1, and ODC1 mRNAs and proteins. Levels of MTOR, RPS6K, RPS6, and EIF4EBP1 mRNAs were not affected by treatment with any of the select nutrients. Similarly, expression of IFNT, NOS2, NOS3, and ODC1 mRNAs were not different. Interestingly, GCH1 mRNA levels increased in response to Arg treatment. Importantly, Arg, Leu, Gln, and glucose increased the abundance of phosphorylated MTOR, RPS6K, RPS6, and EIF4EBP1 proteins as well as NOS and ODC1 proteins, but only Arg increased the abundance of IFNT protein. These findings indicate that Arg, Leu, Gln, and glucose stimulate translation of mRNAs to increase synthesis of proteins through phosphorylation and activation of components of the MTOR signaling pathway. Increases in abundance of IFNT protein (the pregnancy recognition signal), NOS2, NOS3 and GCH1 for conversion of Arg to nitric oxide, and ODC1 for synthesis of polyamines are all important for growth and development of the ovine conceptus during pregnancy.

Role of the cytoskeleton in formation and maintenance of angiogenic sprouts.

Angiogenesis is the formation of new blood vessels from pre-existing structures, and is a key step in tissue and organ development, wound healing and pathological events. Changes in cell shape orchestrated by the cytoskeleton are integral to accomplishing the various steps of angiogenesis, and an intact cytoskeleton is also critical for maintaining newly formed structures. This review focuses on how the 3 main cytoskeletal elements--microfilaments, microtubules, and intermediate filaments--regulate the formation and maintenance of angiogenic sprouts. Multiple classes of compounds target microtubules and microfilaments, revealing much about the role of actin and tubulin and their associated molecules in angiogenic sprout formation and maintenance. In contrast, intermediate filaments are much less studied, yet intriguing evidence suggests a vital, but unresolved, role in angiogenic sprouting. This review discusses evidence for regulatory molecules and pharmacological compounds that affect actin, microtubule and intermediate filament dynamics to alter various steps of angiogenesis, including endothelial sprout formation and maintenance.