Gregory A. Johnson

Amino acids and gaseous signaling.

Gases, such as nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H2S), and sulfur dioxide (SO2) are known toxic pollutants in the air. However, they are now recognized as important signaling molecules synthesized in animals and humans from arginine, glycine (heme), and cysteine, respectively. At physiological levels, NO, CO, and SO2 activate guanylyl cyclase to generate cGMP which elicits a variety of responses (including relaxation of vascular smooth muscle cells, hemodynamics, neurotransmission, and cell metabolism) via cGMP-dependent protein kinases. H2S is also a crucial regulator of both neurological function and endothelium-dependent relaxation through cGMP-independent mechanisms involving stimulation of membrane KATP channels and intracellular cAMP signaling. Additionally, NO, CO, and H2S confer cytoprotective and immunomodulatory effects. Moreover, NH3 is a major product of amino acid catabolism and profoundly affects the function of neurons and the vasculature through glutamine-dependent inhibition of NO synthesis. Emerging evidence shows that amino acids are not only precursors for these endogenous gases, but are also regulators of their production in a cell-specific manner. Thus, recent advances on gaseous signaling have greatly expanded our basic knowledge of amino acid biochemistry and nutrition. These exciting discoveries will aid in the design of new nutritional and pharmacological means to prevent and treat major health problems related to developmental biology and nutrient metabolism, including intrauterine growth restriction, preterm birth, aging, neurological disorders, cancer, obesity, diabetes, and cardiovascular disease.

Interferons and uterine receptivity.

This article focuses on the potential roles of interferons (IFNs) in establishing uterine receptivity to implantation. A common feature of the peri-implantation period of pregnancy in most mammals is production of type I and/or type II IFNs by trophoblasts that induce and/or stimulate expression of an array of IFN-stimulate genes (ISGs). These effects range from pregnancy recognition signaling in ruminants through IFN tau to effects on cellular functions of the uterus and uterine vasculature. For actions of IFNs, progesterone (P4) is permissive to the expression of many effects and to the expression of ISGs that are induced directly by an IFN or induced by P4 and stimulated by an IFN in a temporal and/or cell-specific manner. Uterine receptivity to implantation is P4 dependent; however, implantation events are preceded by loss of expression of progesterone (PGR) and estrogen (ESR1) receptors by uterine epithelia. Therefore, P4 likely acts via PGR-positive stromal cells to induce expression of fibroblast growth factors-7 and -10 and/or hepatocyte growth factor (progestamedins) that then act via their respective receptors on uterine epithelia and trophectoderm to affect expression of ISGs. The permissive effects of P4 on the expression of ISGs and the effects of P4 to induce and IFNs to stimulate gene expression raise the question of whether uterine receptivity to implantation requires P4 and IFN to activate unique, but complementary, cell signaling pathways. Uterine receptivity to implantation, depending on species, involves changes in the expression of genes for the attachment of trophectoderm to the uterine lumenal epithelium (LE) and superficial glandular epithelium (sGE), modification of the phenotype of uterine stromal cells, the silencing of PGR and ESR1 genes, the suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine LE/sGE, mid- to deep-glandular epithelia (GE), and stromal cells in response to P4 and IFNs is likely to influence uterine receptivity to implantation in most mammals. Understanding the roles of IFNs in uterine receptivity for implantation is necessary to develop approaches to enhance reproductive health and fertility in humans and domestic animals.

Protein delivery into live cells by incubation with an endosomolytic agent

We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTAT and a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

Select Nutrients in the Ovine Uterine Lumen. III. Cationic Amino Acid Transporters in the Ovine Uterus and Peri-Implantation Conceptuses

Abstract Arginine is an essential amino acid for conceptus (embryo/fetus and trophoblast/placenta) growth and development; however, the mechanisms for arginine transport into the uterine lumen and uptake by conceptuses are largely unknown. In this study, expression of System y+ (SLC7A1, SLC7A2, and SLC7A3) cationic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses was studied, and effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression were investigated. SLC7A1 mRNA was most abundant in endometrial luminal (LE) and superficial glandular (sGE) epithelia on Day 16 of the estrous cycle and on Days 16–20 of pregnancy, whereas SLC7A2 mRNA was most abundant in LE and mid to deep glandular (GE) epithelia on Days 14–20 of gestation. Expression of SLC7A1 and SLC7A2 was enhanced in pregnant ewes in a cell-specific manner, but abundance of SLC7A3 was not affected by day of the estrous cycle or by pregnancy status. SLC7A1, SLC7A2, and SLC7A3 mRNAs were expressed in trophectoderm and endoderm of conceptuses. In ovariectomized ewes, short-term treatment of ewes with P4 and IFNT did not affect endometrial SLC7A1 mRNA, while long-term treatment with P4 stimulated SLC7A1 in LE and GE, and IFNT tended to increase SLC7A1 abundance in LE. SLC7A2 mRNA abundance increased 4.1-fold in response to short-term P4 treatment and an additional 1.7-fold by IFNT primarily in endometrial LE/sGE, and these effects were ablated by a P4 receptor antagonist. These results indicate that coordinate changes in SLC7A1, SLC7A2, and SLC7A3 expression in uterine endometria and conceptuses are likely important in transport of arginine that is critical to conceptus growth, development, and survival.

Expression of Porcine Endometrial Prostaglandin Synthase During the Estrous Cycle and Early Pregnancy, and Following Endocrine Disruption of Pregnancy

Abstract Porcine trophoblast attachment to the uterine surface is associated with increased conceptus and endometrial production of prostaglandins. Conceptus secretion of estrogen on Day 12 of gestation is important for establishment of pregnancy; however, early (Days 9 and 10) exposure to exogenous estrogens results in embryonic mortality. Present studies established the temporal and spatial pattern of endometrial PTGS1 (prostaglandin-endoperoxide synthase 1) and PTGS2 expression during the estrous cycle and early pregnancy and determined the effect of early estrogen treatment on endometrial PTGS expression in pregnant gilts. Endometrial PTGS1 mRNA expression increased 2- to 3-fold after Day 10 of the estrous cycle and pregnancy, whereas PTGS2 mRNA expression increased 76-fold between Days 5 and 15 of the estrous cycle and pregnancy. Increased expression of the PTGS2 transcript was detected in the lumenal epithelium after Day 10 in both cyclic and pregnant gilts. There was a 10- and 20-fold increase in endometrial PTGS2 protein expression between Days 5 and 18 of the estrous cycle and pregnancy respectively. Administration of estrogen on Days 9 and 10 of gestation increased endometrial PTGS2 mRNA and protein on Day 10, but decreased PTGS2 mRNA and protein in lumenal epithelium (LE) on Day 12 of gestation compared to vehicle-treated gilts. The present study demonstrates that an increase in uterine epithelial PTGS2 expression occurs after Day 10 of the estrous cycle and early pregnancy in the pig. The conceptus-independent increase in the uterine LE indicates that a novel pathway exists for endometrial induction PTGS2 expression before conceptus elongation and attachment to the uterine surface. Epithelial expression of PTGS2 may serve as one of the signals for placental attachment and embryo survival in the pig. Early administration of estrogen on Days 9 and 10 of pregnancy alters endometrial PTGS2 mRNA and protein expression, which may, at least in part, represent a mechanism by which endocrine disruption of pregnancy causes total embryonic loss during implantation in the pig.

Endometrial ISG17 mRNA and a related mRNA are induced by interferon-tau and localized to glandular epithelial and stromal cells from pregnant cows.

The interferon stimulated gene product, ISG17, conjugates to bovine uterine proteins in response to conceptus-derived interferon (IFN)-τ. The objectives of the present experiments were to examine induction of ISG17 (0.65 kb) and a related 2.5 kb mRNA in response to IFN-τ and pregnancy using Northern blotting procedures, and to determine cell types in the endometrium that expressed ISG17 mRNA usingin situ hybridization. RNA was isolated from endometrial explants or from bovine endometrial (BEND) cells cultured in the absence (control) or presence of 25 nM recombinant (r) bolFN-τ for 0, 3, 6, 12, 24, or 48 h. The major ISG17 0.65 kb mRNA and a minor 2.5 kb mRNA were induced (p<0.05) after 6 h (explants) or 3 h (BEND cells) treatment with rbolFN-τ. Both mRNAs were present in endometrium from day 18 pregnant cows, but were absent in endometrium from nonpregnant cows. The ISG17 mRNA was localized to stromal and glandular epithelial cells on d 18 of pregnancy. The 2.5 kb mRNA may encode a novel ISG17 homolog, or a unique polyISG17 repeat that is similar in structure to the polyubiquitin genes. Because ISG17 mRNA is induced in stromal and glandular epithelial cells, it could be assumed that ISG17 has a role in regulating intracellular proteins in both cell types.

Pig blastocyst-uterine interactions.

The litter-bearing pig is an invaluable model for research in reproductive biology. Spherical pig blastocysts on Day 10 of pregnancy undergo rapid morphological changes to tubular and then filamentous forms by Day 12 and a filamentous conceptus of almost 1m in length by Day 16 of pregnancy. Thus, trophectoderm of each conceptus achieves intimate contact with luminal uterine epithelium (LE) for exchange of nutrients, gases, hormones, growth factors and other key molecules for survival and development. Estrogens secreted between Days 11 and 13 of pregnancy signals pregnancy recognition to ensure that nutrients and prostaglandin F2-alpha (PGF) are secreted into the uterine lumen (exocrine secretion) rather than into the uterine vein (endocrine secretion) which would lead to regression of the corpora lutea (CL) and failure to maintain pregnancy. Pigs have a true epitheliochorial placenta. The fluid filled amnion bouys the embryo so that it develops symmetrically. The allantois fills with allantoic fluid to expand contact of the chorioallantois with uterine LE, and the allanotois supports the vascular system of the placenta. The chorion/trophectoderm in direct contact with uterine LE exchanges gases and nutrients and forms unique structures call areolae that absorb nutrient-rich secretions from uterine glands and transports them directly into fetal blood. The period from Days 20 to 70 of pregnancy is for placental growth in preparation for rapid fetal growth between Days 70 and 114 (term) of gestation. Maturation of the fetal hypothalamic-pituitary-adrenal axis leads to increases in secretion of cortisol from the fetal adrenal glands. Cortisol sets in motion secretion of estrogens, oxytocin, relaxin and prolactin, as well as increases in their receptors, which are required for delivery of piglets and for initiation of lactation and expression of maternal behavior. This review provides details of gestation in the pig with respect to uterine biology, implantation, placentation, fetal development and parturition.

Uterine biology in pigs and sheep

There is a dialogue between the developing conceptus (embryo-fetus and associated placental membranes) and maternal uterus which must be established during the peri-implantation period for pregnancy recognition signaling, implantation, regulation of gene expression by uterine epithelial and stromal cells, placentation and exchange of nutrients and gases. The uterus provide a microenvironment in which molecules secreted by uterine epithelia or transported into the uterine lumen represent histotroph required for growth and development of the conceptus and receptivity of the uterus to implantation. Pregnancy recognition signaling mechanisms sustain the functional lifespan of the corpora lutea (CL) which produce progesterone, the hormone of pregnancy essential for uterine functions that support implantation and placentation required for a successful outcome of pregnancy. It is within the peri-implantation period that most embryonic deaths occur due to deficiencies attributed to uterine functions or failure of the conceptus to develop appropriately, signal pregnancy recognition and/or undergo implantation and placentation. With proper placentation, the fetal fluids and fetal membranes each have unique functions to ensure hematotrophic and histotrophic nutrition in support of growth and development of the fetus. The endocrine status of the pregnant female and her nutritional status are critical for successful establishment and maintenance of pregnancy. This review addresses the complexity of key mechanisms that are characteristic of successful reproduction in sheep and pigs and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of livestock species.

Dietary Supplementation with 0.8% l-Arginine between Days 0 and 25 of Gestation Reduces Litter Size in Gilts

In this study, we determined the effects of L-arginine supplementation during early pregnancy on embryonic/fetal survival and growth in gilts. Gilts were housed individually in pens and fed twice daily 1 kg of a corn- and soybean meal-based diet supplemented with 0.0, 0.4, or 0.8% L-arginine (wt:wt) between d 0 and 25 of gestation (10 gilts/treatment). The diets were made isonitrogenous by addition of appropriate amounts of L-alanine. At d 25 of gestation, gilts were fed L-alanine or L-arginine and hysterectomized 30 min later to obtain uteri and conceptuses (embryos and associated fetal membranes and fluids). Dietary supplementation with 0.4 or 0.8% L-arginine enhanced (P < 0.05) its concentrations in maternal plasma (64 and 98%, respectively) as well as the vascularity of chorionic and allantoic membranes, compared with the control group. Reproductive performance [numbers of corpora lutea (CL) and fetuses, placental and fetal weights, and embryonic mortality] did not differ between the 0.4% Arg and control groups. However, supplementation with 0.8% L-arginine decreased (P < 0.05) uterine weight (-20%), total number of fetuses (-24%), CL number (-17%), total fetal weight (-34%), total volume of allantoic and amniotic fluids (-34 to 42%), concentrations of progesterone in maternal plasma (-33%), as well as total amounts of progesterone (-35%), estrone (-40%), and estrone sulfate (-37%) in allantoic fluid, compared with the control group. These results indicate that dietary supplementation with 0.8% L-arginine between d 0 and 25 of gestation, while increasing placental vascularity, adversely affects the reproductive performance of gilts.

Arginine, Leucine, and Glutamine Stimulate Proliferation of Porcine Trophectoderm Cells Through the MTOR-RPS6K-RPS6-EIF4EBP1 Signal Transduction Pathway

ABSTRACT During the peri-implantation and early placentation periods in pigs, conceptuses (embryo and its extra-embryonic membranes) undergo dramatic morphological changes and differentiation that require the exchange of nutrients (histotroph) and gasses across the trophectoderm and a true epitheliochorial placenta. Of these nutrients, arginine (Arg), leucine (Leu), and glutamine (Gln) are essential components of histotroph; however, little is known about changes in their total amounts in the uterine lumen of cyclic and pregnant gilts and their effects on cell signaling cascades. Therefore, we determined quantities of Arg, Leu, and Gln in uterine luminal fluids and found that total recoverable amounts of these amino acids increased in pregnant but not cyclic gilts between Days 12 and 15 after onset of estrus. We hypothesized that Arg, Leu, and Gln have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary porcine trophectoderm (pTr) cells treated with either Arg, Leu, or Gln had increased abundance of phosphorylated RPS6K, RPS6, and EIF4EBP1 compared to basal levels, and this effect was maintained for up to 120 min. When pTr cells were treated with Arg, Leu, and Gln, low levels of pRPS6K and pEIF4EBP1 were detected in the cytosol, but the abundance of nuclear pRPS6K increased. Immunofluorescence analyses revealed abundant amounts of pRPS6 protein in the cytoplasm of pTr cells treated with Arg, Leu, and Gln. These amino acids also increased proliferation of pTr cells. Furthermore, when Arg, Leu, and Gln were combined with siRNAs for either MTOR, RPTOR, or RICTOR, effects of those amino acids on proliferation of pTr cells were significantly inhibited. Collectively, these results indicate that Arg, Leu, and Gln act coordinately to stimulate proliferation of pTr cells through activation of the MTOR-RPS6K-RPS6-EIF4EBP1 signal transduction pathway.