Expression of progesterone receptor in the porcine uterus and placenta throughout gestation: correlation with expression of uteroferrin and osteopontin.

Progesterone (P4) stimulates production and secretion of histotroph, a mixture of hormones, growth factors, nutrients, and other substances required for growth and development of the conceptus (embryo or fetus and placental membranes). Progesterone acts through the progesterone receptor (PGR); however, there is a gap in our understanding of P4 during pregnancy because PGR have not been localized in the uteri and placentae of pigs beyond day 18. Therefore, we determined endometrial expression of PGR messenger RNA (mRNA) and localized PGR protein in uterine and placental tissues throughout the estrous cycle and through day 85 of pregnancy in pigs. Further, 2 components of histotroph, tartrate-resistant acid phosphatase 5 (ACP5; uteroferrin) and secreted phosphoprotein 1 (SPP1; osteopontin) proteins, were localized in relation to PGR during pregnancy. Endometrial expression of PGR mRNA was highest at day 5 of the estrous cycle, decreased between days 5 and 11 of both the estrous cycle and pregnancy, and then increased between days 11 and 17 of the estrous cycle (P < 0.01), but decreased from days 13 to 40 of pregnancy (P < 0.01). Progesterone receptor protein localized to uterine stroma and myometrium throughout all days of the estrous cycle and pregnancy. PGR were expressed by uterine luminal epithelium (LE) between days 5 and 11 of the estrous cycle and pregnancy, then PGR became undetectable in LE through day 85 of pregnancy. During the estrous cycle, PGR were downregulated in LE between days 11 and 15, but expression returned to LE on day 17. All uterine glandular epithelial (GE) cells expressed PGR from days 5 to 11 of the estrous cycle and pregnancy, but expression decreased in the superficial GE by day 12. Expression of PGR in GE continued to decrease between days 25 and 85 of pregnancy; however, a few glands near the myometrium and in close proximity to areolae maintained expression of PGR protein. Acid phosphatase 5 protein was detected in the GE from days 12 to 85 of gestation and in areolae. Secreted phosphoprotein 1 protein was detected in uterine LE in apposition to interareolar, but not areolar areas of the chorioallantois on all days examined, and in uterine GE between days 35 and 85 of gestation. Interestingly, uterine GE cells adjacent to areolae expressed PGR, but not ACP5 or SPP1, suggesting these are excretory ducts involved in the passage, but not secretion, of histotroph into the areolar lumen and highlighting that P4 does not stimulate histotroph production in epithelial cells that express PGR.

Technical note: Isolation and characterization of porcine mammary epithelial cells.

Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), ß-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 µg/mL prolactin to culture medium for 3 d induced the production of ß-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow.

Technical note: Isolation and characterization of ovine brown adipocyte precursor cells.

Brown adipose tissue (BAT) plays a critical role in regulating body temperature in newborn lambs. Availability of a stable BAT cell line would be invaluable for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of fetal BAT growth and development. Ovine brown adipocyte precursor cells (BAPC) were isolated from fetal lambs at d 90 of gestation and cultured to establish a stable cell line. These cells were characterized by adipogenic differentiation and expression of a hallmark gene, (). The BAPC doubled every 24 h. After a 9-d induction with a serum-free Dulbecco's modified Eagle Ham/F12 medium, BAPC differentiated into brown adipocytes with large lipid droplets. The differentiation medium induced expression of mRNA and protein in BAPC. Furthermore, after BAPC were passaged 30 times, they maintained similar cell morphology, the potential for adipogenic differentiation, and the ability to express . Taken together, we have established a stable ovine BAPC cell line for studying nutritional regulation of BAT growth and development in the fetus.

Triennial Growth Symposium: important roles for L-glutamine in swine nutrition and production.

L-Glutamine (Gln) has traditionally not been considered a nutrient needed in diets for livestock species or even mentioned in classic animal nutrition textbooks. This is due to previous technical difficulties in Gln analysis and the unsubstantiated assumption that animals can synthesize sufficient amounts of Gln to meet their needs. Consequently, the current (1998) version of NRC does not recommend dietary Gln requirements for swine. This lack of knowledge about Gln nutrition has contributed to suboptimal efficiency of global pig production. Because of recent advances in research, Gln is now known to be an abundant AA in physiological fluids and proteins and a key regulator of gene expression. Additionally, Gln can regulate cell signaling via the mammalian target of rapamycin pathway, adenosine monophosphate-activated protein kinase, extracellular signal-related kinase, Jun kinase, mitogen-activated protein kinase, and nitric oxide. The exquisite integration of Gln-dependent regulatory networks has profound effects on cell proliferation, differentiation, migration, metabolism, homeostasis, survival, and function. As a result of translating basic research into practice, dietary supplementation with 1% Gln maintains gut health and prevents intestinal dysfunction in low-birth-weight or early-weaned piglets while increasing their growth performance and survival. In addition, supplementing 1% Gln to a corn- and soybean-meal-based diet between d 90 and 114 of gestation ameliorates fetal growth retardation in gilts and reduces preweaning mortality of piglets. Furthermore, dietary supplementation with 1% Gln enhances milk production by lactating sows. Thus, adequate amounts of dietary Gln, a major nutrient, are necessary to support the maximum growth, development, and production performance of swine.

Morphologic and cytokine profile characterization of Salmonella enterica serovar typhimurium infection in calves with bovine leukocyte adhesion deficiency.

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium-induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18-), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18- calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18- calves--including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration--were similar to the ones reported in the literature for CD18- calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18- calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene alpha in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha were poorly correlated with the presence or absence of tissue neutrophils.

Impacts of amino acid nutrition on pregnancy outcome in pigs: mechanisms and implications for swine production.

Pigs suffer up to 50% embryonic and fetal loss during gestation and exhibit the most severe naturally occurring intrauterine growth retardation among livestock species. Placental insufficiency is a major factor contributing to suboptimal reproductive performance and reduced birth weights of pigs. Enhancement of placental growth and function through nutritional management offers an effective solution to improving embryonic and fetal survival and growth. We discovered an unusual abundance of the arginine family of AA in porcine allantoic fluid (a reservoir of nutrients) during early gestation, when placental growth is most rapid. Arginine is metabolized to ornithine, proline, and nitric oxide, and these compounds possess a plethora of physiological functions. Nitric oxide is a vasodilator and angiogenic factor, whereas both ornithine and proline are substrates for placental synthesis of polyamines, which are key regulators of protein synthesis and angiogenesis. Additionally, arginine, leucine, glutamine, and proline activate the mammalian target of rapamycin cell-signaling pathway to enhance protein synthesis and cell proliferation in placentae. To translate basic research on AA biochemistry and nutrition into application, dietary supplementation with 0.83% l-arginine to gilts on d 14 to 28 or d 30 to 114 of gestation increased the number and litter birth weight of live-born piglets. In addition, supplementing the gestation diet with 0.4% l-arginine plus 0.6% l-glutamine enhanced the efficiency of nutrient utilization, reduced variation in piglet birth weight, and increased litter birth weight. By regulating syntheses of nitric oxide, polyamines, and proteins, functional AA stimulate placental growth and the transfer of nutrients from mother to embryo or fetus to promote conceptus survival, growth, and development.

Conceptus-uterus interactions in pigs: endometrial gene expression in response to estrogens and interferons from conceptuses.

This review highlights information on conceptus-uterus interactions in the pig with respect to uterine gene expression in response to estrogens and interferons (IFNs) secreted from elongating conceptuses. Pig conceptuses release estrogens for pregnancy recognition, but also secrete IFNs that do not appear to be antiluteolytic. Estrogens and IFNs induce expression of largely non-overlapping sets of genes, and evidence suggests that pig conceptuses orchestrate essential events of early pregnancy including pregnancy recognition signaling, implantation and secretion of histotroph by precisely controlling temporal and spatial (cell-specific) changes in uterine gene expression through initial secretion of estrogens, followed by cytokines including IFNG and IFND. By Day 12 of pregnancy, estrogens increase the expression of multiple genes in the uterine luminal epithelium including SPP1, STC1, IRF2 and STAT1 that likely have roles for implantation. By Day 15 of pregnancy, IFNs upregulate a large array of IFN responsive genes in the underlying stroma and glandular epithelium including ISG15, IRF1, STAT1, SLAs and B2M that likely have roles in uterine remodeling to support placentation.

Increased susceptibility of aging kidney to ischemic injury: identification of candidate genes changed during aging, but corrected by caloric restriction.

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism underlying the increased susceptibility to injury remains undefined. These experiments were designed to investigate the influence of age on the response of the kidney to ischemic injury and to identify candidate genes that may mediate this response. Renal slices prepared from young (5 mo), aged ad libitum (aged-AL; 24 mo), and aged caloric-restricted (aged-CR; 24 mo) male Fischer 344 rats were subjected to ischemic stress (100% N(2)) for 0-60 min. As assessed by biochemical and histological evaluation, slices from aged-AL rats were more susceptible to injury than young counterparts. Importantly, caloric restriction attenuated the increased susceptibility to injury. In an attempt to identify the molecular pathway(s) underlying this response, microarray analysis was performed on tissue harvested from the same animals used for the viability experiments. RNA was isolated and the corresponding cDNA was hybridized to CodeLink Rat Whole Genome Bioarray slides. Subsequent gene expression analysis was performed using GeneSpring software. Using two-sample t-tests and a twofold cut-off, the expression of 92 genes was changed during aging and attenuated by caloric restriction, including claudin-7, kidney injury molecule-1 (Kim-1), and matrix metalloproteinase-7 (MMP-7). Claudin-7 gene expression peaked at 18 mo; however, increased protein expression in certain tubular epithelial cells was seen at 24 mo. Kim-1 gene expression was not elevated at 8 or 12 mo but was at 18 and 24 mo. However, changes in Kim-1 protein expression were only seen at 24 mo and corresponded to increased urinary levels. Importantly, these changes were attenuated by caloric restriction. MMP-7 gene expression was decreased at 8 mo, but an age-dependent increase was seen at 24 mo. Increased MMP-7 protein expression in tubular epithelial cells at 24 mo was correlated with the gene expression pattern. In summary, we identified genes changed by aging and changes attenuated by caloric restriction. This will facilitate investigation into the molecular mechanism mediating the age-related increase in susceptibility to injury.

Fetal-maternal interactions during the establishment of pregnancy in ruminants.

This review integrates established information with new insights into molecular and physiological mechanisms responsible for events leading to pregnancy recognition, endometrial receptivity, and implantation with emphasis on sheep. After formation of the corpus luteum, progesterone acts on the endometrium and stimulates blastocyst growth and elongation to form a filamentous conceptus (embryo/fetus and associated extraembryonic membranes). Recurrent early pregnancy loss in the uterine gland knockout ewe model indicates that endometrial epithelial secretions are essential for peri-implantation blastocyst survival and growth. The elongating sheep conceptus secretes interferon tau (IFNT) that acts on the endometrium to inhibit development of the luteolytic mechanism by inhibiting transcription of the estrogen receptor alpha (ESR1) gene in the luminal (LE) and superficial ductal glandular (sGE) epithelia, which prevents estrogen-induction of oxytocin receptors (OXTR) and production of luteolytic prostaglandin F2-alpha pulses. Progesterone downregulates its receptors (PGR) in LE and then GE, correlating with a reduction of anti-adhesive MUC1 (mucin glycoprotein one) and induction of secreted LGALS15 (galectin 15) and SPP1 (secreted phosphoprotein one), that are proposed to regulate trophectoderm growth and adhesion. IFNT acts on the LE to induce WNT7A (wingless-type MMTV integration site family member 7A) and to stimulate LGALS15, CTSL (cathepsin L), and CST3 (cystatin C), which may regulate conceptus development and implantation. During the peri-implantation period, trophoblast giant binucleate cells (BNC) begin to differentiate from mononuclear trophectoderm cells, migrate and then fuse with the uterine LE as well as each other to form multinucleated syncytial plaques. Trophoblast giant BNC secrete chorionic somatomammotropin (CSH1 or placental lactogen) that acts on the endometrial glands to stimulate their morphogenesis and differentiated function. The interactive, coordinated and stage-specific effects of ovarian and placental hormones regulate endometrial events necessary for fetal-maternal interactions and successful establishment of pregnancy.

WNTs in the ovine uterus: potential regulation of periimplantation ovine conceptus development.

WNTs (Wingless-type MMTV integration site family member) are involved in critical developmental and growth processes in animals. These studies investigated WNT pathways in the ovine uterus and conceptus during the periimplantation period of pregnancy. WNT2 and WNT2B mRNAs were detected in endometrial stroma. WNT5A and WNT5B mRNAs were most abundant in the stroma and less so in the luminal epithelium, whereas WNT11 mRNA was detected primarily in the glands. WNT7A mRNA was present in the luminal epithelium on d 10, absent on d 12 and 14, and increased between d 16 and 20. Only WNT2, WNT2B, and WNT4 were detected in conceptus trophectoderm. FZD6/8 (frizzled receptor) and GSK3B (glycogen synthase kinase 3beta) mRNAs were detected primarily in endometrial epithelia and conceptus trophectoderm, whereas the LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6) coreceptor was present in all endometrial cells and the trophectoderm. DKK1 (Dickkopf), a WNT signaling inhibitor, increased in the endometrium from d 16-20. CTNNB1 [catenin (cadherin associated protein) beta1] and CDH1 (E-cadherin) mRNAs were most abundant in the endometrial epithelia and trophectoderm. LEF1 (lymphoid enhancer-binding factor 1) mRNA was expressed primarily in uterine epithelia, whereas TCF7L2 [(transcription factor 7-like 2 (T-cell specific, HMG-box)] was primarily in the conceptus. CTNNB1 and TCF7L2 proteins were both abundant in the nuclei of trophoblast giant binucleate cells. WNT7A stimulated a TCF/LEF-luciferase reporter activity in ovine trophectoderm cells that was inhibited by dominant-negative TCF and Sfrp2 (secreted FZD-related protein 2). WNT7A increased trophectoderm cell proliferation as well as MSX2 (msh homeobox 2) and MYC (myelocytomatosis oncogene) mRNA levels. Wnt5a increased trophectoderm cell migration in a Rho kinase-dependent manner. These results support the hypotheses that canonical and noncanonical WNT signaling pathways are conserved regulators of conceptus-endometrial interactions in mammals and regulate periimplantation ovine conceptus development.

Rescuing valuable genomes by animal cloning: a case for natural disease resistance in cattle.

Tissue banking and animal cloning represent a powerful tool for conserving and regenerating valuable animal genomes. Here we report an example involving cattle and the rescue of a genome affording natural disease resistance. During the course of a 2-decade study involving the phenotypic and genotypic analysis for the functional and genetic basis of natural disease resistance against bovine brucellosis, a foundation sire was identified and confirmed to be genetically resistant to Brucella abortus. This unique animal was utilized extensively in numerous animal breeding studies to further characterize the genetic basis for natural disease resistance. The bull died in 1996 of natural causes, and no semen was available for AI, resulting in the loss of this valuable genome. Fibroblast cell lines had been established in 1985, cryopreserved, and stored in liquid nitrogen for future genetic analysis. Therefore, we decided to utilize these cells for somatic cell nuclear transfer to attempt the production of a cloned bull and salvage this valuable genotype. Embryos were produced by somatic cell nuclear transfer and transferred to 20 recipient cows, 10 of which became pregnant as determined by ultrasound at d 40 of gestation. One calf survived to term. At present, the cloned bull is 4.5 yr old and appears completely normal as determined by physical examination and blood chemistry. Furthermore, in vitro assays performed to date indicate this bull is naturally resistant to B. abortus, Mycobacterium bovis, and Salmonella typhimurium, as was the original genetic donor.

Caprine uterine and placental osteopontin expression is distinct among epitheliochorial implanting species.

Osteopontin (OPN) is the most highly up-regulated extracellular matrix/adhesion molecule in the uterus of humans and domestic animals as it becomes receptive to implantation. Studies in sheep and pigs have shown that OPN is a component of ovine and porcine histotroph characterized by a complex temporal and spatial pattern of uterine and conceptus expression involving immune, epithelial, and stromal cells. It is proposed that these expression events are orchestrated to contribute to conceptus attachment and placentation. However, differences in OPN expression between sheep and pigs have been detected that relate to differences in placentation. Therefore, this study examined OPN expression in the caprine uterus and conceptus to gain insight into mechanisms underlying OPN function(s) during pregnancy through comparative analysis of differences in placentation between pigs, sheep, and goats. Goats were hysterectomized (n = 5/day) on Days 5, 11, 13, 15, 17 or 19 of the estrous cycle, and Days 5, 11, 13, 15, 17, 19 or 25 of pregnancy. Slot-blot hybridization showed increases in endometrial OPN mRNA beginning on Day 17 of the estrous cycle and Day 19 of pregnancy. In situ hybridization localized OPN mRNA to endometrial glandular epithelium (GE), Day 25 myometrium, and cells scattered within the placenta hypothesized to be immune. Immunofluorescence microscopy detected OPN protein on the apical surface of endometrial lumenal epithelium (LE), in GE, and on conceptus (Tr). Western blot analysis detected primarily the native 70-kDa OPN protein in endometrial extracts from the estrous cycle and pregnancy, as well as in uterine flushings from pregnant goats. Co-induction of OPN and alpha-smooth muscle actin, but not desmin proteins, was observed in uterine stroma by Day 25 of pregnancy. OPN in cyclic GE, Day 25 myometrium, and desmin-negative endometrial stroma is unique and reflects subtle differences among superficial implanting species that correlate with the depth of Tr invasion.

Conceptus signals for establishment and maintenance of pregnancy.

Establishment and maintenance of pregnancy results from signaling by the conceptus (embryo/fetus and associated extraembryonic membranes) and requires progesterone produced by the corpus luteum. In most mammals, hormones produced by the trophoblast maintain progesterone production by acting directly or indirectly to maintain the corpus luteum. In domestic animals (ruminants and pigs), hormones from the trophoblast are antiluteolytic in that they act on the endometrium to prevent uterine release of luteolytic prostaglandin F2alpha. In cyclic and pregnant sheep, progesterone negatively autoregulates progesterone receptor gene expression in the endometrial luminal and superficial glandular epithelium. In cyclic sheep, loss of the progesterone receptor is closely followed by increases in epithelial estrogen receptors and then oxytocin receptors, allowing oxytocin to induce uterine release of luteolytic prostaglandin F2alpha pulses. In pregnant sheep, the conceptus trophoblast produces interferon tau that acts on the endometrium to inhibit transcription of the estrogen receptor alpha gene directly and the oxytocin receptor gene indirectly to abrogate development of the endometrial luteolytic mechanism. Subsequently, sequential, overlapping actions of progesterone, interferon tau, placental lactogen, and growth hormone comprise a hormonal servomechanism that regulates endometrial gland morphogenesis and terminal differentiated function to maintain pregnancy in sheep. In pigs, the conceptus trophoblast produces estrogen that alters the direction of prostaglandin F2alpha secretion from an endocrine to exocrine direction, thereby sequestering luteolytic prostaglandin F2alpha within the uterine lumen. Conceptus estrogen also increases expression of fibroblast growth factor 7 in the endometrial lumenal epithelium that, in turn, stimulates proliferation and differentiated functions of the trophectoderm, which expresses the fibroblast growth factor 7 receptor. Strategic manipulation of these physiological mechanisms may improve uterine capacity, conceptus survival, and reproductive health.

Production of nuclear transfer horse embryos by Piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract.

We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein) and two activation times (1.5-2 vs. 8-10 h after nuclear transfer) were examined. Cleavage and activation (pseudopronucleus formation) rates of oocytes injected with sperm extract containing 59 microg/ml protein were significantly (P < 0.05) lower than any other dosage. The percentage of embryos cleaving with normal nuclei in oocytes injected with the 1375 microg/ml preparation 1.5-2 h after donor injection was significantly (P < 0.05) higher than that of the 293 microg/ml preparation 8-10 h after donor injection (22 vs. 6%). Embryos developed to a maximum of 10 nuclei. Interspecies nuclear transfer was performed by direct injection of horse nuclei into enucleated bovine oocytes, followed by chemical activation. This resulted in 81% reconstruction (successful injection of the donor cell), 88% cleavage, and 73% cleavage with normal nuclei. These results indicate that direct nuclear injection using the Piezo drill is an efficient method for nuclear transfer in horse and cattle oocytes and that sperm extract can efficiently activate horse oocytes both parthenogenetically and after nuclear transfer

Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation.

Endometrial glands are necessary for conceptus implantation and growth. In the ovine uterine gland knockout (UGKO) model, blastocysts hatch normally but fail to survive or elongate. This peri-implantation defect in UGKO ewes may be due to the absence of endometrial glands or, alternatively, to the lack of certain epithelial adhesion molecules or the inability of the endometrium to respond to signals from the conceptus. Two studies were performed to examine these hypotheses. In study one, normal (n = 8) and UGKO (n = 12) ewes were mated at oestrus (day 0) with intact rams and their uteri were flushed 14 days after oestrus. Normal ewes (n = 4) were also flushed on 14 days after oestrus. Uterine flushes from bred normal ewes contained filamentous conceptuses (n = 7 of 8), whereas those from UGKO ewes contained no conceptus (n = 5 of 12), a growth-retarded, tubular conceptus (n = 6 of 12), or a fragmented, filamentous conceptus (n = 1 of 12). In all groups, expression of mucin 1 and integrin alpha(v), alpha(5), beta(3) and beta(5) was localized at the apical surface of the endometrial luminal epithelium with no detectable differences between normal and UGKO ewes. Uterine flushes from pregnant ewes, but not cyclic or UGKO ewes, contained abundant immunoreactive interferon tau and the cell adhesion proteins, osteopontin and glycosylation-dependent cell adhesion molecule one. In study two, UGKO ewes were fitted with uterine catheters 5 days after oestrus, infused with recombinant ovine interferon tau or control proteins from 11 to 15 days after oestrus, and underwent hysterectomy 16 days after oestrus. Expression of several interferon tau-stimulated genes (ISG17, STAT1, STAT2 and IRF-1) was increased in the endometrium from interferon tau-infused UGKO ewes. These results support the hypothesis that the defects in conceptus elongation and survival in UGKO ewes are due to the absence of endometrial glands and their secretions rather than to alterations in expression of anti-adhesive or adhesive molecules on the endometrial luminal epithelium or to the responsiveness of the endometrium to the conceptus pregnancy recognition signal.

A study of 2,4,6-trinitrotoluene inhibition of benzo[a]pyrene uptake and activation in a microbial mutagenicity assay.

A number of in vitro and in vivo studies have determined that binary and complex mixtures may interact to produce a toxicity that could not be predicted based on the individual chemicals. The present study was conducted with a binary mixture of model compounds to investigate possible interactions affecting their mutagenicity. The compounds included Benzo[a]pyrene (BAP), a polycyclic aromatic hydrocarbon that is an indirect-acting mutagen of great environmental concern, and 2,4,6-Trinitrotoluene (TNT), a nitro-aromatic compound that is a direct-acting mutagen frequently found as a soil contaminant at munitions sites. This study indicated that a binary mixture of BAP and TNT failed to induce the positive mutagenic response in Salmonella typhimurium strain TA98 characteristic of either compound alone. Spectrofluorometric analysis of BAP, and kinetic analyses of 3HBAP uptake in the presence or absence of TNT using TA98 cells that were treated or untreated with activated rat liver microsomes were performed. In cells preloaded with BAP, cellular BAP fluorescence was rapidly suppressed in the presence of TNT. Mass spectroscopy of BAP and TNT mixtures revealed a number of products, believed to be the result of complexation and nitration, that may account for the antagonistic action of TNT on BAP-induced mutagenicity in TA98 cells. Further, kinetic studies indicated that TNT inhibited the incorporation of BAP into cells.

Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus.

Interferon tau (IFNtau) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFNtau on IFN-stimulated gene (ISG) expression are mediated by ISG factor 3 (ISGF3), which is composed of signal transducer and activator of transcription (Stat) 1, Stat 2, and IFN regulatory factor-9 (IRF-9), and by gamma-activated factor (GAF), which is a Stat 1 homodimer. Induction of ISGs, such as ISG17 and 2',5'-oligoadenylate synthetase, by IFNtau during pregnancy is limited to the endometrial stroma (S) and glandular epithelium (GE) of the ovine uterus. The IRF-2, a potent transcriptional repressor of ISG expression, is expressed in the luminal epithelium (LE). This study determined effects of the estrous cycle, pregnancy, and IFNtau on expression of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in the ovine endometrium. In cyclic ewes, Stat 1, Stat 2, IRF-1, and IRF-9 mRNA and protein were detected at low levels in the S and GE. During pregnancy, expression of these genes increased only in the S and GE. Expression of IRF-2 was detected only in the LE and superficial GE (sGE) of both cyclic and pregnant ewes. In cyclic ewes, intrauterine administration of IFNtau stimulated Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Ovine IRF-2 repressed transcriptional activity driven by IFN-stimulated response elements that bind ISGF3, but not by gamma-activation sequences that bind GAF. These results suggest that IRF-2 in the LE and sGE restricts IFNtau induction of ISGs to the S and GE. In the S and GE, IFNtau hyperactivation of ISG expression likely involves formation and actions of the transcription factors ISGF3 and, perhaps, IRF-1.

Mares with delayed uterine clearance have an intrinsic defect in myometrial function.

Persistent, postmating endometritis affects approximately 15% of mares and results in reduced fertility and sizable economic losses to the horse-breeding industry. Mares that are susceptible to postmating endometritis have delayed uterine clearance associated with reduced uterine contractility. Unfortunately, the mechanism for reduced uterine contractility remains an enigma. The present study examined the hypothesis that mares with delayed uterine clearance have an intrinsic contractile defect of the myometrium. Myometrial contractility was evaluated in vitro by measuring isometric tension generated by longitudinal and circular uterine muscle strips in response to KCl, oxytocin, and prostaglandin F(2alpha) (PGF(2alpha)) for young nulliparous mares, older reproductively normal mares, and older mares with delayed uterine clearance. In addition, intracellular Ca(2+) regulation was evaluated using laser cytometry to measure oxytocin-stimulated intracellular Ca(2+) transients of myometrial cells loaded with a Ca(2+)-sensitive fluorescent dye, fluo-4. For all contractile agonists, myometrium from mares with delayed uterine clearance failed to generate as much tension as myometrium from older normal mares. Oxytocin-stimulated intracellular Ca(2+) transients were similar for myometrial cells from mares with delayed uterine clearance and from older normal mares, suggesting that the contractile defect did not result from altered regulation of intracellular Ca(2+) concentration. Furthermore, no apparent age-dependent decline was observed in myometrial contractility; KCl-depolarized and oxytocin-stimulated longitudinal myometrium from young normal mares and older normal mares generated similar responses. However, circular myometrium from young normal mares failed to generate as much tension as myometrium from older normal mares when stimulated with oxytocin or PGF(2alpha), suggesting possible age-related alterations in receptor-second messenger signaling mechanisms downstream of intracellular Ca(2+) release. In summary, for mares with delayed uterine clearance, an intrinsic contractile defect of the myometrium may contribute to reduced uterine contractility following breeding.

Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep.

The extracellular matrix protein osteopontin (OPN) is a component of histotroph that increases in uterine flushings from pregnant ewes during the peri-implantation period and is localized on the apical surfaces of the uterine luminal epithelium (LE) and conceptus trophectoderm (Tr). The potential involvement of OPN in the implantation adhesion cascade in sheep was investigated by examining temporal, spatial, and potential functional relationships between OPN, Muc-1, and integrin subunits during the estrous cycle and early pregnancy. Immunoreactive Muc-1 was highly expressed at the apical surfaces of uterine luminal (LE) and glandular epithelium (GE) in both cycling and pregnant ewes but was decreased dramatically on LE by Day 9 and was nearly undetectable by Day 17 of pregnancy when intimate contact between LE and Tr begins. In contrast, integrin subunits alpha(v), alpha(4), alpha(5), beta(1), beta(3), and beta(5) were constitutively expressed on conceptus Tr and at the apical surface of uterine LE and GE in both cyclic and early pregnant ewes. The apical expression of these subunits could contribute to the apical assembly of several OPN receptors including the alpha(v)beta(3), alpha(v)beta(1), alpha(v)beta(5), alpha(4)beta(1), and alpha(5)beta(1) heterodimers on endometrial LE and GE, and conceptus Tr in sheep. Functional analysis of potential OPN interactions with conceptus and endometrial integrins was performed on LE and Tr cells in vitro using beads coated with OPN, poly-L-lysine, or recombinant OPN in which the Arg-Gly-Asp sequence was replaced with RGE or RAD. Transmembrane accumulation of talin or alpha-actinin at the apical surface of uterine LE and conceptus Tr cells in contact with OPN-coated beads revealed functional integrin activation and cytoskeletal reorganization in response to OPN binding. These results provide a physiological framework for the role of OPN, a potential mediator of implantation in sheep, as a bridge between integrin heterodimers expressed by Tr and uterine LE responsible for adhesion for initial conceptus attachment.