Maternal nutrient restriction alters thyroid hormone dynamics in placentae of sheep having small for gestational age fetuses.

Thyroid hormones regulate a multitude of metabolic and cellular processes involved in placental and fetal growth, while maternal nutrient restriction (NR) has the potential to influence these processes. Those fetuses most impacted by NR, as categorized by weight, are termed small for gestational age (SGA), but the role of thyroid hormones in these pregnancies is not fully understood. Therefore, the aims of the present study were to determine effects of NR during pregnancy on maternal and fetal thyroid hormone concentrations, as well as temporal and cell-specific expression of mRNAs and proteins for placental thyroid hormone transporters, thyroid hormone receptors, and deiodinases in ewes having either SGA or normal weight fetuses. Ewes with singleton pregnancies were fed either a 100% NRC (n = 8) or 50% NRC (NR; n = 28) diet from Days 35 to 135 of pregnancy with a single placentome surgically collected on Day 70. Fetal weight at necropsy on Day 135 was used to designate the fetuses as NR NonSGA (n = 7; heaviest NR fetuses) or NR SGA (n = 7; lightest NR fetuses). Thyroid hormone levels were lower in NR SGA compared to NR NonSGA ewes, while all NR fetuses had lower concentrations of thyroxine at Day 135. Expression of mRNAs for thyroid hormone transporters SLC16A2, SLC16A10, SLCO1C1, and SLCO4A1 were altered by day, but not nutrient restriction. Expression of THRA mRNA and protein was dysregulated in NR SGA fetuses with protein localized to syncytial and stromal cells in placentomes in all groups. The ratio of deiodinases DIO2 and DIO3 was greater for NR SGA placentae at Day 70, while DIO3 protein was less abundant in placentae from NR SGA than 100% NRC ewes. These results identify mid-gestational modifications in thyroid hormone-associated proteins in placentomes of ewes having SGA fetuses, as well as a potential for placentomes from NonSGA pregnancies to adapt to, and overcome, nutritional restrictions during pregnancy.

RAS-related protein 1: an estrogen-responsive gene involved in development and molting-mediated regeneration of the female reproductive tract in chickens.

It is important to identify molecular candidates involved in morphological and functional changes in the female reproductive system. We have discovered several candidate genes that were significantly altered in chick oviducts by exogenous estrogen and those candidates included dexamethasone (DEX)-induced RAS-related protein 1 (RASD1). RAS-related protein 1, a member of the Ras family of monomeric G proteins, is involved in various cellular processes including cell growth, proliferation and differentiation, as well as a cell-signaling protein regulating hormonal actions. Although the RASD1 gene was first identified as a DEX (a corticosteroid) inducible gene, there is evidence that it is also an estrogen-responsive gene. However, hormone-mediated expression and biological functions of RASD1 in the avian female reproductive system are poorly understood. Therefore, we tested the hypothesis that RASD1 may be involved in the development and remodeling of the chicken reproductive system as an estrogen-responsive gene. Here we demonstrate differential expression of RASD1 gene and candidate microRNAs (miRNAs) targeting chicken RASD1 transcripts in chicken oviducts in response to diesthylstilbestrol (DES, a synthetic non-steroidal estrogen) and the estrogen-mediated molting process. Result of the present study indicated that expression of RASD1 messenger RNA (mRNA) increased in the developing oviducts of chicks treated with DES, particularly in the glandular (GE) and luminal (LE) epithelia of the magnum and the shell gland. Also, during induced molting by zinc feeding, RASD1 expression changed in concert with changes in concentrations of estrogen in blood of laying hens. Our results revealed that expression of RASD1 mRNA decreased as the oviduct regressed and then increased as the oviduct underwent re-growth and recrudescence in hens. Furthermore, RASD1 mRNA was expressed predominantly in GE and LE of the oviduct of laying hens during regeneration of the oviduct after induced molting, but not during the period of regression of the oviduct during molting. In addition, the relative expression of candidate miRNAs (miR-30a-5p, miR-30b-5p, miR-30c-5p and miR-30d) regulating RASD1 transcripts changed in response to estrogen stimulation of chick oviducts. These results indicate that transcription of the RASD1 gene and miRNAs regulating post-transcriptional aspects of expression of RASD1 are modulated by estrogen which is critical for growth, development, remodeling and maintenance of function of the chicken oviduct.

Differential expression of neuregulin 1 (NRG1) and candidate miRNA regulating NRG1 transcription in the chicken oviduct in response to hormonal changes.

Neuregulin 1 (NRG1), a signaling protein that binds to members of the erythroblastoma (ErbB) family, is known to play essential biological roles in mediating cell-cell interactions and orchestrating vital cell functions in the heart and nervous system. NRG1 is closely associated with developmental processes in various organs and expression of several NRG1 isoforms are regulated by estrogen. However, expression and its hormonal regulation during development of the female reproductive tract remain unknown. The reproductive system of chickens undergoes dynamic morphological and functional changes in response to estrogen and the molting process. Therefore, in the current study, we found differential expression of and candidate microRNA regulating chicken transcription in response to estrogen stimulation and/or the molting process. The results revealed that relative expression of mRNA increased in the oviducts of chicks treated with diesthylstilbestrol (DES; a synthetic non-steroidal estrogen). Additionally, abundant NRG1 mRNA expression was primarily in the glandular (GE) and luminal (LE) epithelia of the magnum in the developing chick oviduct in response to exogenous estrogen. Also, expression decreased during regression of the oviduct following induced molting by feeding high levels of zinc in the diet, and then gradually resurged as the oviduct underwent remodeling and recrudescence in laying hens. In addition, the present results revealed changes in relative expression of candidate chicken microRNA (, and ) targeting transcription in chicken oviducts in response to exogenous estrogen. In conclusion, these results provide the first evidence that is a novel estrogen-responsive gene closely correlated with the estrogen-dependent development of the oviduct of chicks and regeneration of the oviduct after molting. Further, regulation of expression involving at least 3 chicken miRNA is likely a prerequisite for those estrogen regulated developmental events.

Rapid Communication: MicroRNA co-expression network reveals apoptosis in the reproductive tract during molting in laying hens.

The aim of this study was to determine the regulatory mechanisms of molting and recrudescence via studying the micro-RNA (miRNA) expression in the oviduct of laying hens. We performed a cDNA microarray analysis in the magnum tissue from the oviduct to identify the whole miRNA profiles through the molting and recrudescence periods. A total of 35 laying hens (47-wk-old) were divided into 7 groups (0 d: a control group; 6 and 12 d: 2 molting-period groups fed on a high-zinc diet; and 20, 25, 30, and 35 d: 4 recrudescence-period groups fed on a normal diet after a 12-d period on a high-zinc diet). An miRNA co-expression network (miRCN) was generated using the differentially expressed miRNA (DEM) according to the entire data integration. The significantly co-expressed miRNA ( = 111) were highly differentially expressed from 12 to 20 d, which was a transition period between molting and recrudescence, while their expression patterns were contrary to the estrogen changes. The targets of highly connected miRNA ( = 12) indicated the significant biological pathways and gene ontology (GO) terms, such as MAPK and Wnt signaling and magnesium-ion binding, which are associated with apoptotic activities. These results suggest that the miRNA of the miRCN might play a role in the apoptotic progression of the reproductive tract during molting.

Adiponectin: A prosurvival and proproliferation signal that increases bovine mammary epithelial cell numbers and protects them from endoplasmic reticulum stress responses.

Cell-cell interactions between epithelial and stromal cells are predominant in the mammary gland, and various stromal cell-derived factors can elicit mitogenic responses in adjacent epithelial cells. Adiponectin is a hormone secreted mainly by adipocytes that mediates stromal-epithelial interactions in a number of tissues. Adiponectin receptors are expressed by bovine mammary epithelial cells, but the regulatory effects of adiponectin on the development and function of the mammary gland remain unclear. We therefore sought to investigate the effects of adiponectin on bovine mammary epithelial (MAC-T) cells and the regulatory mechanisms that underlie these adiponectin-induced actions. Our results revealed an increase in MAC-T cell proliferation and cell cycle progression in response to adiponectin. The expression of nuclear proliferating cell nuclear antigen (PCNA) and cyclin D1 was induced in MAC-T cells, and intracellular signaling molecules such as serine/threonine protein kinase (AKT), 70 kDa ribosomal S6 kinase (P70S6K), ribosomal protein S6 (S6), extracellular signal-regulated kinases 1 and 2 (ERK1/2), 90 kDa ribosomal S6 kinase (P90S6K), and cyclin D1 were activated in a dose-dependent manner. The abundance of adiponectin-induced signaling proteins was suppressed following inhibition of AKT or ERK1/2 mitogen-activated protein kinase (MAPK) signaling. In addition, inhibition of AKT or ERK1/2 signaling significantly reduced adiponectin-stimulated MAC-T cell proliferation. Furthermore, adiponectin reduced tunicamycin-induced expression and activation of endoplasmic reticulum stress-related proteins in MAC-T cells and attenuated the repressive effect of tunicamycin on proliferation of MAC-T cells. Collectively, these results suggest that adiponectin-mediated signaling may affect the development and function of the mammary gland in dairy cows by increasing mammary epithelial cell numbers. These findings may result in important implications for improving our fundamental understanding of lactation physiology in livestock species.

The functional effects and mechanisms by which fibroblast growth factor 2 (FGF2) controls bovine mammary epithelial cells: Implications for the development and functionality of the bovine mammary gland.

Fibroblast growth factor (FGF) signaling plays essential roles in tissue development and homeostasis. Accumulating evidence reveals that fibroblast growth factor 2 (FGF2) regulates ductal elongation, which requires cell proliferation and epithelial expansion in the mammary gland. However, the function and mechanisms by which FGF2 controls functionality of epithelial cells is less well defined. Here, we demonstrate the functional effects of FGF2 on bovine mammary epithelial (MAC-T) cells and the intracellular signaling mechanisms for these FGF2-induced actions. The current results show that treatment of MAC-T cells with a recombinant FGF2 induced cell proliferation and cell-cycle progression with increased expression of proliferating cell nuclear antigen and cyclin D1. Moreover, FGF2 increased phosphorylation of serine/threonine protein kinase (protein kinase B [AKT]), extracellular signal-regulated kinases 1 and 2 (ERK1/2), Jun N-terminal kinase (JNK), 70 kDa ribosomal S6 kinase (P70S6K), 90 kDa ribosomal S6 kinase (P90S6K), ribosomal protein S6 (S6), and cyclin D1 proteins. These FGF2-induced activations of signaling pathway proteins were inhibited by blocking AKT, ERK1/2, or JNK phosphorylation. The effect of FGF2 to stimulate MAC-T cell proliferation was mediated by activation of FGF receptors (FGFR) and AKT, ERK1/2, and JNK mitogen-activated protein kinase pathways in response to FGF2 stimulation. Furthermore, expression and activation of endoplasmic reticulum (ER) stress-related factors and ER stress-induced MAC-T cell death was reduced by FGF2. Together, these results suggest that the FGF2-FGFR-intracellular signaling cascades may contribute to maintaining and/or increasing numbers of mammary epithelial cells by inducing proliferation of mammary epithelial cells and by protecting cells from ER stress responses. Therefore, this study provides evidence that FGF2 signaling is a positive factor for mammary gland remodeling and for increasing persistency of milk production.

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.

Diethylstilbestrol regulates expression of avian apolipoprotein D during regression and recrudescence of the oviduct and epithelial-derived ovarian carcinogenesis.

Apolipoprotein D (APOD) is a glycoprotein which is widely expressed in mammalian tissues. It is structurally and functionally similar to the lipocalins which are multiple lipid-binding proteins that transport hydrophobic ligands and other small hydrophobic molecules, including cholesterol and several steroid hormones. Although multiple functions for APOD in various tissues have been reported, its expression, biological function, and hormonal regulation in the female reproductive system are not known. Thus, in this study, we focused on correlations between APOD and estrogen during development, differentiation, regression, and regeneration of the oviduct in chickens and in the development of ovarian carcinogenesis in laying hens. Results of the present study indicated that APOD messenger RNA (mRNA) expression increased (P < 0.001) in the luminal and glandular (GE) epithelia of the chicken oviduct in response to diethylstilbestrol (a nonsteroidal synthetic estrogen). In addition, the expression of APOD mRNA and protein decreased (P < 0.001) as the oviduct regressed during induced molting, and gradually increased (P < 0.001) with abundant expression in GE of the oviduct during recrudescence after molting. Furthermore, APOD mRNA and protein were predominantly localized in GE of cancerous, but not normal ovaries from laying hens. Collectively, results of the present study suggest that APOD is a novel estrogen-stimulated gene in the chicken oviduct which likely regulates growth, differentiation, and remodeling of the oviduct during oviposition cycles. Moreover, up-regulated expression of APOD in epithelial cell-derived ovarian cancerous tissue suggests that it could be a candidate biomarker for early detection and treatment of ovarian cancer in laying hens and in women.

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 Reproduction Symposium: limitations in uterine and conceptus physiology that lead to fetal losses.

Conceptus losses in livestock occur throughout gestation. The uterus and the embryo-placenta-fetus play interconnected roles in these losses, the details of which depend on the period of gestation and the species. Studies in sheep and pigs have indicated that the uterine glands are essential for full fertility, based on experiments where gland development was reduced through the use of exogenous hormones. In sheep and cattle, normally the uterus is well able to support more than a single fetus although these species differ in the consequences of multiple births. When 2 conceptuses are present, the placentas of cattle often anastomose, putting 1 fetus at risk if the other is lost. One likely reason this does not occur in sheep is because sheep embryos undergo intrauterine migration, similar to pigs. In pigs, the relatively equidistant separation of conceptuses is likely to be essential for optimizing conceptus survival as is the simultaneous and uniform elongation of blastocysts that occurs during the time of maternal recognition of pregnancy. Other studies in pigs have indicated that the size of the uterus influences litter size and therefore fetal losses. In response to crowded intrauterine conditions in the pig, increased conceptus losses begin to occur between d 30 and 40 of pregnancy, and further losses occur sporadically during later gestation. There is evidence that improved fetal erythropoiesis can reduce these losses. Other studies indicated that profound changes in placental development occurred under crowded intrauterine conditions that may contribute to losses during late gestation. Reductions in placental stroma formation may compromise the ability of the pig placenta to adapt to reduced uterine space. Consistent with this, both hyaluronan and hyaluronidase activity are decreased in the placentas of small compared with large fetuses. These results indicate that improvements in placental stroma formation could improve placental ability to compensate for reduced intrauterine space, resulting in increased placental function and reduced fetal losses during late gestation.

Physiology and Endocrinology Symposium: biological role of interferon tau in endometrial function and conceptus elongation.

This review integrates established and new information on the biological role of ovarian progesterone (P4) and interferon tau as well as conceptus- and endometrial-derived factors, PG and cortisol, in endometrial function and conceptus elongation during the periimplantation period of pregnancy in ruminants. Interferon tau is the maternal recognition of pregnancy signal that inhibits production of luteolytic pulses of PGF2α by the endometrium to maintain corpora lutea and their production of P4, the unequivocal hormone of pregnancy. Conceptus-endometrial interactions in ruminants are complex and involve carefully orchestrated temporal and spatial alterations in endometrial gene expression during pregnancy. Available results from studies in sheep support the idea that the individual, interactive, and coordinated actions of P4, interferon tau, PG, and cortisol regulate expression of elongation- and implantation-related genes in the endometrial epithelia and that P4 and PG are essential regulators of conceptus elongation. The outcome of these gene expression changes is alterations in endometrial secretions that govern conceptus elongation via effects on trophectoderm proliferation, migration, attachment, and adhesion. An increased knowledge of conceptus-endometrial interactions during early pregnancy in ruminants is necessary to understand and elucidate the causes of recurrent pregnancy loss and to provide a basis for new strategies to improve pregnancy outcome and reproductive efficiency.

Growth and development of the ovine conceptus.

This study was designed to characterize development of the ovine conceptus throughout gestation to establish the temporal relationships in metabolites, electrolytes, fluid volumes within the placenta, and hormonal changes with fetal growth. Length and weight of placentae, weight of cotyledons, and uterine weight increased between d 25 and 80 of gestation in advance of increases in fetal growth between d 80 and 140 of gestation. Allantoic fluid volumes changed (P < 0.01) between d 25 (21 mL) and 40 (91 mL), decreased to d 70 (32 mL), and then increased to d 140 (438 mL). Concentrations and total amounts of proteins in allantoic fluid were reduced between d 25 and 50, but total protein increased (P < 0.01) from d 40 (63 mg) to d 140 (2,991 mg). Concentrations of fructose in allantoic fluid varied between 2 and 6 mg/mL throughout gestation, but total fructose increased (P < 0.01) between d 25 (46 mg) and d 120 (679 mg). Concentrations of glucose ranged from 0.1 to 0.3 mg/mL, and total glucose increased (P < 0.05) from d 25 (3 mg) to d 140 (63 mg) of gestation. Amniotic fluid volume increased (P < 0.01) between d 30 and 140. Concentrations of estrogens in allantoic fluid, maternal uterine artery, and uterine vein increased (P < 0.01) with advancing pregnancy, and concentrations of progesterone in allantoic fluid (P < 0.07) and plasma (P < 0.05) were affected by day of gestation. Concentrations of glucose were greater (P < 0.05) in uterine artery than uterine vein, but concentrations of electrolytes and osmolarity of plasma were not affected by day of gestation. Increases in weights of fetal organs were proportional to increases in fetal weight during gestation. Results of the present study of conceptus growth and development highlight areas of needed research and provide benchmarks for comparisons when evaluating effects of various treatments, environmental conditions, and epigenetics on successful outcomes of pregnancy in sheep.

Morpho-functional studies regarding the fertility prognosis of mares suffering from equine endometrosis.

The aim of the present study was to characterize the morpho-functional features of endometrosis in barren and foaling mares, using both conventional histopathological and immunohistochemical methods. Endometrial biopsy samples were collected during the physiological breeding season from 159 estrous, clinically healthy mares (mean age 12 years), and the quality and degree of endometrosis was histomorphologically defined. The mares were bred and those that foaled were put in the foaling group whereas those that did not foal were placed in the barren group. Foaling mares were then compared with barren mares. Sixty-four percent (101/159) of uterine samples showed varying degrees of endometrosis and were used for this study. The sample population consisted of 51 barren and 50 foaling mares suffering from endometrosis. Expression of steroid hormone receptors (estrogen receptor, progesterone receptor) and endometrial protein secretion patterns (uteroglobin [UG], uterocalin [UC], calbindin(D9k) [CAL], uteroferrin [UF]) was evaluated by immunohistochemistry (barren mares N = 51, foaling mares N = 31). In comparison with unaffected glands, fibrotic glands generally showed a cycle-asynchronous, partially patchy protein expression pattern which is interpreted as a sign of endometrial maldifferentiation within fibrotic areas. In barren mares (N = 51) more than half of biopsy samples (27/51) showed a destructive mostly moderate (20/27) type of endometrosis. In affected glands, staining for UG (17/21) was decreased (P < 0.001). Foaling mares (N = 50) frequently showed a mild, nondestructive endometrosis (35/50). Compared with barren mares, foaling mares had statistically (P < 0.05) more often a cycle-synchronous or increased UG expression pattern within fibrotic glands. Obvious deviations of either UG or UC rarely occurred. Within fibrotic foci, UF often demonstrated a cycle-synchronous or more intense expression pattern in both foaling (28/31) and barren mares (41/51), compared with healthy glands. Mares of both groups showed a cycle-asynchronous staining for estrogen receptor and progesterone receptor in the stromal cells in areas of periglandular fibrosis and the glandular epithelia. These findings indicate that affected areas become independent of the uterine control mechanisms and exhibit specific differentiation dynamics. Immunohistochemical investigations showed that the secretory patterns differ between barren and foaling mares. The findings in this study should be considered as a useful addition to the "classical" Kenney categorization.

Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant?

This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.

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.

Pregnancy and interferon tau regulate N-myc interactor in the ovine uterus.

In ruminants, interferon tau (IFNT) is synthesized and secreted by the mononuclear trophectoderm cells of the conceptus and maintains the corpus luteum and its secretion of progesterone for successful implantation and maintenance of pregnancy. In this study, we examined regulation of the expression of N-myc interactor (NMI) gene by IFNT in the ovine uterus based on results of microarray data from a study that compared gene expression by human 2fTGH and U3A (STAT1-null 2fTGH) cell lines in response to treatment with IFNT or vehicle. In the present study, semiquantitative reverse transcription-polymerase chain reaction analyses verified that IFNT stimulated expression of NMI mRNA in 2fTGH (ie, in a STAT1-dependent manner), but not in U3A (STAT1-null) cells. Furthermore, results of western blot analyses indicated that immunoreactive NMI proteins in 2fTGH and U3A cell lines increased in a time-dependent manner only in response to IFNT. In ovine endometria, steady-state levels of NMI mRNA increased between days 14 and 16 of pregnancy and then decreased slightly by day 20, but there was no effect of day of the estrous cycle. Expression of NMI mRNA was most abundant in endometrial stromal cells, glandular epithelium, and conceptus trophectoderm. Intrauterine infusion of IFNT in cyclic ewes increased expression of NMI in the endometrium. Expression of NMI in ovine and bovine uterine cell lines increased in response to IFNT. Collectively, the results of the present study indicate that IFNT regulates expression of NMI mRNA and protein in ovine endometria during pregnancy via a STAT1-dependent cell signaling pathway.

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.

Effect of pregnancy and progesterone concentration on expression of genes encoding for transporters or secreted proteins in the bovine endometrium.

The objective of this study was to determine the temporal and spatial expression patterns of genes encoding transporters, as well as selected secreted proteins that may be regulated by progesterone (P4) and/or the presence of the conceptus in the bovine endometrium. Estrus-synchronized beef heifers were randomly assigned to either: 1) pregnant, high P4; 2) pregnant, normal P4; 3) cyclic, high P4; or 4) cyclic, normal P4. Uteri were collected on days 5, 7, 13, and 16 of the estrous cycle or pregnancy. Localization of mRNAs for ANPEP, CTGF, LPL, LTF, and SLC5A1 in the uteri was determined by radioactive in situ hybridization, and expression quantified in the endometria by quantitative real-time PCR. ANPEP localized to luminal (LE) and superficial glandular (sGE) epithelia of all heifers on days 5 and 7 only. SLC5A1 mRNA was detected in the LE and sGE on days 13 and 16 in all heifers, and expression increased on day 16 in pregnant groups. CTGF localized weakly to the LE and GE on days 5 and 7 but increased on days 13 and 16 with an increase (P < 0.05) in CTGF expression in high P4 (day 7) and pregnant heifers (day 16). Both LPL and LTF localized to the GE only on days 5 and 7. In conclusion we have characterized the temporal expression pattern of these genes and modulation of their transcript abundance by P4 (CTGF, LPL) and/or the conceptus (CTGF, SLC5A1) likely modifies the uterine microenvironment, enhancing histotroph composition and contributing to advanced conceptus elongation.

Transplacental transfer of iron in the water buffalo (Bubalus bubalis): uteroferrin and erythrophagocytosis.

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental-maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl's reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal-placental tissues in buffalo throughout pregnancy.