Gary R. Newton

Inhibition of lymphocyte proliferation by ovine trophoblast protein-1 and a high molecular weight glycoprotein produced by the peri-implantation sheep conceptus

ABSTRACT: Sheep conceptuses were flushed from uteri on day 16 of pregnancy and cultured in vitro. Three peaks of immunosuppressive activity (i.e., ability to inhibit [3H]thymidine incorporation into mitogen‐stimulated lymphocytes) were localized in conceptus‐conditioned culture medium: one corresponding to a high‐molecular‐weight glycoprotein (HMWG; Mr = 800–900 kDa), one corresponding to ovine trophoblast protein‐1 (oTP‐1), the antiluteolytic, interferon‐like molecule of the sheep conceptus, and a third fraction containing a previously undescribed molecule with a molecular weight between 10 and 14 kDa. Both HMWG and oTP‐1 inhibited lymphocyte proliferation in a dose‐dependent manner. Delaying the addition of HMWG or oTP‐1 until 24 h after the addition of phytohemagglutinin (PHA) reduced the degree of suppression relative to cultures where HMWG and oTP‐1 were added at the time of PHA addition. Nonetheless, the molecules were still capable of inhibiting proliferation. Addition of human recombinant IL‐2 (0–65 U/ml) to PHA‐stimulated lymphocyte cultures could not reverse HMWG‐ or oTP‐1‐induced suppression of [3H]thymidine uptake by PHA‐stimulated cells. Furthermore, treatment of lymphocytes with HMWG or oTP‐1 suppressed IL‐2‐induced proliferation. Therefore, HMWG and oTP‐1 affect both early and later events in the in vitro proliferative response of mitogen‐stimulated lymphocytes, including responsiveness to IL‐2.

Galectin 15 (LGALS15): a gene uniquely expressed in the uteri of sheep and goats that functions in trophoblast attachment.

Abstract Galectins are a family of secreted animal lectins with biological roles in cell adhesion and migration. In sheep, galectin 15 (LGALS15) is expressed specifically in the endometrial luminal (LE) and superficial glandular (sGE) epithelia of the uterus in concert with blastocyst elongation during the peri-implantation period. The present study examined LGALS15 expression in the uterus of cattle, goats, and pigs. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels, and all contained a conserved carbohydrate recognition domain and RGD recognition sequence for integrin binding. Endometrial LGALS15 mRNA levels increased after Day 11 of both the estrous cycle and pregnancy, and were considerably increased after Day 15 of pregnancy in goats. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Immunoreactive LGALS15 protein was present in endometrial epithelia and conceptus trophectoderm of goat uteri and detected within intracellular crystal structures in trophectoderm and LE. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypothesis that LGALS15 is uniquely expressed in Caprinae endometria and functions as an attachment factor important for peri-implantation blastocyst elongation.

Protein restriction in early pregnancy alters fetal and placental growth and allantoic fluid proteins in swine

This study was designed to determine the impact of protein malnutrition during early pregnancy on fetal and placental growth and on the protein synthesis capacity of placental and endometrial tissues. Twelve crossbred sows received 1.8 kg/d of a control (13% protein) or protein-restricted (0.5% protein) diet from the day of breeding to Day 63 of pregnancy, when dissections were performed on each conceptus unit. The de novo protein synthetic rate of placental and endometrial explants was measured using (35)S-methionine. These proteins and the proteins from amniotic and allantoic fluids were separated by polyacrylamide gel electrophoresis. Placental weight was significantly reduced in the sows fed the restricted diet, with a tendency for decreased fetal weight as well. No differences were found due to dietary treatment in de novo protein synthesis or in the electrophoretic patterns of secreted proteins of the placenta or endometrium. The apparent quantity of 3 proteins in the allantoic fluid of the restricted diet fetuses decreased, while 1 protein increased in comparison with that of the control fetuses. These data suggest that protein malnutrition in early pregnancy decreases placental growth, thereby decreasing both fetal growth and the opportunity for compensatory growth upon nutritional rehabilitation.

secretion of the major progesterone-induced proteins of the sheep uterus by caruncular and intercaruncular endometrium of the pregnant ewe from days 20–140 of gestation

An experiment was performed to evaluate the types and quantity of proteins secreted by intercaruncular endometrium at days 20, 60, 100 and 140 of gestation and caruncular endometrium from days 100 and 140 of gestation. Tissues were obtained from ewes made unilaterally pregnant. Based on SDS polyacrylamide gel electrophoresis (SDS-PAGE), the major proteins present in uterine fluid at days 60-140 of gestation were the uterine milk proteins (UTM-proteins), a pair of structurally-related, progesterone-induced polypeptides with molecular weights of 55,000 and 57,000. These proteins were also present in uterine fluid at day 20, but the major protein at this time migrated coincident with albumin. Cultured explants of endometrium at all days of pregnancy produced UTM-proteins as their major radiolabelled product for both caruncular and intercaruncular endometrium. The amount of protein secretion in vitro was greater (P less than .04) for intercaruncular endometrium than for caruncular endometrium but was not significantly affected by stage of gestation or local presence of the conceptus. Immunohistochemical analysis revealed that UTM-proteins were present in glandular and luminal epithelium of intercaruncular endometrium and in both epithelial and stromal elements of caruncular endometrium. It was concluded that the UTM-proteins are produced earlier than previously described (i.e., day 20). In addition, caruncles contribute to the uterine secretory protein milieu through the secretion of proteins that are similar to that produced by the glandular intercaruncular epithelium.

Uterine secretions of the cow contain proteins that are immunochemically related to the major progesterone-induced proteins of the sheep uterus.

Proteins that cross-react with antiserum to the major progesterone-induced proteins found in the pregnant sheep uterus, the uterine milk proteins (UTM-proteins), were detected as radiolabelled secretory products of endometrium from pregnant cows. Cross-reactive proteins included a form at 57,000 molecular weight as well as other lower-molecular-weight variants found in lower amounts. Similar proteins were also detected in uterine fluid from a cow at day 270 of gestation. Using immunohistochemical procedures, proteins that cross-reacted with antiserum to sheep UTM-proteins could be localized to the epithelial cells of endometrial glands in the cow. Results indicate that UTM-protein-like molecules are secreted by the endometrium of the cow during pregnancy.

Biochemical and immunological properties of related small ruminant trophoblast interferons.

The objectives of this study were 1) to evaluate the biochemical and immunological properties of caprine interferon tau (cIFNtau), 2) to determine if intrauterine injection of recombinant ovine interferon tau (roIFNtau) extends CL life span in goats, and 3) to evaluate potential side effects of intramuscular (i.m.) administration roIFNtau. Caprine IFNtau was purified, and its effects on lymphocyte proliferation were evaluated. Incorporation of 3H-thymidine into newly synthesized DNA was suppressed (P<0.05) by cIFNtau. Spanish goats were also fitted with bilateral uterine catheters at Day 7 or 8 postestrus. The goats received twice-daily intrauterine injections of 100 microg roIFNtau (n = 4) or caprine serum proteins (n = 4) from Days 14 to 18 postestrus. Intrauterine injection of roIFNtau extended CL life span compared with that of control goats (26.4 +/- 1.7 vs 17.8 +/- 1.9 d, respectively; P<0.01). Potential side effects of intramuscular injections of roIFNtau were also evaluated. Goats received 0, 1, 2 or 4 mg of roIFNt on Days 10, 13, 16 or 19 of the estrous cycle. Treatment of goats with roIFN resulted in hyperthermia (P<0.01), with rectal temperatures of 40.5 degrees C recorded after 4 h and returning to normal (38.5 degrees C) after 24 h. Concomitant with the increase in rectal temperatures was a decrease (P<0.01) in plasma progesterone concentrations. Therefore, the tau interferons of goats and sheep have similar biological properties and roIFNtau has side effects associated with other classes of interferons.

Binding of immunoglobulins to the major progesterone-induced proteins secreted by the sheep uterus☆

We examined the binding of immunoglobulins to the uterine milk proteins, the major progesterone-induced proteins secreted by uterine endometrium of pregnant ewes. Binding was ascertained by measuring binding of 125 I-immunoglobulin to uterine milk proteins that were Western or dot-blotted to nitrocellulose or were coupled to Sepharose. The magnitude of binding was greatest for sheep IgM, intermediate for sheep secretory IgA, low for human secretory and serum IgA, and barely detectable for sheep IgG. Binding of IgA and IgM to uterine milk proteins was time and concentration dependent, saturable, inhibited by high ionic strength buffers, and lost due to enzymatic destruction of the Fc portion of the immunoglobulin molecule. In conclusion, the uterine milk proteins preferentially bind IgA and IgM in a species-dependent manner. Such binding may be related to the role of these proteins in the uterus and may make the uterine milk proteins a useful tool for studying or purifying sheep immunoglobulins.

Expression of Carbohydrate Antigens in the Goat Uterus During Early Pregnancy and on Steroid-Treated Polarized Uterine Epithelial Cells In Vitro

Abstract Our objectives were to determine whether specific fucosylated carbohydrate antigens, associated with uterine receptivity in rodents, are expressed in pregnant caprine uterine tissues and polarized uterine luminal epithelial (ULE) cells in culture. Immunofluorescence microscopy on frozen endometrium revealed that expression of the H-type 1 antigen, confined to epithelial cells, was regulated during early pregnancy. Staining was high on Day 5 and low on Days 11 and 13. Strong, uniform apical staining was characteristic of ULE cells between Days 15 and 19 but declined markedly by Day 25. Immunofluorescence analysis of the apical surface of polarized ULE cells cultured in steroid-free medium revealed weak and diffuse staining for the H-type 1 antigen, while progesterone (P4) treatment resulted in the formation of aggregates of punctate staining along the apical surface. Domain-specific biotinylation of polarized ULE cells, coupled with streptavidin precipitation and Western blotting, revealed that six apical surface proteins (31, 33, 42, 55, 60, and 70 kDa) carry the H-type 1 antigen. Therefore, H-type 1 antigen expression is up-regulated in vivo during the periimplantation period, stimulated by P4 on polarized ULE cells in culture, and may be a useful marker for uterine receptivity in this species.

REGULATION OF PROTEIN AND PROSTAGLANDIN SECRETION IN POLARIZED PRIMARY CULTURES OF CAPRINE UTERINE EPITHELIAL CELLS

SummaryCaprine uterine epithelial (UE) cells were cultured on Matrigel-coated filters. Transmission electron microscopy revealed polarized UE cells characterized by basally located nuclei, apical microvilli, convoluted lateral membranes, and junctional complexes. Domain-specific secretion of prostaglandins and radiolabeled proteins provide further evidence of functional epithelial cell polarity. Two experiments were conducted to evaluate factors controlling prostaglandin E2 (PGE) and prostaglandin F2α (PGF) secretion. In experiment one, steroid-treated (estradiol, progesterone, or estradiol + progesterone) polarized UE cells were treated with interferon tau (IFNτ) and/or oxytocin (OT). Steroid treatment did not influence PGE or PGF secretion. However, analysis of variance revealed an IFNτ by OT interaction (P<.01) for both PGE and PGF. This interaction was caused by a reduction in PGE and PGF secretion by cultures receiving only IFNτ and the inability of IFNτ to block OT-induced release of PGE or PGF. In experiment 2, polarized UE cells were cultured in progesterone, with or without IFNτ, and sequentially challenged with estradiol and OT. Oxytocin stimulated the release of both PGE and PGF by polarized cUE cells (P<.01) and resulted in an increased accumulation of PGE (OT*domain; P<.01) in the basal compartment. Interferon tau did not influence PGE (P<.1) secretion. However, further analysis revealed that IFNτ reduced PGF secretion and was unable to block OT-induced PGF secretion (IFNτ*OT; P<.05) by polarized UE cells. Therefore, caprine UE cells form polarized monolayers and retain responsiveness to IFNτ and OT in vitro.

Temporal changes in histomorphology and gene expression in goat testes during postnatal development.

Testicular cell proliferation and differentiation is critical for development of normal testicular function and male reproductive maturity. The objective of the current study was to evaluate histoarchitecture and expression of genes marking specific cells and important functions as well as testosterone production of the developing goat testes. Testes were harvested from Alpine bucks at 0, 2, 4, 6, and 8 mo of age (n = 5/age group). Paired testes weight increased from 2 to 4 (P < 0.001) and 4 to 6 mo (P < 0.01). The greatest increases in seminiferous tubule and lumen diameters and height of the seminiferous epithelium occurred between 2 and 4 mo (P < 0.001). Genes expressed in haploid germ cells (Protamine1 [PRM1], Outer Dense Fiber protein 2 [ODF2], and Stimulated by Retinoic Acid gene 8 [STRA8]) increased dramatically at the same time (P < 0.001). Expression of other genes decreased (P < 0.05) during testicular maturation. These genes included P450 side chain cleavage (CYP11A1), Sex determining region Y-box 9 (SOX9), Insulin-like Growth Factor 1 Receptor (IGF1R), and Heat Shock Protein A8 (HSPA8). The Glutathione S-Transferase A3 (GSTA3) gene, whose product was recently recognized as a primary enzyme involved in isomerization of androstenedione in man and livestock species including goats, sheep, cattle, pigs, and horses, uniquely peaked in expression at 2 mo (P < 0.05). Follicle-Stimulating Hormone Receptor (FSHR) mRNA abundance tended to steadily decrease with age (P = 0.1), while Luteinizing Hormone Receptor (LHCGR) mRNA abundance in testes was not significantly different across the ages. Testosterone content per gram of testicular tissue varied among individuals. However, testosterone content per testis tended to increase at 6 mo (P = 0.06). In conclusion, major changes in cellular structure and gene expression in goat testes were observed at 4 mo of age, when spermatogenesis was initiated. Male goats mature rapidly and represent a good model species for the study of agents that enhance or impair development of testicular functions.