Effects of 3-nitrooxypropanol on rumen fermentation, lactational performance, and resumption of ovarian cyclicity in dairy cows.

This study examined the effect of 3-nitrooxypropanol (3-NOP), a substance under investigation, on enteric methane (CH4) emission, rumen fermentation, lactational performance, sensory properties of milk, and the resumption of ovarian cyclicity in early-lactation dairy cows. Fifty-six multi- and primiparous Holstein cows, including 8 that were rumen cannulated, were used in a 15-wk randomized complete block design experiment. Cows were blocked based on parity and previous lactation milk yield (MY) or predicted MY, and within each block were randomly assigned to one of 2 treatments: (1) control (CON), administered no 3-NOP, or (2) 3-NOP applied at 60 mg/kg of feed dry matter (3-NOP). Enteric CH4 emission was measured during experimental wk 2, 6, 9, and 15, using the GreenFeed system. Dry matter intake (DMI) and MY data were collected daily throughout the experiment, and milk composition samples were collected 7 times during the experiment. Milk samples were collected from 14 to 60 (±2) d after calving, 3 d per week, and assayed for progesterone concentration to determine resumption of ovarian activity. Compared with CON, 3-NOP decreased daily CH4 emission by 26%, CH4 yield (CH4 per kg of DMI) by 21%, and CH4 emission intensity [CH4 per kg of MY or energy-corrected milk (ECM)] by 25%. Enteric emission of carbon dioxide was decreased by 5%, and hydrogen emission was increased 48-fold by 3-NOP. Inclusion of 3-NOP decreased concentration of total volatile fatty acids (by 9.3%) and acetate but increased butyrate molar proportion, ethanol, and formate concentrations in ruminal fluid. Dry matter intake was lower for 3-NOP compared with CON, but DMI expressed as a percentage of body weight was not different between treatments. Treatment had no effect on milk and ECM, body weight change, or body condition score. Milk composition and milk fat and protein yields were not affected by treatment, except that concentrations of short-chain fatty acids in milk were increased by 3-NOP. Nutrient digestibility and blood metabolites and hormones were not affected by 3-NOP, except that insulin was decreased by 3-NOP. There was no effect of 3-NOP on postpartum resumption of ovarian activity, including days to first and second luteal phases, length of first and second luteal phases, and interval from first to second luteal phase. Sensory properties of milk from cows fed 3-NOP and cheese made from that milk were not affected by treatment. In this experiment, 3-NOP decreased daily enteric CH4 emission, emission yield, and emission intensity, improved feed efficiency, and did not affect lactational performance or onset of ovarian activity in early-lactation dairy cows.

Impact of preovulatory estradiol concentrations on conceptus development and uterine gene expression.

This experiment was conducted to determine the effect of altering preovulatory estradiol concentrations, through manipulation of length of proestrus, on peripheral progesterone concentrations, conceptus development, interferon tau (IFNT) production and uterine gene expression in cattle. Approximately 6 days after a time-synchronized ovulation, all antral follicles (≥5 mm) were ablated from the ovaries in beef heifers. To manipulate preovulatory estradiol concentrations, the length of proestrus prior to the GnRH-induced LH surge was altered between treatments. Heifers were administered PGF(2α) either -2.5 days (2.5 days of proestrus; HiE2; n=5) or -1.5 days (1.5 days of proestrus; LoE2; n=5) prior to GnRH (Day 0 of the experiment; 6.75 days after follicle ablation). Follicular dynamics and estradiol concentrations were evaluated during proestrus and progesterone concentrations were analyzed in the subsequent estrous cycle. On Day 7, embryos were transferred into all heifers using standard procedures. On Day 15.5 heifers were slaughtered, the reproductive tract was flushed to collect the conceptus and uterine flush media, and the uterine tissue was processed for subsequent analyses. Peripheral progesterone concentrations, conceptus development and IFNT production were similar between treatments. However, amount of nuclear progesterone receptor in the deep glandular epithelium and mRNA concentrations for estradiol receptor alpha (ESR1) in the uterine endometrium were less in the LoE2 than HiE2 treatment. These changes in uterine characteristics in heifers with lower preovulatory estradiol concentrations were not related to aspects of conceptus development monitored, however, it is speculated that the alterations in mRNA and receptor protein detected may contribute to pregnancy failure subsequent to day 15.5 of gestation.

Receptor transporter protein 4 (RTP4) in endometrium, ovary, and peripheral blood leukocytes of pregnant and cyclic ewes.

Interferon-tau (IFNT) is secreted by the conceptus trophoblast and signals pregnancy recognition in ruminants. IFNT regulates expression of genes in the endometrium, peripheral blood leukocytes (PBLs), and corpus luteum (CL). Microarray analysis identified that expression of (chemosensory) receptor transporter protein 4 (RTP4) increased in PBLs during early pregnancy in cows. In the present study, we cloned and characterized RTP4 transcription during early pregnancy in ewes. Endometrium, PBLs, and CL were collected on Days 11, 13, and 15 of the cycle and on Days 11, 13, 15, 17, and 19 of pregnancy. Northern blot analysis revealed an expected 1.6-kb mRNA and an unexpected 2.6-kb mRNA. In endometria, RTP4 mRNA levels in cyclic ewes remained low, whereas RTP4 mRNA increased from Day 11 to Day 17 in pregnant ewes. Levels of RTP4 mRNA also increased from Day 15 to Day 19 in CL and PBL samples from pregnant ewes only. The RTP4 mRNA was located in the glandular epithelium, stratum compactum, and caruncular stroma. Ovine glandular epithelial cells were treated with IFNT to determine if IFNT alone could induce RTP4. IFNT increased RTP4 more than 70-fold at 1.5 h after treatment, with maximal induction of nearly 300-fold above values observed in nontreated controls at 6 h after treatment. These results indicate that RTP4 mRNA levels are induced in the ovine endometrium, PBLs, and CL by IFNT during early pregnancy and in cell culture in response to IFNT. If RTP4 expression affects G protein-coupled receptor function, it may be important for establishment of pregnancy in domestic ruminants.

Correlation between reproductive status and steady-state messenger ribonucleic acid levels of the Myxovirus resistance gene, MX2, in peripheral blood leukocytes of dairy heifers.

The objectives of the current study were to evaluate the correlation between reproductive status and steady-state levels of Myxovirus resistance gene (MX2) mRNA in peripheral blood leukocytes (PBL) of dairy heifers and the reliability of using change in MX2 messenger RNA (mRNA) for identification of nonpregnant heifers 18 to 19 d after AI. Holstein heifers (n = 266), 13 +/- 1 mo of age, were assigned randomly to be inseminated (BRED; n = 214) or not (NONBRED; n = 52). Estrous cycles of all heifers were synchronized with an intravaginal insert containing progesterone for 7 d. At insert removal, heifers received an injection of PGF2alpha. Heifers in the BRED group were inseminated on detection of estrus or at a fixed time, 72 h after insert removal concomitant with a GnRH treatment. Heifers in the NONBRED group received an injection of GnRH 48 h after insert removal. Blood samples collected on d 0 (d of AI or estrus) and 18 were used to determine steady-state levels of MX2 mRNA. Samples collected on d 0, 7, 14, and 21 were analyzed for progesterone concentration. Pregnancy was determined retrospectively by progesterone concentration on d 21 and was diagnosed at 30 +/- 1 and 60 +/- 3 d after AI. The fold change in levels of MX2 mRNA from d 0 to 18 was greater for heifers classified and diagnosed as pregnant on d 21 (P < 0.05) and 30 +/- 1 (P < 0.05) and 60 +/- 3 (P < 0.05) d after AI compared with nonpregnant (bred but not pregnant) and NONBRED heifers. Heifers that experienced pregnancy loss from 21 to 30 +/- 1 (P = 0.11) or 21 to 60 +/- 3 (P = 0.08) d of gestation tended to have smaller fold increases in MX2 mRNA expression than those that maintained pregnancy. The sensitivity (range 57.1 to 65.6%) and negative predictive values (range 47.9 to 57.1%) of determining reproductive status on d 18 according to the change in the level of MX2 mRNA expression in PBL were low, and the correlation between diagnosis of pregnancy by fold change in MX2 mRNA expression and other methods was small (r = 0.20 to 0.36). The current study indicates that increased expression of MX2 mRNA in PBL is related to pregnancy approximately 21, 30, and 60 d after AI in dairy heifers and that losses that occurred later in pregnancy were associated with lower fold increases in MX2 mRNA. However, using the change in MX2 mRNA expression was not a reliable method for diagnosis of pregnancy at 18 d after AI because of the low sensitivity and negative predictive value.

Cloning and characterizing of the ovine MX1 gene promoter/enhancer region.

Ovine MX1 (MX1) is expressed in the uterus during the estrous cycle and is strongly up-regulated during early pregnancy in the uterus and peripheral blood leukocytes. In this study we cloned the MX1 gene promoter/enhancer, and tested its response to interferon tau (IFN-tau). To address the role of IFN tau in regulating MX1 expression, serial deletion mutants were prepared along with a clone that contained a full-length promoter including the two proximal ISREs but lacking an intronic ISRE site. Promoter deletions showed the two proximal ISRE sites, but not the intronic ISRE site, were required for maximal response to IFN tau. Interestingly, MX1 promoter deletion mutants revealed the presence of distal positive (-920 to -715) and negative (-715 to -437) regulatory regions. Identifying positive and negative regulatory regions in MX1 promoter will help define the complex regulation of MX1 during early pregnancy in ruminants.

Regulation of interferon-stimulated genes in peripheral blood leukocytes in pregnant and bred, nonpregnant dairy cows.

In ruminants, pregnancy results in up-regulation of a large number of IFN-stimulated genes (ISG) in the uterus. Recently, one of these genes was also shown to increase in peripheral blood leukocytes (PBL) during early pregnancy in sheep. Our working hypothesis is that conceptus signaling activates maternal gene expression in PBL in dairy cattle. The objectives of this study were to characterize ISG expression in PBL from pregnant (n = 20) and bred, nonpregnant (n = 30) dairy cows. Steady-state levels of mRNA for Mx1, Mx2, beta2-microglobulin, ISG-15, IFN regulatory factor-1, and IFN regulatory factor-2 were quantified. Holstein cows were synchronized to estrus and artificially inseminated (d 0). Blood samples were collected (coccygeal venipuncture) on d 0 and 16, 18, and 20 d after insemination for progesterone analysis and PBL isolation. Pregnancy was confirmed by transrectal ultrasonography at approximately 40 d after breeding. A status x day interaction was detected for Mx1, Mx2, and ISG-15 gene expression. When analyzed within day, levels of mRNA for ISG-15 and Mx1 were greater in pregnant compared with bred, nonpregnant cows on d 18 and 20, respectively. Expression of the Mx2 gene increased in the pregnant group compared with bred, nonpregnant cows on d 16, 18, and 20 after insemination. beta2-Microglobulin, IFN regulatory factor-1, and IFN regulatory factor-2 were not different between groups. The results clearly indicated that components of the innate immune response are activated in PBL during the period of pregnancy recognition and early embryo signaling. The physiological implications of these changes on maternal immune function are as yet unknown; however, they do provide a unique opportunity to identify bred, nonpregnant, cows 18 d after insemination in dairy cattle.

Temporal and spatial associations of oestrogen receptor alpha and progesterone receptor in the endometrium of cyclic and early pregnant mares.

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.

Phenotypic and ultrastructural characteristics of bronchoalveolar lavage cells of lentivirus-infected lambs treated with recombinant ovine IFN-tau.

Ovine lentivirus (OvLV) belongs to the family Retroviridae and closely resembles the human immunodeficiency virus (HIV). Pulmonary lesions in OvLV-infected sheep consist of lymphoid interstitial pneumonia (LIP) and lymphocytic alveolitis. Similar pulmonary lesions occur in up to 40% of HIV-infected children and in some adults with AIDS. Interferon-tau (IFN-tau), a type I IFN, is produced by trophectoderm of ruminant conceptuses and is the pregnancy recognition signal in these species. To evaluate changes in phenotypes of bronchoalveolar lavage (BAL) cells of OvLV-infected lambs treated with recombinant ovine IFN-tau (rOvIFN-tau), 24 lambs were randomly allocated to one of four groups (n = 6 per group): 1, no virus + placebo (NVP); 2, no virus + rOvIFN-tau (NVI); 3, virus + placebo (VP); 4, virus + rOvIFN-tau (VI). The BAL cells from 3 lambs in each group were labeled with monoclonal antibodies (mAb) to cell surface markers at 16 weeks of treatment, and cells from the remaining 3 lambs in each group were labeled with mAb at 34 weeks of treatment. After labeling, BAL cells were analyzed by flow cytometry. The morphology of BAL cells from all experimental lambs was examined by transmission electron microscopy (TEM). At week 16, no differences in the relative proportions of BAL cell phenotypes were detected among the experimental groups. At week 34, VI lambs had higher proportions of CD8(+), gammadelta(+), MHC class II(+), and L-selectin (LS(+)) BAL cells compared with VP lambs. Higher proportions of CD14(+) and CD44(+) cells were found in VP lambs compared with NVP lambs at 34 weeks. OvLV-like particles were detected only in bronchoalveolar macrophages of VP lambs. In this study, rOvIFN-tau increased the proportions of primary antiviral gammadelta(+) and CD8(+) immune cells in OvLV-infected lambs. This may represent a cellular mechanism to explain the antiviral and therapeutic efficacy of this cytokine, in addition to its direct antiviral effect. However, because the actual number of cells labeled with mAb CD8 was low and some subsets of gammadelta cells may coexpress the CD8 marker, further studies are necessary to better define the role of rOvIFN-tau in the modulation of these cells in vivo.

Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred, non-pregnant ewes.

Interferon-tau (IFN tau) acts locally on the endometrium to suppress estrogen and oxytocin receptor expression and block luteolysis in ruminants. Systemic administration of conceptus homogenates or recombinant ovine IFN tau does not block luteolysis or enhance pregnancy rates in sheep or cattle, respectively. However, IFN tau up-regulates expression of the antiviral protein Mx throughout the entire uterine wall during early pregnancy. These studies determined if conceptus-derived IFN tau also up-regulates Mx expression in components of the circulating immune system that migrate through the endometrial wall. In experiment one, peripheral blood mononuclear cells (PBMC) were isolated from ewes at D26 post-artificial insemination (AI) and Mx mRNA levels examined by Northern and slot-blot hybridization. Pregnancy resulted in a two-fold increase in Mx mRNA levels compared to bred, non-pregnant ewes at D26. In experiment two, PBMC were isolated from ewes at AI, and every three days from D9 to D30. Results showed a four-fold increase in Mx mRNA levels in PBMC from pregnant versus bred, non-pregnant ewes at D15. Increased Mx mRNA, which remained elevated through D30, was accompanied by increased levels of Mx protein. These results show that pregnancy recognition signaling rapidly induces Mx gene expression in PBMC, and are the first to suggest that IFN tau activates gene expression in components of the circulating immune system.

Interferon tau-induced hepatocyte apoptosis in sheep.

Clinical applications of Type I interferon (IFN) are limited by adverse side effects mediated largely by unknown mechanisms. This study examined the mechanisms of acute hepatic injury in lambs treated with systemic administration of IFN-tau, a Type I IFN. Liver tissues were collected at 24, 48, or 96 hours after treatment with either IFN-tau or saline. Histopathology revealed acute hepatopathy including cellular swelling, cytoplasmic aggregates, and apoptosis in all IFN-tau-treated lambs, which were accompanied by elevation of aspartate transaminase (AST) (P <.01). The number of apoptotic hepatocytes in IFN-tau-treated lambs was higher than for control lambs (P <.001). Immunohistochemistry for proliferating cell nuclear antigen (PCNA) revealed that IFN-tau induced hepatocyte growth arrest at the G0/G1 phase of the cell cycle and that the majority of hepatocytes in S or G2 phase were eliminated by apoptosis. We investigated expression of bax-alpha and bcl-2, acting as pro- and antiapoptotic molecules, in IFN-tau-induced apoptosis. Northern blot analysis revealed increased expression of bax-alpha messenger RNA (mRNA) in IFN-tau-treated lambs (P <.01) compared with control lambs, consistent with the expression pattern for bax-alpha protein. However, there was no detectable difference in expression of bcl-2 proteins between control and IFN-tau-treated lambs. The levels of bax-alpha associated with the mitochondria also increased during IFN-tau treatment. Bax-alpha immunostaining showed scattered immunoreactive hepatocytes with morphological hallmarks of apoptosis. These results suggest that IFN-tau induces growth arrest as well as apoptosis by regulating bax-alpha expression. These pathological effects of IFN-tau on sheep liver indicate potential mechanisms of Type 1 IFN-induced hepatotoxicity in animals and humans.

Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle.

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy. Ewe lambs (n = 22) received a Norgestomet (Nor) implant at birth and every two weeks thereafter for 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. Control ewe lambs (n = 13) received no Nor treatment (Groups V and VI). Ewes in Groups I, II, III, and VI were hemihysterectomized (Hhx) at 16 weeks of age. After puberty, the remaining uterine horn in Hhx ewes was removed on either Day 9 or 15 of the estrous cycle (Day 0 = estrus). Histological analyses of uteri indicated that progestin exposure for 8, 16, or 32 weeks prevented endometrial adenogenesis and produced the UGKO phenotype in adult ewes. Three endometrial phenotypes were consistently observed in Nor-treated ewes: 1) no glands, 2) slight glandular invaginations into the stroma, and 3) limited numbers of cyst- or gland-like structures in the stroma. Overall patterns of uterine progesterone, estrogen, and oxytocin receptor expression were not different in uteri from adult cyclic control and UGKO ewes. However, receptor expression was variegated in the ruffled luminal epithelium of uteri from UGKO ewes. Intact UGKO ewes displayed altered estrous cycles with interestrous intervals of 17 to 43 days, and they responded to exogenous prostaglandin F(2 approximately ) (PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.

Effects of recombinant ovine interferon tau, placental lactogen, and growth hormone on the ovine uterus.

Studies were conducted to determine effects of intrauterine administration of recombinant ovine interferon tau (IFNtau), placental lactogen (PL), and growth hormone (GH) on endometrial function. In the first study, administration of IFNtau to cyclic ewes for one period (Days 11-15) resulted in an interestrous interval (IEI) of approximately 30 days, whereas administration for two periods (Days 11-15 and Days 21-25) extended the IEI to greater than 50 days. Administration of IFNtau from Days 11 to 15 and of PL or GH from Days 21 to 25 failed to extend the IEI more than for IFNtau alone. In the second study, effects of IFNtau, PL, and GH on endometrial differentiation and function were determined in ovariectomized ewes receiving ovarian steroid replacement therapy. Endometrial expression of mRNAs for estrogen receptor (ER), progesterone receptor (PR), and oxytocin receptor (OTR) were not affected by PL or GH treatment; however, uterine milk protein mRNA levels and stratum spongiosum gland density were increased by both PL and GH treatments. Collectively, results indicated that 1) PL and GH do not regulate endometrial PR, ER, and OTR expression or affect corpus luteum life span; 2) down-regulation of epithelial PR expression is requisite for progesterone induction of secretory gene expression in uterine glandular epithelium; 3) effects of PL and GH on endometrial function require IFNtau; and 4) PL and GH regulate endometrial gland proliferation and perhaps differentiated function.

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.

Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cyclic ewes.

Interferon tau (IFNtau) is the antiluteolytic signal produced by the conceptus of ruminants. Intrauterine administration of recombinant ovine IFNtau suppresses expression of endometrial estrogen receptor (ER) and oxytocin receptor (OTR) in the luminal and superficial glandular epithelia to abrogate the production of luteolytic prostaglandin F(2alpha) (PGF(2alpha)) pulses. Subcutaneous (s.c.) injections of recombinant ovine (o) IFNtau appear to extend the interestrous interval by altering uterine PGF(2alpha) response to oxytocin. The present study tested the hypothesis that antiluteolytic effects of roIFNtau injected into the uterine lumen (paracrine) or s.c. (endocrine) are equivalent in suppressing expression of endometrial ER and OTR and inducing uterine expression of type I IFN-regulated Mx and ubiquitin cross-reactive proteins (UCRP). Sixteen cyclic ewes were fitted with uterine catheters on Day 5 (Day 0 = estrus), were assigned randomly to receive treatment with control proteins or roIFNtau (2 x 10(7) antiviral units/day) by either intrauterine or s.c. injections from Days 11 to 15, and were ovariohysterectomized on Day 16. Results indicated that expression of ER and OTR mRNAs in endometrial epithelium was suppressed by intrauterine but not by s.c. injections of roIFNtau. Intrauterine injections of roIFNtau increased expression of Mx and UCRP mRNA in the endometrium. Subcutaneous injections of roIFNtau increased endometrial Mx mRNA levels but not UCRP mRNA. Unexpectedly, intrauterine and s.c. injections of roIFNtau were equally effective in inducing expression of Mx and UCRP mRNA in the corpus luteum. Although s.c. injections of roIFNtau induced Mx mRNA in the endometrial epithelium, s.c. injections of roIFNtau did not abrogate activation of the uterine luteolytic mechanism by suppressing epithelial ER and OTR expression. Therefore, results of this study failed to support the assumption that endocrine roIFNtau mimics antiluteolytic effects of paracrine IFNtau to improve pregnancy rates in sheep.

Estradiol up-regulates estrogen receptor-alpha messenger ribonucleic acid in sheep endometrium by increasing its stability.

During the preovulatory period, estrogen up-regulates estrogen receptor-alpha (ER) gene expression in endometrium in female mammals of all species examined. The purpose of this study was to determine directly whether estradiol up-regulates ER mRNA by increasing the stability of the message. Endometrial tissue was collected from ovariectomized ewes 18 h after the ewes were injected with 50 microg estradiol. Previous work indicated rapid accumulation of ER mRNA at this time. Estradiol increased uterine weights (to 157 +/- 15%) as well as steady-state concentrations of ER (to 309 +/- 37%), progesterone receptor (PR; to 165 +/- 19%), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; to 374 +/- 32%) mRNAs in endometrium, compared to control levels of 100%. The effects of estradiol on ER mRNA stability in endometrium were measured in explants cultured with the transcription inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole, as well as by labeling RNA in vivo with 4-thiouridine. Both assays indicated that estradiol enhanced ER mRNA stability (half-life increased from 9 h to >/= 24 h). The estradiol effect was specific, because the stabilities of PR, GAPDH, and c-fos mRNAs were unaffected by treatment. Thus, estradiol up-regulates steady-state concentrations of ER mRNA in endometrium by a novel posttranscriptional mechanism.

Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation.

Little is known about the mechanism responsible for retarded placental and fetal growth induced by maternal dietary protein malnutrition. On the basis of the recent finding that nitric oxide (NO) and polyamines (products of L-arginine) play an important role in embryonic and placental development, the present study was designed to determine whether protein deficiency decreases placental and endometrial activities of NO synthase (NOS) and ornithine decarboxylase (ODC) (the first and key regulatory enzyme in polyamine synthesis). Primiparous gilts selected genetically for low or high plasma total cholesterol concentrations (low line and high line, respectively) were mated and then fed 1.8 kg/d of isocaloric diets containing 13% or 0.5% crude protein. At d 40 or 60 of gestation, they were hysterectomized, and placenta and endometrium were obtained for incubations, NOS and ODC assays, and measurements of free amino acids and polyamines. Maternal dietary protein restriction decreased arginine and ornithine concentrations, constitutive and inducible NOS activities and NO production, as well as ODC activity and polyamine concentrations in placenta and endometrium of both lines of gilts. Placental NO synthase activity and NO generation were lower in high line gilts than in low line gilts. ODC activities and polyamine concentrations in placenta and endometrium were decreased at d 60 compared with d 40 of gestation. These changes in placental and endometrial synthesis of NO and polyamines during early gestation may be a mechanism responsible for reduced placental and fetal growth in protein-deficient gilts and for altered conceptus development in high line gilts.

Expression of interferon regulatory factors one and two in the ovine endometrium: effects of pregnancy and ovine interferon tau.

Availàble evidence suggests that interferon tau (IFNtau), the signal for pregnancy recognition in ruminants, suppresses transcription of the estrogen receptor (ER) gene in the endometrial lumenal epithelium (LE) and superficial glandular epithelium (sGE) to prevent oxytocin receptor (OTR) expression and pulsatile release of luteolytic prostaglandin F2alpha. The IFN regulatory factors one (IRF-1) and two (IRF-2) are transcription factors induced by type I IFNs that activate and silence gene expression, respectively. The objectives of these studies were to determine effects of pregnancy and IFNtau on expression of immunoreactive IRF-1 and IRF-2 proteins in the ovine endometrium. In study one, IRF-1 and IRF-2 were not detected in the LE or sGE of cyclic ewes. In pregnant ewes, IRF-1 expression was detected transiently in the LE and sGE only on Days 11 and 13, and IRF-2 was detected in these same epithelia on Days 13, 15, 17, and 20. In study two, 36 ewes were fitted with uterine catheters on Day 5 of the estrous cycle, and one uterine horn was double-ligated at the base. Uterine horns of each ewe received twice-daily injections of either recombinant ovine IFNtau or control proteins beginning on Day 11 until hysterectomy at 1, 3, 6, 12, 24, 48, 72, 96, or 120 h after initial injection. The IRF-1 was detected transiently in the endometrial LE and sGE only at 12 and 24 h in the uterine horn receiving IFNtau but not in those tissues receiving control proteins. The IRF-2 was expressed in the LE and sGE at 24 h and thereafter in the IFNtau-treated, but not control uterine horns. In control uterine horns, ER and OTR were first detected in the LE at 48 h and 72 h, respectively, and remained abundant thereafter. In horns receiving IFNtau, ER and OTR expression was not detected in the endometrial LE and sGE. Results suggest that IFNtau acts directly on the LE and sGE during pregnancy to sequentially induce IRF-1 and then IRF-2 gene expression, which is correlated temporally with an absence of ER and OTR. The ovine ER gene may contain an IFNtau-responsive element(s) that binds negative-acting, IFNtau-inducible transcription factors, such as IRF-2, which silences transcription of the ER gene in the endometrial epithelium during maternal recognition of pregnancy.

Effects of exogenous recombinant ovine interferon tau on circulating concentrations of progesterone, cortisol, luteinizing hormone, and antiviral activity; interestrous interval; rectal temperature; and uterine response to oxytocin in cyclic ewes.

Interferon tau (IFN tau) is the conceptus-produced antiluteolytic signal in ruminants. Three experiments examined the effects of s.c. administration of recombinant ovine (ro)IFN tau on interestrous interval (IEI), oxytocin (OT)-induced uterine prostaglandin F2alpha metabolite (PGFM) production, rectal temperature (RT), respiration rate (RR), and plasma concentrations of progesterone, cortisol, LH, and antiviral activity (AVA) in plasma and uterine flushings. In experiment I, 20 ewes were treated s.c. with either 0, 1, 2, or 4 mg/day roIFN tau (0.7 x 10(8) U/mg; 5 ewes/dosage) from Days 11 to 15 of the estrous cycle (estrus = Day 0) and were challenged with OT (30 IU) on Day 15. Jugular blood samples were collected at -10, 0, 10, 20, 30, 40, 50, and 60 min relative to the OT challenge and assayed for PGFM. Recombinant oIFN tau increased IEI (16.7, 18.7, and 22.6 +/- 0.6 days for 0, 2, and 4 mg roIFN tau, respectively, p < 0.01). Recombinant oIFN tau did not affect peak PGFM response to OT (2309 +/- 172 pg/ml; p > 0.1). However, the 4 mg/day dosage delayed the time to peak PGFM (32.4 vs. 47.5 +/- 3.4 min; p < 0.01, 0 vs. 4 mg) and resulted in approximately 200% higher concentrations of PGFM at 60 min post-OT (0 vs. 4 mg/day, p < 0.07). Experiment II was similar to experiment I, except that only the 0- and 4-mg/day dosages of roIFN tau were administered. Ewes were hysterectomized on Day 16, and assay of uterine flushes detected no AVA from ewes treated with either 0 or 4 mg/day roIFN tau. In experiment III, 20 ewes were treated s.c. with either 0, 2, 4, or 6 mg roIFN tau on Day 12. Blood samples, RT, and RR were obtained at frequent intervals for 24 h, and plasma was assayed for progesterone, cortisol, LH, and AVA. Plasma AVA, which increased in a dose-dependent manner, was detectable within 60 min and remained elevated at 24 h compared to control values. RT (elevated 0.5-1.0 degrees C), RR, and cortisol increased in response to all dosages of roIFN tau, with peak values occurring 150-180 min postinjection. For all dosages of roIFN tau, plasma progesterone declined from 120 to 360 min posttreatment and then returned to pretreatment values by 24 h (p < 0.01) as compared to controls. Overall, exogenous roIFN tau altered uterine PGFM response to OT from a pulse to a gradual and sustained elevation and extended IEI with only a transient decline in progesterone and mild hyperthermia, effects that are not expected to compromise pregnancy.

Interferon tau: a novel pregnancy recognition signal.

PROBLEM: Trophectoderm of ruminant conceptuses (embryo and associated membranes) secretes tau interferons (IFNtau) as the pregnancy recognition signal. How does it act? METHOD: Review of current data. RESULTS: IFNtau acts on uterine epithelium to suppress transcription of the genes for estrogen receptor and oxytocin receptor. This blocks development of the uterine luteolytic mechanism and, therefore, release of luteolytic pulses of prostaglandin F2alpha, but it has no effect on expression of the progesterone receptor. Maintenance of progesterone secretion by the corpus luteum ensures establishment and maintenance of pregnancy. Secretion of IFNtau on days 12-15 for sheep and days 14-17 for cows and goats is essential for pregnancy recognition. CONCLUSION: We propose that IFNtau affects endometrial gene expression by activating the Jak/Stat pathway, which results in formation of the ISGF3alpha transcription factor complex. ISGF3alpha binds to interferon-stimulated response elements and activates transcription of interferon-responsive genes such as interferon regulatory factor-1 (IRF-1) which, in turn, activates expression of the negative-acting transcription factor IRF-2. Pregnancy (or intrauterine injection of roIFNtau) results in a transient increase in endometrial IRF-1 expression followed 36-48 hr later by a sustained increase in IRF-2. We propose that IRF-2, or an IFNtau-induced negative regulatory factor like IRF-2, suppresses expression of the estrogen receptor gene and directly or indirectly blocks expression of the gene for oxytocin receptor to abrogate the uterine luteolytic mechanism and ensure the establishment of pregnancy.

Placental interferons.

Trophectoderm of ruminant conceptuses (embryo and associated membranes) secrete tau interferons (IFN tau) as the pregnancy recognition signal. Secretion of IFN tau on gestational days 12-13 for sheep and gestation days 14-17 for cows and goats is critical for pregnancy recognition. IFN tau acts on uterine epithelium to suppress estrogen receptor and oxytocin receptor gene expression, which prevents uterine release of luteolytic pulses of prostaglandin F2 alpha (PGF). Expression of the progesterone receptor (PR) gene in uterine endometrium is not affected by oIFN tau. Maintenance of progesterone secretion by the corpus luteum (CL) ensures establishment of pregnancy. Pig conceptuses secrete both IFN alpha and IFN gamma between days 15-21 of gestation, but their role(s) in early pregnancy is unknown. Estrogen secreted by pig trophoblast between gestational days 11-13 and 15-25 increases endometrial receptors for prolactin and causes exocrine secretion of PGF into the uterine lumen to prevent luteolysis. Shared cell-signaling mechanisms by IFNs and lactogenic hormones through Janus kinases (JAK) 1 and 2 may provide a common pathway to abrogate luteolytic mechanisms to ensure establishment of pregnancy. The role(s) of IFNs produced by human and rodent placentae is not known.