Swine leukocyte antigen-DQ expression and its regulation by interferon-gamma at the maternal-fetal interface in pigs

Successful pregnancy requires an appropriate intrauterine immune response to the conceptus, which is a semiallograft within the uterus. We reported that swine leukocyte antigen-DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene, is expressed in the uterine endometrium at the time of conceptus implantation in pigs. Because MHC molecules play critical roles in the immune system, SLA-DQ was hypothesized to be involved in immune regulation during pregnancy. Therefore, we examined expression of SLA-DQ in uterine endometrial tissues obtained during the estrous cycle and pregnancy. SLA-DQA and SLA-DQB mRNAs were detected as 1.3-kb and 1.2-kb bands, respectively. Real-time RT-PCR analysis indicated that SLA-DQA and SLA-DQB mRNA expression was affected by day and pregnancy status, with the highest expression on Day 15 of pregnancy. SLA-DQ was localized primarily to subepithelial stromal cells and endothelial cells of the uterus. Using endometrial explant cultures from Day 12 of the estrous cycle, we determined that expression of SLA-DQA and SLA-DQB mRNAs increased in response to interferon-gamma (IFNG), which is produced by pig conceptus trophectoderm between Days 14 and 18 of pregnancy. The abundance of SLA-DQ protein was less in endometria from gilts with conceptuses resulting from somatic cell nuclear transfer compared with endometria from gilts with conceptuses resulting from natural mating. These results support our hypothesis that SLA-DQ is expressed in response to IFNG from the conceptus, and likely regulates immune response at the maternal-fetal interface to support the maintenance of pregnancy in pigs.     

Differential expression of secreted phosphoprotein 1 in response to estradiol-17β and in ovarian tumors in chickens

Roundabout 2 (Robo2) is a member of the membrane protein receptor family. The chemorepulsive effect of Slit2-Robo2 signaling plays vital roles in nervous system development and neuron migration. Slit2-Robo2 signaling is also important for maintaining the normal morphogenesis of the kidney and urinary collecting system, especially for the branching of the ureteric bud (UB) at the proper site. Slit2 or Robo2 mouse mutants exhibit multilobular kidneys, multiple ureters, and dilatation of the ureter, renal pelvis, and collecting duct system, which lead to vesicoureteral reflux. To understand the effect of Robo2 on kidney development, we used microinjection and electroporation to overexpress GFP-Robo2 in an in vitro embryonic kidney model. Our results show reduced UB branching and decreased glomerular number after in vitro Robo2 overexpression in the embryonic kidneys. We found fewer metanephric mesenchymal (MM) cells surrounding the UB but no abnormal morphology in the branching epithelial UB. Meanwhile, no significant change in MM proliferation or apoptosis was observed. These findings indicate that Robo2 is involved in the development of embryonic kidneys and that the normal expression of Robo2 can help maintain proper UB branching and glomerular morphogenesis. Overexpression of Robo2 leads to reduced UB branching caused by fewer surrounding MM cells, but MM cell apoptosis is not involved in this effect. Our study demonstrates that overexpression of Robo2 by microinjection in embryonic kidneys is an effective approach to study the function of Robo2.     

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.     

Proline and hydroxyproline metabolism: implications for animal and human nutrition

Proline plays important roles in protein synthesis and structure, metabolism (particularly the synthesis of arginine, polyamines, and glutamate via pyrroline-5-carboxylate), and nutrition, as well as wound healing, antioxidative reactions, and immune responses. On a per-gram basis, proline plus hydroxyproline are most abundant in collagen and milk proteins, and requirements of proline for whole-body protein synthesis are the greatest among all amino acids. Therefore, physiological needs for proline are particularly high during the life cycle. While most mammals (including humans and pigs) can synthesize proline from arginine and glutamine/glutamate, rates of endogenous synthesis are inadequate for neonates, birds, and fish. Thus, work with young pigs (a widely used animal model for studying infant nutrition) has shown that supplementing 0.0, 0.35, 0.7, 1.05, 1.4, and 2.1% proline to a proline-free chemically defined diet containing 0.48% arginine and 2% glutamate dose dependently improved daily growth rate and feed efficiency while reducing concentrations of urea in plasma. Additionally, maximal growth performance of chickens depended on at least 0.8% proline in the diet. Likewise, dietary supplementation with 0.07, 0.14, and 0.28% hydroxyproline (a metabolite of proline) to a plant protein-based diet enhanced weight gains of salmon. Based on its regulatory roles in cellular biochemistry, proline can be considered as a functional amino acid for mammalian, avian, and aquatic species. Further research is warranted to develop effective strategies of dietary supplementation with proline or hydroxyproline to benefit health, growth, and development of animals and humans.     

Identification of bovine trophoblast protein-1, a secretory protein immunologically related to ovine trophoblast protein-1

This paper demonstrates that a group of proteins, representing a major secretory component of cattle conceptuses, is immunologically related to ovine trophoblast protein-1 (oTP-1), a principal product of culture Day 13 to 21 sheep conceptuses. Conceptuses from cows (Day 17-18) and ewes (Day 16-17) were cultured for 24 h in the presence of L-[3H]leucine. By using a rabbit antiserum to oTP-1 and Ouchterlony double-immunodiffusion analysis it was shown that material in the bovine conceptus culture medium was serologically related, but not identical, to oTP-1. A solid-phase radiobinding assay indicated that the cross-reacting bovine secretory component had an affinity for anti-oTP-1 antibody similar to that of oTP-1. Anti-oTP-1 antiserum specifically immunoprecipitated a group of 6-8 polypeptides from culture medium of cow conceptuses which, when analysed by two-dimensional gel electrophoresis, fell into two major molecular weight classes (22,000 and 24,000) with isoelectric points between 6.5 and 6.7. These immunoprecipitated polypeptides, defined as bTP-1, constituted the major secretory products of Day 16-25 cow conceptuses. They were larger and more basic than oTP-1 polypeptides (Mr about 18,000; pI 5.4-5.7). Anti-oTP-1 antiserum also recognized the major translation product of Day 17 bovine conceptus mRNA, a polypeptide significantly smaller (Mr approximately 18,000) than the secreted protein. It is suggested that oTP-1 and the homologous bovine protein may play similar roles in the phenomenon of maternal recognition of pregnancy in the two species.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.