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Stem cell factor (SCF) is a growth factor known to have profound effects on the proliferation, migration, differentiation, and survival of numerous cell types, including those of the ovary. The objectives of the present study were to identify and characterize expression of this growth factor in the ovine corpus luteum (CL). A 952-bp cDNA was amplified from Day 3 (Day 0 = estrus) ovine luteal total cellular (tc) RNA by reverse transcriptase-polymerase chain reaction and determined to encode SCF. Northern analysis of Day 10 luteal poly(A) + RNA indicated one major transcript of approximately 6.5 kb. SCF mRNA was localized within Day 3 and Day 10 CL by in situ hybridization and was expressed throughout luteal tissue on both days examined. To assess expression throughout the luteal phase, SCF mRNA was quantified by ribonuclease protection assay in tcRNA collected on Days 3, 7, 10, 13, and 16; values did not differ across days (p > 0.10). Similarly, SCF mRNA was quantified in tcRNA isolated from pools of Day 10 large and small steroidogenic cells (n = 4 and 3, respectively); levels did not differ (p > 0.10) between cell types. In addition, SCF protein was detected in CL on Days 3 and 10, and was expressed in a cell-specific manner in cells with morphological characteristics of large and small luteal cells. These data indicate that SCF may be involved in communication among steroidogenic cells and/or between-steroidogenic and nonsteroidogenic cells of the CL.

Specific binding of ovine GH (oGH) to microsomal membranes isolated from fetal sheep liver is slight to nonexistent. The complementary DNA sequence encoding the oGH receptor (oGHR) has been reported, and Northern blot analysis has indicated that oGHR messenger RNA (mRNA) is present in fetal liver and skeletal muscle from mid- to late gestation. In human tissues, the GHR mRNA exists in multiple forms, including the deletion of exon 3 and variable 5'-untranslated regions. In rodents, the GHR mRNA exists in two forms, one encoding the membrane-bound receptor and the other encoding the soluble GH-binding protein. To further characterize the oGHR mRNA transcript present in ovine fetal liver during gestation, we designed a series of primers to be used in reverse transcriptase-polymerase chain reactions (RT-PCR), which generate products that span from the 5'-untranslated region through the coding region of the oGHR mRNA. Nucleotide sequences of the resulting complementary DNAs revealed that an oGHR mRNA is present from mid- to late gestation (days 60-135) which contains the region analogous to exon 3 of the human GHR gene. However, the 5'-untranslated region previously reported in adult tissues was not present until day 135 of gestation in fetal liver, nor was it present in day 100 fetal skeletal muscle. Northern hybridization analysis indicates that the major oGHR transcript in day 105 fetal liver is 5.8 kilobases (kb) in size, with minor transcripts observed at 4.7 kb and three transcripts greater than 6.5 kb. By day 135 of gestation, the transcript size is the same as that observed in day 100 pregnant ewe liver (5.5 kb). We conclude that the oGHR mRNA present in midgestation fetal liver differs structurally from the transcript present in late gestation fetal liver and adult liver, and this difference may explain the lack of specific GH binding to ovine fetal liver membranes. Furthermore, our results suggest that there is a developmental switch in the structure of oGHR mRNA that occurs shortly before term, potentially preparing the fetus to respond to GH postnatally.

The objective of this study was to evaluate the effects of overfeeding naturally-mated adolescent ewes (Ovis aries) on maternal, fetal, and postnatal lamb growth, hormone concentrations, and lamb carcass characteristics. Two experiments were conducted in which singleton-bearing adolescent ewes were fed a diet containing 2.72 Mcal/kg ME at a rate which met NRC gestational age requirements (MN; n = 10 in Exp. 1, n = 7 in Exp. 2) or were fed the same diet ad libitum (15% refusal rate) throughout gestation (HN; n = 7 in Exp. 1, n = 6 in Exp. 2). Ewe BW was greater (P0.05) for HN than MN ewes beginning on 75 d and 52 d of gestation for Exp. 1 and 2, respectively. Final BCS was greater (P ≤ 0.05) for HN than MN ewes in both experiments; 3.5 vs. 3.0, respectively, for Exp. 1, and 4.8 vs. 2.9, respectively, for Exp. 2. Fasting maternal blood insulin concentrations were greater (P ≤ 0.05) in HN ewes near term (135 d of gestation), whereas fasting maternal glucose concentrations were greater (P ≤ 0.05) during most of the second half of gestation in HN ewes, for both experiments. Gestation length did not differ (P = 0.69) between treatments in Exp. 1, but in Exp. 2, HN ewes had shorter (P = 0.01) gestation lengths (144 vs. 149 d) and had increased (P = 0.002) dystocia scores. Fetal abdominal circumference was greater (P0.05) in lambs from MN than HN ewes at 97 d of gestation in Exp. 1 (20.8 vs. 17.4 cm) but did not differ (P = 0.94) between treatments at 95 d of gestation in Exp. 2 (averaging 20.5 cm). There were no differences (P ≥ 0.15) in lamb BW, abdominal circumference, crown-rump length, and biparietal distance at birth; or in postnatal BW and plasma concentrations of glucose, insulin, and lactate in either experiment. There were no differences (P ≥ 0.18) in HCW, dressing percentage, LM area, fat thickness, or KPH between treatments in Exp. 2. Although there was no difference (P ≥ 0.31) between treatments in concentrations of IGF1 or IGF2 mRNA in liver samples collected at harvest, lambs from MN ewes had greater (P ≤ 0.05) concentrations of IGF1R and INSR mRNA, suggesting long-term effects of maternal diet on postnatal hepatic function. In conclusion, excess nutrition during gestation in naturally-mated adolescent ewes did not affect birth weight or postnatal performance of offspring.

Stem cell factor (SCF), or c-kit ligand, is a multipotent growth factor that has been implicated in an important role in various aspects of animal development, including maintenance of the viability of primordial germ cells. A porcine SCF (pSCF) cDNA was generated from porcine uterine endometrial mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), and its nucleotide sequence was determined. The 952-bp pSCF cDNA contained an open reading frame encoding 274 amino acids. The deduced amino acid sequence of pSCF is approximately 86%, 83%, and 82%, identical to human, rat, and mouse SCFs, respectively; and it contains the four conserved cysteine residues and Asn-linked glycosylation sites. One additional amino acid was identified in pSCF, Glu130, which is not in the human (hSCF), rat (rSCF), or mouse (mSCF) sequences. Northern analysis of poly(A)+ RNA obtained from Day 16 pregnant endometrium revealed a transcript of approximately 6.5 kb. The size of this transcript is consistent with the size of full-length SCF mRNA, and the occurrence of alternatively spliced pSCF mRNAs were not detected by RT-PCR/Southern hybridization analysis of endometrial and ovarian total cellular RNA (tcRNA). Porcine SCF mRNA has been localized by in situ hybridization in porcine endometrial stromal tissue. Pregnancy does not appear to be a prerequisite for pSCF mRNA expression in endometrial tissue since it was detectable in tissue and tcRNA obtained from pregnant and nonpregnant gilts. The biological significance of uterine pSCF expression is currently unclear, but it probably participates in intracellular communication within the uterus.

The placenta provides the means for nutrient transfer from the mother to the fetus, waste transfer from the fetus to the mother, protection of the fetus from the maternal immune system, and is an active endocrine organ. While many placental functions have been defined and investigated, assessing the function of specific genes expressed by the placenta has been problematic, since classical ablation-replacement methods are not feasible with the placenta. The pregnant sheep has been a long-standing animal model for assessing in vivo physiology during pregnancy, since surgical placement of indwelling catheters into both maternal and fetal vasculature has allowed the assessment of placental nutrient transfer and utilization, as well as placental hormone secretion, under unanesthetized-unstressed steady state sampling conditions. However, in ruminants the lack of well-characterized trophoblast cell lines and the inefficiency of creating transgenic pregnancies in ruminants have inhibited our ability to assess specific gene function. Recently, sheep and cattle primary trophoblast cell lines have been reported, and may further our ability to investigate trophoblast function and transcriptional regulation of genes expressed by the placenta. Furthermore, viral infection of the trophoectoderm layer of hatched blastocysts, as a means for placenta-specific transgenesis, holds considerable potential to assess gene function in the ruminant placenta. This approach has been used successfully to "knockdown" gene expression in the developing sheep conceptus, and has the potential for gain-of-function experiments as well. While this technology is still being developed, it may provide an efficient approach to assess specific gene function in the ruminant placenta.

A study was conducted to examine the influence of ovarian steroids on relaxin-induced changes in distensibility and composition of the uterine cervix in gilts. Ovariectomized, prepubertal gilts received (i.m.) either 1) 0.5 ml corn oil (CO) for 16 days; 2) 0.5 ml CO for 16 days and estradiol benzoate (EB; 200 pig) twice daily on Days 13-14; 3) EB on Days 0-1 followed by CO on Days 3-16 (EB + CO); 4) EB on Days 0-1 followed by CO on Day 2 and twice daily injections of 100 mg progesterone (P) on Days 3-16 (EB + P); or 5) EB on Days 0-1 followed by CO on Day 2 and P on Days 3-16 with additional EB treatment on Days 13-14 (EB + P + EB). One half of each group also received (i.m.) 0.5 ml 0.1 M PBS or 0.5 mg relaxin (RLX) every 6 h for 54 h coinciding with the final 2 days of the experimental period. Administration of RLX increased (p < 0.05) cervical distensibility compared to PBS.treatment, with the response being greater (p < 0.05) in EB-, EB + CO-, and EB + P + EBtreated gilts compared to CO- or EB + P-treated gilts. Water content of cervical tissue was greater (p < 0.05) in EB-, EB + P, and EB + P + EB-treated gilts compared to CO- or EB + CO-treated gilts; and in all treatments, RLX enhanced (p < 0.05) imbibition of water by cervical tissues compared to that in PBS-treated gilts. Compared to administration of CO and EB + CO, administration of steroids reduced (p < 0.05) the cervical concentrations of glycosaminoglycans (GAG) in EB- and EB + Ptreated gilts and those of collagen in EB- and EB + P + EB-treated gilts. Co-administration of RLX depressed (p < 0.05) the concentration of GAGs in cervical tissues from gilts in all steroid treatments except EB + P + EB. Additionally, administration of RIX reduced (p < 0.05) concentrations of collagen in CO- and EB + CO-treated gilts but did not affect concentrations of collagen in those treated with EB, EB + P, or EB + P + EB. These data demonstrate that RLX stimulates cervical dilation in gilts, a response augmented by exposure to estrogen. RLX-induced compositional changes in cervical tissues associated with increased distensibility included the enhanced imbibition of water and reduced concentrations of GAGs and collagen. To a lesser extent, administration of EB or P increased water content and reduced the concentrations of GAGs and collagen in cervical tissues, but these compositional changes were not associated with changes in cervical distensibility.

Two experiments were conducted to evaluate effects of ractopamine (RAC) and steroidal implant treatments on performance, carcass traits, blood metabolites, and lipogenic enzyme activity in feedlot cattle. In Exp. 1, yearling steers (n = 486; initial BW = 305 kg) were used in a 3 × 3 factorial arrangement of RAC doses of 0 (R0), 100 (R100), or 200 (R200) mg·steer(-1)·d(-1) fed for 28 d and implant regimens (implant-reimplant) of no implant-no reimplant (NI-NI), 120 mg of trenbolone acetate (TBA) and 24 mg of estradiol-17β (E17B)-no implant (RS-NI), or 80 mg of TBA and 16 mg of E17B followed by 120 mg of TBA and 24 mg of E17B (RI-RS). Except for KPH and skeletal maturity score, no RAC × implant interactions were noted (P0.10). Carcasses from R200 were 6.3 kg (P = 0.042) heavier than those from R0. Marbling, calculated empty body fat (EBF), and USDA quality grade did not differ (P0.10) among RAC treatments. The RI-RS steers had 12.6 kg (P = 0.001) and 41.1 kg (P0.001) greater HCW than RS-NI and NI-NI, respectively. Despite no difference (P0.10) in EBF, marbling score was decreased for RI-RS (P0.001) and RS-NI (P = 0.001) relative to NI-NI, resulting in 14.6 and 11.4 percentage unit fewer USDA Prime and Choice carcasses with RI-RS (P = 0.008) and RS-NI (P = 0.039) than with NI-NI. In Exp. 2, heifers (n = 48; initial BW = 347 kg) were used in a 3 × 2 factorial arrangement of RAC doses of 0 (R0) or 250 (R250) mg·heifer(-1)·d(-1) and implant regimens of none (NI), 200 mg of TBA (TO), or 200 mg of TBA and 20 mg of E17B (TE). Blood samples were collected at various times during the feeding period, and subcutaneous adipose samples were collected on d 119. For growth and carcass measurements, no RAC × implant interactions (P0.10) were detected. The RAC-supplemented heifers had greater HCW (P0.10) with no difference in marbling score. For implant regimens, TE heifers had greater HCW than the NI (P = 0.001) and TO (P = 0.037) heifers. Although EBF did not differ among implant treatments (P0.10), TE (P = 0.021) and TO (P = 0.039) had fewer Choice carcasses than NI. Heifers with implants had decreased cortisol and increased IGF-1 and NEFA (P0.10) compared with NI heifers. An implant × RAC interaction was detected (P = 0.001) for serum urea nitrogen (SUN), with TE and RAC-supplemented heifers having decreased SUN. These data suggest that the effects of implant and RAC on growth and carcass traits are independent and that USDA quality grade and marbling score can differ significantly among carcasses with similar calculated EBF values.

Ovine placental lactogen (oPL) is structurally similar to PRL, is a product of the chorionic epithelium, and has been implicated in playing a supportive role in fetal growth. This study examined the concentration and cellular location of oPL mRNA at five stages of pregnancy (days 60, 90, 105, 120, and 135) in 21 cross-bred ewes, and results were compared to maternal and fetal serum oPL concentrations, cotyledonary DNA and actin mRNA concentrations, and total fetal weight. The concentration of oPL mRNA in fetal cotyledonary tissue increased (Por = 0.05) from day 60 (15.4 pg/micrograms total cellular RNA) to day 120 (73.7 pg/micrograms total cellular RNA) of gestation and then plateaued, whereas no significant changes occurred in the concentration of actin mRNA over the gestational ages examined. The concentration of DNA in cotyledonary tissue (micrograms per mg wet tissue) increased (Por = 0.05) from days 60 through 120 and remained constant through day 135, such that when oPL mRNA was expressed on a picogram per microgram DNA basis, no stage of gestation effect (Por = 0.10) was observed. The maternal serum oPL concentration increased (Por = 0.05) from day 60 (7.1 ng/ml) to day 105 (417.7 ng/ml), followed by a large but nonsignificant (Por = 0.10) increase in maternal serum oPL occurring on day 135 (902.0 ng/ml). Fetal serum oPL concentrations increased (Por = 0.05) from day 60 (11.0 ng/ml) to day 90 (29.0 ng/ml) and then remained relatively constant. Maternal serum oPL (r = 0.68; Por = 0.01) and cotyledonary oPL mRNA levels (r = 0.61; Por = 0.05) were correlated with total fetal weight when adjusted for fetal number and gestational age, and together accounted for 80.6% (r2 value) of the variation found in total fetal weight. The correlation between fetal serum oPL concentrations and total fetal weight was nonsignificant (Por = 0.10). Examination of placentome cross-sections by immunocytochemistry and in situ hybridization at the five gestational ages indicated that the chorionic binucleate cell was the sole source of oPL. These data provide evidence that, like maternal serum concentrations of oPL, oPL mRNA expression by chorionic binucleate cells increases until late gestation, whereas fetal serum concentrations of oPL plateau during midgestation.(ABSTRACT TRUNCATED AT 400 WORDS)

This study examined the influence of ovarian steroids on the uterotropic actions of relaxin (RLX) in ovariectomized prepubertal gilts. Ovariectomized gilts received (im) corn oil (CO), estradiol benzoate (EB), or EB and progesterone (P) for 0-16 days. Steroid administration was patterned to approximate the plasma concentrations of endogenous ovarian steroids observed during 1) the follicular phase (EB), 2) luteal phase (EB+P), and 3) early pregnancy (EB+P+EB). Half of each group also received PBS or 0.5 mg RLX every 6 h for 54 h, coinciding with the final 2 days of the experimental period. After hysterectomy, uterine tissues were analyzed for water, dry matter, protein, DNA, glycosaminoglycans (GAGs), and collagen contents. Administration of EB or P increased uterine weight 5- to 6-fold, but no differences were observed between EB+P- and EB+P+EB-treated gilts. Cotreatment with RLX enhanced steroid-induced uterine growth 40-70%, and RLX stimulated growth in CO- and EB+CO control gilts 2- to 3-fold. The water content of uterine tissues was greater in EB-, EB+P-, and EB+P+EB-treated gilts than in their respective controls, and this response was augmented by RLX in all treatment groups. Administration of steroids stimulated a 4- to 5-fold increase in uterine dry weight compared to that in controls, with responses not differing between EB+P- and EB+P+EB-treated gilts. In all groups, RLX increased uterine dry weight. Protein and DNA contents of uterine tissue increased with steroid treatment, but neither variable differed between EB+P- and EB+P+EB-treated gilts. Administration of RLX, alone or in combination with steroids, increased protein and DNA contents of uterine tissues. The tissue content of GAGs increased in response to steroids, and coadministration of RLX did not alter this response. Although the uterine tissue concentration of collagen was reduced in steroid- and RLX-treated gilts, the collagen content of the uterus was not affected by the various treatments. The results of this study indicate that RLX is a potent stimulator of uterine growth under a variety of steroidal environments. RLX- or steroid-induced uterine growth was manifest by increased water, dry matter, protein, and DNA and GAG contents, but the uterine content of collagen was not affected. The overall growth-promoting effects of EB and the stimulation of DNA accretion by RLX were not observed when gilts were cotreated with P.(ABSTRACT TRUNCATED AT 400 WORDS)

The endometrium of the pig secretes retinol-binding protein (RBP) under the influence of progesterone (P4). The objective of this study was to determine how conceptus-derived estrogen might modulate this production of RBP around days 11-13 of pregnancy when conceptuses elongate from spheres to long thread-like forms. Concentrations of retinol and RBP were low (35 +/- 7 ng/ml) in uterine flushings obtained on days 10-12 of the estrous cycle or from pregnant gilts in which conceptuses had not elongated. Concentrations of retinol and RBP increased 7- to 8-fold (P less than 0.01) in flushings where filamentous conceptuses were present. Size exclusion and ion exchange chromatography demonstrated that virtually all retinol assayed in uterine flushings was associated with RBP. Northern blot analysis with a cDNA representing uterine RBP revealed a single endometrial mRNA 1.1 kilobases in length. Expression of RBP mRNA in uterine endometrium was measured in ovariectomized prepubertal gilts after the administration of steroids according to the following regimens: I, corn oil (days 0-16; n = 10); II, estradiol benzoate (EB; days 13-14; n = 11); III, EB (days 1-2; n = 12); IV, EB (days 1-2) plus P4 (days 3-16; n = 12); and V, EB (days 1-2) plus P4 (days 3-16) plus EB (days 13-14; n = 12). EB (200 micrograms) and P4 (100 mg) were administered twice daily. Treatment IV was designed to stimulate the estrous cycle, and treatment V simulated early pregnancy. All gilts were hysterectomized on day 16, and total uterine mRNA (3 micrograms) was analyzed by Northern blotting. No RBP mRNA was detected in groups I, II, or III. In group IV, 5 of 12 gilts had detectable RBP mRNA, as measured by densitometric scanning (OD = 0.35 +/- 0.14). RNA isolated from all gilts in group V (12 of 12) gave a strong hybridization signal (OD = 1.58 +/- 0.22) for RBP. Finally, RBP mRNA was examined in the uterine endometrium of mature gilts on day 13 of the estrous cycle (n = 4), day 13 of pseudopregnancy (2.5 mg EB given on days 11-12; n = 4), or day 13 of pregnancy after conceptuses had elongated (n = 4). RBP mRNA was present in all groups, but was enhanced approximately 12-fold (P less than 0.01) in pregnant and pseudopregnant gilts compared to that in control gilts.(ABSTRACT TRUNCATED AT 400 WORDS)

A randomized complete block design experiment with 360 single-source black yearling steers (average BW = 316.1 +/- 9.1 kg) fed a 91% concentrate (steam-flaked corn base) diet was conducted to evaluate the effects of supplemental vitamin A (0, 1,103, 2,205, 4,410, or 8,820 IU/kg of dietary DM) on plasma and liver vitamin A and E concentrations, lipogenic enzyme activity, marbling score, and performance of yearling steers. Final BW (586, 580, 590, 585, and 584 kg for 0, 1,103, 2,205, 4,410, and 8,820 IU of supplemental vitamin A/kg of DM, respectively) did not differ (P = 0.39) among treatments. Feed efficiency, ADG, and daily DMI did not differ (P0.10) among treatments within each 28-d period or for the overall experiment. From d 57 to slaughter, average DMI (10.33, 10.28, 10.57, 9.75, and 10.22 kg/steer daily for 0, 1,103, 2,205, 4,410, and 8,820 IU of vitamin A/kg of DM, respectively) was less (P0.02) by steers receiving 4,410 IU of supplemental vitamin A/kg of dietary DM than for steers in the other treatments. Furthermore, DMI was greater (P = 0.06) for steers receiving 2,205 IU of supplemental vitamin A/kg of dietary DM than for steers receiving 8,820 IU of supplemental vitamin A/kg of DM. Marbling score, HCW, LM area, and 12th-rib fat thickness did not differ (P0.10) among treatments. Similarly, the percentage of carcasses gradingor=USDA Choice (62.6, 52.8, 64.0, 58.4, and 58.4% for 0, 1,103, 2,205, 4,410, and 8,820 IU of vitamin A/kg of DM, respectively), Select, oror=Standard did not differ (P0.10) among treatments. Except for d 56 (P = 0.050; r = 0.18 for liver retinol), no correlations (P0.10) between marbling score and any plasma or liver tissue retinol or alpha-tocopherol concentrations or vitamin A intake were found, and no differences (P0.10) in lipogenic enzyme activity were detected among treatments. Taken together with previous and concurrent research, results of this experiment suggest that vitamin A supplementation at a concentration up to twice the NRC recommendation has little effect on performance, marbling, or lipogenic enzyme activity in adipose tissue samples in yearling feedlot steers, and that 2,205 IU of supplemental vitamin A/kg of DM (20,000 IU/steer daily) or less is adequate to meet the vitamin A requirements of finishing beef cattle.

Twenty-three gilts were assigned io receive a 1.0 mi injection of either saline (C) or 0.5 mg purified porcine relaxin in saline (pRLX) every 6 h on Days 6–11 of gestation. All 12 gilts assigned to relaxin treatment were pregnant on Day 11. Following hysterectomy on Day 11, mean uterine wet weight was greater (P 0.10) in pRLX and C gilts, respectively. Uterine volumes and circumferences were also increased (P

Pregnancy in cattle and sheep can be diagnosed by the presence of a conceptus-derived antigen in maternal serum that is secreted by trophoblast and placental tissue primarily as an acidic component of Mr 67,000. Molecular cloning of its cDNA reveals that the antigen belongs to the aspartic proteinase family and has greater than 50% amino acid sequence identity to pepsin, cathepsin D, and cathepsin E. The inferred sequences of the ovine and bovine polypeptides show approximately 73% identity to each other. Critical amino acid substitutions at the active site regions suggest that both proteins are enzymatically inactive. The antigen is a product of trophoblast binucleate cells that invade maternal endometrium at implantation sites.

Crossbred beef heifers (N = 36) were assigned to one of three treatment groups: untreated controls (C; N = 15); Group R4, treated with pig relaxin (1.0 mg i.m.) 4 days pre partum (N = 11); or Group R7, treated with pig relaxin (1.0 mg i.m.) 7 days pre partum (N = 10). Bioactivity of the pig relaxin (UMC-R-P8) was determined by the mouse interpubic ligament assay to be greater than or equal to 3000 U/mg, both before and after the experiment was conducted. Peripheral serum immunoreactive relaxin values were 7.5, 3.4, 2.5, and 1.5 ng/ml at 2, 4, 6 and 8 h after injection of relaxin, respectively. Gestation lengths were 282.9 +/- 1.1, 285.5 +/- 1.3 and 285.6 +/- 1.5 days for Groups C, R4 and R7 (C vs R4 + R7; P congruent to 0.08). Calving difficulty score (1 to 4) tended to be greater (P congruent to 0.08) for Group R4 and R7 heifers (C vs R4 + R7; 1.3 +/- 0.24 vs 1.75 +/- 0.28 + 2.04 +/- 0.32), but the incidences of dystocia and retained placentae were not influenced by treatment (P greater than or equal to 0.10). The mean concentration and concentration profile of daily serum progesterone, oestradiol-17 beta, dihydroprostaglandin F-2 alpha and relaxin were not affected by treatment from 6 days pre partum through 2 days post partum. Cervical diameter, cervical softness score, pelvic measurements, and vulva opening length during the periparturient period were not affected (P greater than or equal to 0.10) by treatment, but all of these characteristics changed over time (P less than or equal to 0.01), relative to calving. We conclude that i.m. administration of pig relaxin (greater than or equal to 3000 U) does not effectively alter periparturient characteristics of beef heifers. Discrepancies between these results and those reported for intracervical administration cannot be readily explained.

Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4·2 mg of oPL, with an ∼ 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0·18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4·1 nmol/l) and oPRL (ED50 = 1·1 μmol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a λZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides). The predicted amino acid sequence derived from the nucleotide sequence confirmed homology to bPL (67%) and oPRL (48%). Little amino acid sequence existed with other PLs (≤29%) or GH proteins (≤27%). These results suggest that oPL and oGH are more biologically similar in their ability to compete for fetal liver binding sites, but that oPL is structurally more similar to oPRL. Elucidation of exact structure–function relationships for oPL will, however, require further investigation. Journal of Endocrinology (1990) 126, 141–149

Uterotropic actions of relaxin (RLX) have been demonstrated in rats and mice but not in other species. Experiments were conducted to assess (1) if RLX has uterotropic effects in prepubertal gilts and (2) if RLX-induced imbibition of water by uterine tissues is mediated by histamine. Prepubertal gilts received (i.m.) either 0.5 mg pRLX (greater than or equal to 3000 U/mg; n = 10) in 0.1 M phosphate-buffered saline (PBS) or PBS vehicle (n = 8) every 6 h for 54 h. Additionally, one-half of each group received either (1) the histamine receptor antagonists (300 mg i.m. of each), pyrilamine maleate (H1) and cimetidine (H2), in PBS and corn oil, respectively, or (2) control vehicles every 6 h for 66 h beginning 12 h prior to initiation of RLX treatment. Treatment with RLX stimulated a twofold increase (p less than or equal to 0.05) in uterine weight in prepubertal gilts. Increases in total uterine weight were manifested by greater (p less than or equal to 0.05) uterine water content and dry weight in RLX-treated gilts. Cotreatment with histamine receptor antagonists did not alter RLX-induced changes in uterine water content or dry weight. Uterine contents of soluble protein and DNA were greater (p less than or equal to 0.05) in RLX-treated gilts compared to controls. Treatment with RLX tended (p approximately 0.08) to reduce protein; DNA ratio of uterine tissues and increased (p less than or equal to 0.05) uterine DNA as a percentage of uterine dry weight.(ABSTRACT TRUNCATED AT 250 WORDS)

The definition of hormone function was classically accomplished by ablation-replacement studies. However, as our knowledge of the complexity of hormones and growth factors has grown, it has become increasingly difficult to clearly define the necessity and function of many of the hormones, growth factors, and regulatory proteins under investigation. The use of homologous recombination within mouse embryonic stem cell lines allows functional gene ablation and has been used extensively during the past 15 yr to define specific gene function. The use of similar methodologies in livestock species has yet to yield an efficient approach. In contrast, the parallel development of our understanding of naturally occurring RNA interference, along with the development of efficient virus-based vectors for gene transfer, holds great potential for effectively "knocking down" specific gene function. Short-hairpin (sh) RNA-encoding cassettes, typically consisting of inverted repeats separated by a loop sequence and followed by a short poly(T) string to terminate transcription, are inserted downstream of an RNA polymerase III promoter within the viral vector of choice. Several viral vectors are useful for delivery of shRNA expression cassettes, each with particular attributes. Adenovirus- and lentivirus-derived vectors provide a high rate of infectivity in most mammalian cell types, with lentiviral vectors allowing stable integration into the host genome if the study of long-term effects is needed. Upon transcription, a shRNA is generated, and the loop is recognized by the processing enzyme Dicer, generating guide sequences. Guide sequences are incorporated into the RNA-induced silencing complex, which targets mRNA for degradation if recognized by the guide sequence. For each mRNA of interest, design and testing of a number of shRNA, along with adequate controls, are required to identify the most efficient construct before proceeding to in vivo use. This technology may become the method of choice for defining gene function in livestock.

Intrauterine growth restriction (IUGR) is a significant health issue that not only affects infant mortality and morbidity, but may also predispose individuals to coronary heart disease, diabetes, hypertension and stroke as adults. The majority of IUGR pregnancies in humans are characterized by asymmetric fetal growth, resulting from inadequate nutrient transfer to the fetus. Furthermore, most of these pregnancies involve functional placental insufficiency, and may also show altered umbilical velocimetry. As the severity of IUGR increases, the fetus becomes increasingly hypoxic, hypoglycaemic and acidotic. In addition, placental transfer or utilization of some amino acids is known to be altered in IUGR pregnancies. Although a great deal has been learned from clinical studies of human IUGR, appropriate animal models are required to define completely the mechanisms involved in the development of IUGR. The pregnant sheep is a long-standing model for placental-fetal interactions, and fetal growth restriction can be induced in pregnant sheep by maternal nutrient restriction, maternal nutrient excess, administration of glucocorticoid, utero-placental embolization, carunclectomy and maternal hyperthermia. Although all of these sheep models are capable of inducing fetal growth restriction, the degree of restriction is variable. This review compares these sheep models of IUGR with the characteristics of human IUGR.

Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2alpha was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2alpha, but variations in the predicted N-terminus were observed, and three unique AP-2alpha splice-variants were identified. Expression of AP-2alpha variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP-2alpha splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2alpha transcript, which shares identity to Xenopus AP-2alpha, was expressed in BeWo cells. Immunoblot analysis confirmed AP-2alpha variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2alpha variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2alpha splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.

Ovine placental lactogen (oPL), a member of the growth hormone/prolactin gene family, is produced by chorionic binucleate cells at the maternal-fetal interface, and is thought to modulate metabolic processes and enhance fetal growth. We have determined that the oPL gene contains five exons and four introns, and the transcriptional start site was mapped 91 bp 5' of the initiation codon (AUG). An additional 4.5 kb of 5'-flanking sequence was sequenced and used for transient transfection analysis in human (BeWo) and rat (Rcho-1) choriocarcinoma cell lines to examine trophoblast cell-specific activity. Trophoblast cell-specific transactivation of the reporter gene was conferred by the proximal 1. 1 kb of oPL gene 5'-flanking sequence. Transfection of deletion constructs derived from the 1.1 kb of 5'-flanking sequence resulted in varying profiles of transactivation between the two choriocarcinoma cell lines, but maximal activation in both cell lines resided within the proximal 383 bp of oPL gene 5'-flanking sequence. DNase I protection analysis using ovine chorionic binucleate cell nuclear protein, identified 19 footprints within the 1.1-kb sequence, six of which are located within the 383-bp region. Electrophoretic mobility-shift assays and mutational analysis identified two functional GATA (-67, -102) sequences as transactivators of the oPL gene. However, a previously undefined element (GAGGAG) residing at -338 and -283 is required for full transactivation, and mutation of either significantly reduces reporter activity. In addition, an AP-2 site (-58) and an E-box (-163) were identified and may coordinate oPL transactivation. Transcriptional regulation of human and rodent PL genes has been previously characterized, and our results indicate that tissue-specific regulation of oPL expression may result from cis-acting elements in common with human and rat genes expressed within the placenta. However, our data indicate that regulation of oPL also results from novel cis-acting elements.

While several studies have documented the presence of plasminogen activator (PA) activity in hen ovarian follicle granulosa and theca tissues, to date it has not been possible to conclusively distinguish between the urokinase (u) and the tissue-type (t) form of the enzyme; this inability is due, in part, to lack of the cloned or characterized chicken tPA gene or gene product. Thus, the present studies were conducted to identify a partial cDNA for chicken tPA and subsequently to evaluate expression of uPA and tPA mRNA in granulosa and theca tissues in vivo and in vitro. Urokinase PA and mRNA levels were highest in prehierarchical-follicle granulosa (3- to 5- and 6- to 8-mm follicles) and theca (6- to 8-mm follicles) tissue compared to hierarchical (9-12 mm through largest preovulatory) follicles. In vitro treatment with a phorbol ester (phorbol 12-myristate, 13-acetate), but not a cAMP analogue (8-bromo-cAMP), significantly increased uPA mRNA levels in both granulosa and theca tissue from the largest and second-largest preovulatory follicles. Of special interest was the finding that levels of uPA mRNA were 10.9-fold higher in atretic compared to morphologically normal 3- to 5-mm follicles. Moreover, 4- to 8-mm-follicle granulosa cells, which spontaneously undergo apoptosis in vitro, demonstrated a rapid increase in uPA mRNA levels after 1 h of incubation (prior to the detection of oligonucleosome formation) while levels in preovulatory-follicle granulosa cells, which do not undergo spontaneous apoptosis, were not altered after 18 h of incubation. By contrast, while tPA mRNA can be identified in granulosa and theca tissues from prehierarchical and preovulatory follicles following polymerase chain reaction amplification, constitutively expressed levels of the transcript were too low to reliably measure by Northern blot analysis. These data indicate that while the chicken expresses a tPA gene that is homologous to the mammalian tPA, uPA is the predominant PA expressed in the hen ovary. In addition, the higher levels of uPA mRNA found in granulosa cells actively undergoing apoptosis and in follicles most susceptible to atresia (4-8 mm) suggest a role for this protease in mediating processes both during the early stages of programmed cell death and in the later stages of follicle atresia and resorption.

Stem cell factor (SCF) is a growth factor known to have profound effects on the proliferation, migration, differentiation, and survival of numerous cell types, including those of the ovary. The objectives of the present study were to identify and characterize expression of this growth factor in the ovine corpus luteum (CL). A 952-bp cDNA was amplified from Day 3 (Day 0 = estrus) ovine luteal total cellular (tc) RNA by reverse transcriptase-polymerase chain reaction and determined to encode SCF. Northern analysis of Day 10 luteal poly(A)+ RNA indicated one major transcript of approximately 6.5 kb. SCF mRNA was localized within Day 3 and Day 10 CL by in situ hybridization and was expressed throughout luteal tissue on both days examined. To asses expression throughout the luteal phase, SCF mRNA was quantified by ribonuclease protection assay in tcRNA collected on Day 3, 7, 10, 13, and 16; values did not differ across days (p0.10). Similarly, SCF mRNA was quantified in tcRNA isolated from pools of Day 10 large and small steroidogenic cells (n = 4 and 3, respectively); levels did not differ (p0.10) between cell types. In addition, SCF protein was detected in CL on Days 3 and 10, and was expressed in a cell-specific manner in cells with morphological characteristics of large and small luteal cells. These data indicate that SCF may be involved in communication among steroidogenic cells and/or between steroidogenic and nonsteroidogenic cells of the CL.

Ovine placental lactogen (oPL) exerts actions in sheep and rodent fetal tissues that growth hormone (GH) does not. However, in postnatal tissues, both oPL and GH possess these activities. Although a high-affinity binding site for oPL in ovine fetal liver has been reported, some investigators believe this to be the GH receptor. It was our objective to discriminate between oPL and GH binding to fetal liver microsomes using competitive saturation analyses. Microsomal membranes from fetal liver (Days 60, 90, 105, 120, and 135 of gestation) and postnatal liver (1 wk of age) were incubated with increasing amounts of [ 125 I]oPL in the absence or presence of a 100-fold molar excess of unlabeled oPL. Saturable binding of [ 125 I]oPL was observed with fetal liver and postnatal liver microsomes. The K d of the oPL-binding site in fetal liver was 122.1 ± 8.2 pM (mean ± standard error), and receptor concentrations remained relatively constant (9.8 ± 1.1 fmol/mg of membrane protein) across gestation. The highest concentration of oPL binding was detected in 1-wk postnatal liver microsomes (53.0 fmol/mg of membrane protein). Saturation analyses using [ 125 I]GH and [ 125 I] prolactin (PRL) were also conducted with fetal liver membrane preparations. Although specific binding for these two radiolabeled ligands was observed in control tissues, no specific binding was observed in fetal liver. These data are in agreement with earlier reports that a high-affinity binding site for oPL exists in fetal tissues. The fact that saturable binding could not be demonstrated for either GH or PRL with fetal liver microsomes contradicts recent suggestions that oPL is binding the GH receptor.

Ruminant placentae produce at least two distinct subclasses of the growth hormone/prolactin gene family, the placental lactogens and prolactin-related proteins. Placental lactogens have been purified from cattle, goat and sheep placentae, and the amino acid sequences of bovine and ovine placental lactogen are known. Bovine and ovine placental lactogens are structurally more similar to prolactin than they are to growth hormone. In addition, six unique mRNAs have been described in cattle that encode prolactin-related proteins that are structurally distinct from ruminant placental lactogens. All characterized ruminant placental lactogens and prolactin-related proteins are products of chorionic binucleate cells, but specific biological functions of these placental hormones have not been elucidated. Ovine placental lactogen may modify maternal and fetal intermediary metabolism to provide energy substrates to the fetus. Bovine placental lactogen has been implicated as a luteotropic agent, and is also capable of stimulating mammogenesis and lactogenesis. No ruminant placental lactogen receptor has been structurally characterized, although they are presumed to be similar to either the growth hormone or prolactin receptor. Available technologies will allow many of the questions regarding the regulation, mechanism of action and function of these placental hormones to be addressed.

The corpus luteum is the primary source of circulating relaxin (RLX) in female pigs. However, a preliminary experiment in our laboratory identified RLX in the uterine endometrium of a dav 16 nreanant ailt that had been ovariectomized on day 8 of pregnancy, Hnd the pregnancy maintained via progesterone replacement therapy. Therefore, our objective was to examine the uterus as a potential extraovarian source of RLX during the estrous cycle and early pregnancy in pigs. Reproductive tissues were collected from pregnant (n 2 3/day) and nonpregnant (n 2 S/day) gilts on days 10 (n = 6), 12 (n = 6), 14 (n = 6), 16 (n = 8), 18 (n = 6), and 20 (n = 6) of the estrous cycle and pregnancy and on day 42 (n = 2) of gestation. To verify that the RLX identified in uterine tissues was not of ovarian origin, three additional pregnant gilts were ovariectomized on day 8, and the pregnancy was maintained by progesterone replacement therapy until day 16, when the reproductive tissues were collected for immunohistochemistry. The uterine tissues were scored for specific RLX immunostaining and analyzed for the effects day and pregnancy by analysis of variance. Within the uterine endometrium, the luminal epithelium of pregnant pigs contained more RLX than that of nonpregnant pigs (P 5 0.001). However, there was no difference in either the glandular epithelium or stromal tissues (P 2 0.10). Increased RLX in the luminal epithelium of pregnant pigs was detected on both days 18 (P 5 0.05) and 20 (P 5 0.001) of gestation compared to that in nonpregnant pigs on similar days of the estrous cycle. An effect of day was also observed for RLX immunostaining in the luminal epithelium (P 5 0.005), but not in glandular or stromal tissues (P 2 0.10). In both pregnant and nonpregnant pigs, luminal epithelial RLX immunostaining was faint on days 10-12. Thereafter, RLX immunostaining increased on day 14 to reach peak levels on day 16. In nonpregnant pigs, RLX immunostaining dropped to low levels on day 18 and became faint to absent by day 20 of the estrous cycle. In pregnant pigs, RLX immunostaining remained elevated, but appeared to decline slightly by day 20 and became undetectable by day 42 of gestation. RLX immunolocalization in the glandular epithelium was detected on all days in pregnant and nonpregnant pigs, whereas stromal-specific immunostaining was not observed. In addition, RLX was immunolocalized in both the luminal and glandular epithelium of uteri from pregnant gilts 8 days after ovariectomy. Northern blot analysis identified the approximately l-kilobase RLX messenger RNA in RNA isolated from luteal and endometrial tissues. Reverse transcription of endometrial messenger RNA and subsequent polymerase chain amplification with porcine RLX gene-specific primers identified RLX gene expression in both pregnant and nonpregnant endometrial samples between days 10 and 20. We conclude that the porcine uterine endometrium is an additional source of RLX production during early pregnancy and the estrous cycle. Local uterine production may allow RLX to exert its uterotropic actions during the period of pregnancy establishment. (Endocrinology 135: 2517-2525, 1994)

Previous binding studies indicated that there is little to no specific prolactin binding in ovine fetal liver and adult ovary. Therefore, we sought to determine if ovine prolactin receptor (PRLR) mRNA is present in those tissues. Primers were designed from the bovine PRLR cDNA sequence for use in reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR analysis of ovine fetal liver total cellular RNA (tcRNA) isolated from days 60, 90, 105, 120 and 135 of gestation, and luteal tcRNA isolated from days 3, 7, 10, 13 and 16 of the estrous cycle revealed that PRLR mRNA was present in these tissues. However, two RT-PCR products were generated from both tissues. The two RT-PCR products did not differ between the two tissue sources in sequence, and were designated oPRLR-1 and oPRLR-2. Ovine PRLR-1 is 513 bp in length and is 96.4% identical to the bovine cDNA. Ovine PRLR-2 is identical to oPRLR-1 until nucleotide (nt) 420 at which point a 39 bp insertion occurs. This insertion occurs between Homology Boxes 1 and 2 within the cytoplasmic domain of the receptor, resulting in an 11 amino acid divergent sequence, followed by two stop codons. Ribonuclease-protection assay revealed that oPRLR-1 mRNA is the most abundant in these tissues. Our data indicate that two forms of oPRLR mRNA are Present in fetal liver and adult ovary, and that one form (oPRLR-2) is predicted to encode a truncated PRLR.

Secretion of a tissue inhibitor of metalloproteinases (TIMP-1) is initiated by ovine preovulatory follicles after the gonadotropin surge. In addition, TIMP-1 is a major secretory product of ovine corpora lutea. We have isolated an approximately full-length cDNA clone for TIMP-1 from an ovine luteal cDNA library. The 887-basepair cDNA obtained was 95%, 86%, and 77% identical to the reported nucleotide sequences of bovine, human, and mouse TIMP-1 cDNAs, respectively. Total cellular RNA was isolated from preovulatory follicles collected before (presurge; n = 5) or 12-14 h after (postsurge; n = 4) a LHRH-induced gonadotropin surge (36 h after prostaglandin F2 alpha-induced luteolysis); from luteal tissue collected on days 3, 7, 10, 13, and 16 postestrus (n = 5, 5, 4, 5, and 5, respectively); and from purified populations of small (n = 4) and large (n = 3) luteal cells. Concentrations of TIMP-1 mRNA (picograms per micrograms tissue DNA) were increased in preovulatory follicles after exposure to a gonadotropin surge (Por = 0.01). TIMP-1 mRNA was localized primarily to the granulosa layer of postsurge follicles by in situ hybridization. Concentrations of TIMP-1 mRNA in luteal tissue did not differ throughout the luteal phase (P = 0.07). However, TIMP-1 mRNA was localized predominantly to specific cells located in the connective tissue surrounding and within day 3 corpora lutea. In situ hybridization of day 10 corpora lutea localized TIMP-1 mRNA predominantly to specific cells that were randomly dispersed throughout the luteal tissue. TIMP-1 mRNA was expressed by purified populations of both small and large luteal cells collected from day 10 corpora lutea. Concentrations of TIMP-1 mRNA (picograms per micrograms DNA) were greater in the large luteal cell populations (Por = 0.0001). We conclude that 1) expression of TIMP-1 mRNA by the granulosa layer of ovine preovulatory follicles increased after the gonadotropin surge, whereas TIMP-1 mRNA concentrations during the luteal phase remained constant; 2) during the luteal phase, TIMP-1 mRNA was localized to specific cells surrounding (day 3) or located within (day 10) the corpus luteum; and 3) expression of TIMP-1 mRNA was greatest in large luteal cells.

Stem cell factor (SCF), or c-kit ligand, is a multipotent growth factor that has been implicated in an important role in various aspects of animal development, including maintenance of the viability of primordial germ cells. A porcine SCF (pSCF) cDNA was generated from porcine uterine endometrial mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), and its nucleotide sequence was determined. The 952-bp pSCF cDNA contained an open reading frame encoding 274 amino acids. The deduced amino acid sequence of pSCF is approximately 86%, 83%, and 82%, identical to human, rat, and mouse SCFs, respectively; and it contains the four conserved cysteine residues and Asn-linked glycosylation sites. One additional amino acid was identified in pSCF, Glu130, which is not in the human (hSCF), rat (rSCF), or mouse (mSCF) sequences. Northern analysis of poly(A)+ RNA obtained from Day 16 pregnant endometrium revealed a transcript of approximately 6.5 kb. The size of this transcript is consistent with the size of full-length SCF mRNA, and the occurrence of alternatively spliced pSCF mRNAs were not detected by RT-PCR/Southern hybridization analysis of endometrial and ovarian total cellular RNA (tcRNA). Porcine SCF mRNA has been localized by in situ hybridization in porcine endometrial stromal tissue. Pregnancy does not appear to be a prerequisite for pSCF mRNA expression in endometrial tissue since it was detectable in tissue and tcRNA obtained from pregnant and nonpregnant gilts. The biological significance of uterine pSCF expression is currently unclear, but it probably participates in intracellular communication within the uterus.

A study was conducted to examine the influence of ovarian steroids on relaxin-induced changes in distensibility and composition of the uterine cervix in gilts. Ovariectomized, prepubertal gilts received (i.m.) either 1) 0.5 ml corn oil (CO) for 16 days; 2) 0.5 ml CO for 16 days and estradiol benzoate (EB; 200 micrograms) twice daily on Days 13-14; 3) EB on Days 0-1 followed by CO on Days 3-16 (EB + CO); 4) EB on Days 0-1 followed by CO on Day 2 and twice daily injections of 100 mg progesterone (P) on Days 3-16 (EB + P); or 5) EB on Days 0-1 followed by CO on Day 2 and P on Days 3-16 with additional EB treatment on Days 13-14 (EB + P + EB). One half of each group also received (i.m.) 0.5 ml 0.1 M PBS or 0.5 mg relaxin (RLX) every 6 h for 54 h coinciding with the final 2 days of the experimental period. Administration of RLX increased (p0.05) cervical distensibility compared to PBS treatment, with the response being greater (p0.05) in EB-, EB + CO-, and EB + P + EB-treated gilts compared to CO- or EB + P-treated gilts. Water content of cervical tissue was greater (p0.05) in EB-, EB+P- and EB + P + EB-treated gilts compared to CO- or EB + CO-treated gilt: and in all treatments, RLX enhanced (p0.05) imbibition of water by cervical tissues compared to that in PBS-treated gilts.(ABSTRACT TRUNCATED AT 250 WORDS)

We used DNA probes to study dietary effects on the prokaryotic population in the rumen. Procedures used to isolate and quantify prokaryotic 16S ribosomal RNA (rRNA) from the rumen using universal and species-specific DNA probes were evaluated. In this experiment, three ruminally fistulated steers were fed orchard-grass hay, and ruminal digesta were collected at 0, 3, and 9 h after offering hay (0800). Samples of ruminal digesta were taken from the interior portion of the digesta mat and from the fluid below the mat in the dorsal rumen. Freezing (-65 degrees C) and blending samples both increased (P less than .07) the yield of 16S rRNA from ruminal digesta. Extraction of prokaryotic rRNA was greater (P less than .04) when phenol buffered with sodium acetate was used than when it was buffered with hydroxymethyl-amino-methane. Prokaryotic 16S rRNA concentration of the fluid phase was similar (P greater than .10) at 0, 3, and 9 h after offering hay. Prokaryotic 16S rRNA concentration of the mat phase increased up to the 9 h after feeding. The proportion of Fibrobacter succinogenes remained constant in both digesta phases at all times measured. From these data we concluded that DNA probes can be used to monitor bacterial population shifts in the rumen.

Pregnant pony mares in Group A (n = 4) received i.m. injections at 07:00 and 17:00 h of 0.8 mg bromocriptine/kg body weight 0.75 per day beginning on Day 295 of gestation and continuing until parturition. Group B (n = 4) was treated similarly, but perphenazine was administered orally at 0.375 mg/kg body weight twice a day beginning on Day 305 of gestation and continuing until parturition. Mares in Group C (n = 3) received i.m. injections of saline. Mean plasma prolactin and progesterone concentrations were greater (P less than 0.05) for mares in Group C than in Groups A and B from 295 to 309 days of gestation. From 305 days of gestation, plasma prolactin and progesterone concentrations were greater (P less than 0.05) in Group B and C than in Group A mares. Progesterone and prolactin concentrations increased over this period for Group B and Group C mares, but remained constant in Group A mares. From 10 days pre partum through foaling, mares in Group A had lower progesterone (P less than 0.05) and prolactin (P less than 0.01) concentrations than Group B and C mares. All mares in Group A were agalactic at foaling, while all mares in Groups B and C had normal milk secretion. Gestation was longer (P less than 0.05) in Group A than in Group C mares. In Group A, 2 mares retained the placenta for greater than 3 h, 3 mares had dystocia and all 4 mares had thickened, haemorrhagic placentae.(ABSTRACT TRUNCATED AT 250 WORDS)

Uterotropic actions of relaxin (RLX) have been demonstrated in rats and mice but not in other species. Experiments were conducted to assess (1) if RLX has uterotropic effects in prepubertal gilts and (2) if RLX-induced imbibition of water by uterine tissues is mediated by histamine. Prepubertal gilts received (i.m.) either 0.5 mg pRLX (greater than or equal to 3000 U/mg; n = 10) in 0.1 M phosphate-buffered saline (PBS) or PBS vehicle (n = 8) every 6 h for 54 h. Additionally, one-half of each group received either (1) the histamine receptor antagonists (300 mg i.m. of each), pyrilamine maleate (H1) and cimetidine (H2), in PBS and corn oil, respectively, or (2) control vehicles every 6 h for 66 h beginning 12 h prior to initiation of RLX treatment. Treatment with RLX stimulated a twofold increase (p less than or equal to 0.05) in uterine weight in prepubertal gilts. Increases in total uterine weight were manifested by greater (p less than or equal to 0.05) uterine water content and dry weight in RLX-treated gilts. Cotreatment with histamine receptor antagonists did not alter RLX-induced changes in uterine water content or dry weight. Uterine contents of soluble protein and DNA were greater (p less than or equal to 0.05) in RLX-treated gilts compared to controls. Treatment with RLX tended (p approximately 0.08) to reduce protein; DNA ratio of uterine tissues and increased (p less than or equal to 0.05) uterine DNA as a percentage of uterine dry weight.(ABSTRACT TRUNCATED AT 250 WORDS)

The trophoblast of the sheep conceptus secretes a low mol wt, acidic polypeptide called ovine trophoblast protein-1 (oTP-1) from days 13-21 of pregnancy. This protein has been proposed to have an antiluteolytic role during pregnancy, since infusion of oTP-1 into the uterine lumen of nonpregnant ewes lengthened their estrous cycles. In the present study, mRNA from day 16 conceptuses was translated using a cell-free wheat germ lysate, and oTP-1 was immunoprecipitated from the translation mixture by means of a specific rabbit antiserum. The predominant protein synthesized under these conditions was oTP-1, which had a mol wt (21,000) higher than that of oTP-1 secreted by intact conceptuses into culture medium (mol wt, 17,000). In a separate experiment, in vitro secretion of oTP-1 from conceptuses on days 13, 17, and 21 of pregnancy was examined. Conceptuses were cultured for 24 h in the presence of [3H]leucine, and oTP-1 was immunoprecipitated from the culture medium. Incorporation of [3H]leucine into secreted macromolecules (mol wt, greater than 3500) increased 12-fold from days 13 to 21, while release of immunoprecipitable 3H-labeled oTP-1 increased 27-fold from days 13 to 17 and declined by over 50% from days 17 to 21. When results were expressed per microgram DNA, secretion of total macromolecules declined from days 13 to 17 and then remained constant until day 21. The pattern of oTP-1 secretion held whether results were expressed per conceptus or per unit DNA. The interestrous intervals for the donor ewes were shorter for ewes which had their conceptuses removed on day 13 than for ewes flushed on day 17 or 21. Thus, the period of oTP-1 synthesis corresponds closely to the time at which the conceptus acts to extend luteal lifespan.

In Study 1, explants of caruncular and intercaruncular endometrium and fetal membrane were collected from ewes (5-6/day) on Days 11-13, 16-18 and 21-23 after mating and Days 10-12 after oestrus, and incubated for 24 h. Explant-conditioned media were evaluated for their effects on endothelial cell proliferation. Both caruncular and intercaruncular endometrium secreted factor(s) which stimulated endothelial cell proliferation, and which appeared to be greater than 100 x 10(3) Mr and heat-labile. In Study 2, conditioned media from explant incubations of caruncular and intercaruncular endometrium, cotyledon and intercotyledonary fetal membrane obtained from ewes (6-7/day) on Days 40, 65, 90, 115 and 140 after mating were evaluated for their effects on endothelial cell proliferation. Caruncular and intercaruncular endometrium and intercotyledonary fetal membrane secreted factor(s) which inhibited endothelial cell proliferation. Media from cotyledonary explants tended to stimulate endothelial cell proliferation on Day 115. Conditioned media from cotyledonary explants obtained from 3 additional ewes at Day 120 of gestation stimulated endothelial cell proliferation, and this activity also appeared to be greater than 100 x 10(3) Mr. Placental angiogenesis in ewes therefore appears to be modulated by both maternal and fetal placental tissues via stimulatory and inhibitory factors.

Summary At approximately 100 days of gestation, 45 Angus x Hereford and 10 Hereford heifers were assigned either to be implanted with 36 mg zeranol or to serve in a control group. Monthly measurements revealed no effect of treatment on body weight. Increased pelvic area was observed during the first (P

Ovine trophoblast protein-1 (oTP-1), an interferon of embryonic origin, is produced during the peri-implantation period of early pregnancy. Secretion of oTP-1 is detectable between days 13 and 21, but not beyond. In this study, the levels of oTP-1 mRNA in embryos were analyzed to determine if they reflected the transient nature of oTP-1 production. Total cellular RNA (tcRNA) was isolated from embryos representing day 12 (n = 5), 14 (n = 7), 16 (n = 5), 18 (n = 6), 20 (n = 4), and 22 (n = 5) of pregnancy and spotted on nylon membranes. Complementary RNA was transcribed from a specific oTP-1 cDNA (550 base pairs) template and applied (16-1000 pg) to nylon membranes to develop a standard curve. The fixed RNA samples were then allowed to hybridize with the 32P-labeled oTP-1 cDNA. oTP-1 mRNA was not detectable on day 12, increased to high levels (3.6 +/- 1.6 ng/ug of embryo DNA) on day 14, decreased about 5-fold by day 16, 15-fold by day 18, 170-fold by day 20, and 200-fold by day 22 of pregnancy. At day 14 oTP-1 mRNA comprised 0.060 +/- 0.019% of the tcRNA and was more abundant than actin mRNA. Northern analyses of pooled tcRNA representing each day of pregnancy showed that the oTP-1 probe hybridized to a single class of mRNA (approximately 1.1 kilobases) and confirmed the results obtained with dot blots.

Ovine and bovine trophoblast protein-1 (oTP-1 and bTP-1) are newly discovered proteins produced by embryonic tissues for a limited period in early gestation. They appear to act as agents that prevent regression of the corpus luteum during early pregnancy in the ewe and cow. Ovine TP-1 [mol wt (Mr), 17,000] consists of three or four isoelectric variants (pI 5.4-5.7), whereas bTP-1, which cross-reacts with antiserum to oTP-1, is found as two predominant Mr classes (Mr, 22,000 and 24,000), each with several isoelectric variants (in the pI range 6.3-6.8). Cell-free translation of ovine conceptus mRNA yields pre-oTP-1 also consists of three or four isoelectric variants, assumed to have arisen by translation of multiple mRNA species. Ovine TP-1 is not glycosylated. When bovine conceptus mRNA is translated, a group of four or five isoforms of pre-bTP-1 are generated, each with a Mr of 19,000. In the presence of microsomes the Mr shifts upward to about 21,500. Bovine conceptuses cultured in presence of either [3H]glucosamine or [3H]mannose incorporate label into both size classes of bTP-1 (Mr, 22,000 and 24,000). Culture in presence of [35S]methionine and tunicamycin gave rise to a nonglycosylated form of bTP-1 with an apparent Mr of 18,000. Treatment of [35S]methionine-labeled bTP-1 with either endoglycosidase-H or peptide:N-glycosidase F yielded products with Mr of 17,000 and 16,000, respectively. bTP-1, although functionally and structurally related to oTP-1, appears to be a glycoprotein carrying at least two Asn-linked oligosaccharides. The two Mr classes of bTP-1 arise as a result of differences in either the number or structure of the carbohydrate chains. Like oTP-1, bTP-1 is probably translated from multiple mRNA species.

Bovine trophoblast protein-1 (bTP-1) is a secreted glycoprotein that consists of several forms differing slightly in mol wt and isoelectric point. It is produced by bovine conceptuses after about day 15 of pregnancy and is believed to play a key role in signalling the presence of an embryo to the mother. In this study, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18-19 bovine conceptus poly(A)+ mRNA. Base sequencing of several cDNAs indicated that multiple mRNAs for bTP-1 exist. Northern blotting and primer extension experiments showed that the mRNAs average about 1 kilobase in length. One apparently full-length cDNA clone consisted of 1035 bases up to the beginning of the poly(A) tail. It contained an open reading frame of 195 codons which began at a position 79 bases from the 5' end. Its entire sequence was 85% identical to that of a cDNA for the immunologically related ovine trophoblast protein-1 (oTP-1) and about 79% identical to that for a bovine interferon-alpha II (IFN alpha II). The highest conservation of sequence (greater than 90%) was noted in the 3'-untranslated sequences of the bTP-1 and oTP-1 cDNAs. The deduced amino acid sequence of bTP-1 shared 80% identity with oTP-1, between 45-55% with human, rodent, porcine, and bovine IFNs of the alpha 1 subfamily and about 70% with a bovine IFN alpha II. A single potential site for N-glycosylation was noted at Asn78. These results show that bTP-1, like its ovine counterpart oTP-1, is structurally related to the IFN alpha S. We suggest that these embryonic IFNs play a role in controlling immunoreactions at the trophoblast-uterus interface as well as triggering other maternal responses to pregnancy.

Sheep (Ovis aries) that are not pregnant have recurring estrous cycles of about 17 days’ duration during the breeding season, i.e., August through December to January. The first day of behavioral estrus is designated Day 0 with ovulation occurring about 30h after onset of estrus and the ovulatory surge of Luteinizing Hormone (LH). The estrous cycle is characterized by growth (Days 4–12) and then regression (luteolysis) of the corpus luteum (CL). Luteolysis begins on about Day 15 after onset of estrus. Prostaglandin F2-alpha (PGF), produced by the uterine endometrium, is the uterine luteolytic hormone in sheep (1). Beginning on Days 14 to 15 and continuing until the onset of the next period of estrus, PGF is released in a pulsatile manner. About 5 episodes of PGF release per 24h are required for luteolysis (2,3).

Fifty-nine crossbred heifers (427 kg) bred to one Hereford sire were randomly assigned at 75 d prepartum to two diets. Heifers were individually fed, and diets were isocaloric but contained either a low (LP = 81% NRC, .56 kg/d) or high (HP = 141% NRC, .98 kg/d) level of crude protein. Jugular vein cannulae were inserted into 16 LP and 16 HP heifers at 10 prepartum. Daily preprandial blood samples that were collected until parturition were analyzed for serum estradiol-17 beta (E2), progesterone (P4), glucose (G) and urea nitrogen (UN). Heifers fed LP gained slower than HP-fed heifers before calving (.73 vs 1.02 kg/d; P less than .01); immediate post-calving weights and condition scores were 418 vs 444 kg (P less than .01) and 5.4 vs 6.1 (P less than .01; LP vs HP, respectively). Calf birth weights (35.3 vs 36.1 kg), average calving difficulty score (1.6 vs 1.6) and percent assisted births (35.5 vs 35.7%) did not differ (P greater than .10; LP vs HP, respectively). Prepartum concentrations of UN (6.2 vs 13.5 mg/dl) and G (52.9 vs 58.2 mg/dl) were lower (P less than .05) and P4 (5.94 vs 4.26 ng/ml) was higher (P approximately equal to .07) in LP heifers. Prepartum concentration profiles were related to calving difficulty score (CD, 1 = no assistance to 3 = hard pull) for E2 (CD1 vs CD2 + CD3, P less than .01; CD2 vs CD3, P approximately equal to .01), P4 (CD1 vs CD2 + CD3, P less than .05), G (CD1 vs CD2 + CD3, P less than .05) and UN (CD2 vs CD3, P less than .05). After calving, all dams were maintained together on pasture and supplemented with alfalfa hay and grain mix until adequate range forage was available to maintain weight gains. Dams that were fed LP prepartum gained faster than HP dams during this period (.49 vs .15 kg/d; P less than .01). Prebreeding weights (443 vs 453 kg; LP vs HP) and condition scores (5.1 vs 5.1) did not differ, nor was the postpartum interval affected (44 vs 40 d; LP vs HP). There was no effect of dietary protein on dystocia or postpartum interval, although there were diet-induced differences in body weight and condition of the dams at calving. Results indicate that differences in prepartum profiles of serum steroid hormones and metabolites may be related to dystocia, in addition to relative fetal oversize.

In most species the length of a pregnancy exceeds that of the luteal phase of the ovarian cycle. The conceptus within the uterus, therefore, is believed to produce a substance or substances which directly or indirectly prolong the lifespan of the corpus luteum and prevent a return to ovarian cyclicity. This phenomenon is known as maternal recognition of pregnancy1. The active substance implicated in signalling maternal recognition of pregnancy in the sheep is an embryonic secretory protein, known as ovine trophoblast protein-1 (oTP-1) (refs 2–4), which is targeted in a paracrine manner to the uterine epithelium of the mother5. We report here the primary amino-acid sequence of oTP-1 as inferred from a cloned complementary DNA and demonstrate that the protein is most probably an interferon-alpha.

Fifteen Hereford and 47 crossbred heifers were allotted by breed and body weight to be artificially inseminated to one of two Angus sires selected for progeny birth weights (L = low; H = high). Forty-two of the heifers were randomly assigned to be slaughtered at 200, 215, 230, 245 or 260 d of gestation for measurement of fetal and placental characteristics. Twenty heifers were allowed to go to term and five calves from each sire group were randomly assigned to be euthanized and dissected within 24 h after birth. Sire differences in birth weight (BW) and dystocia score (32.9 vs 35.4 kg; 1.8 vs 3.1, L vs H sires, respectively) existed (P less than .01), and there was a sire effect (P less than .01) for fetal calf weights (FW) and eviscerated calf weights (EW). However, there was a sire X calf sex interaction for BW (P less than .05), EW (P less than .01), FW (P less than .01), femur length (P less than .05), heart weight (P less than .05), kidney weight (P less than .01) and pituitary weight (P less than .01). Weight differences suggested these interactions were a result of the relationship of the organ weights to fetal body weights and the interaction effects on calf weights resulted from limitations in the maternal environment which restricted growth of H-sired male calves in utero. Sire X fetal age interaction effects were all nonsignificant (P greater than .10) except for cerebrum weight. This finding indicates that fetus and calf growth rates were parallel for the L and H sires. A sire effect was found for biceps (P less than .01) and liver weights (P less than .01), but not for cerebrum weights (P greater than .10). Increasing weight due to fetal age was attributed to hypertrophy for the cerebrum (P less than .05) and liver (P approximately equal to .01), while the biceps increased (P less than .05) by both hypertrophy and hyperplasia, as determined from deoxyribonucleic acid and protein analyses. All measured fetal organ weights except heart, when expressed as a ratio with EW, decreased (P less than .05) with increasing fetal age. Brain (cerebrum + cerebellum):liver weight ratios were higher (P less than .01) in L-sired calves (.32 vs. .28) than in H-sired calves. Total placentome weight (b' = 91; P less than .01) and placental fluid volume (b' = .32; P less than .01) were highly associated with FW, accounting for 84% of the variation in FW.(ABSTRACT TRUNCATED AT 400 WORDS)

This paper demonstrates that a group of proteins, representing a major secretory component of cattle conceptuses, is immunologically related to ovine trophoblast protein-1 (oTP-1), a principal product of culture Day 13 to 21 sheep conceptuses. Conceptuses from cows (Day 17-18) and ewes (Day 16-17) were cultured for 24 h in the presence of L-[3H]leucine. By using a rabbit antiserum to oTP-1 and Ouchterlony double-immunodiffusion analysis it was shown that material in the bovine conceptus culture medium was serologically related, but not identical, to oTP-1. A solid-phase radiobinding assay indicated that the cross-reacting bovine secretory component had an affinity for anti-oTP-1 antibody similar to that of oTP-1. Anti-oTP-1 antiserum specifically immunoprecipitated a group of 6-8 polypeptides from culture medium of cow conceptuses which, when analysed by two-dimensional gel electrophoresis, fell into two major molecular weight classes (22,000 and 24,000) with isoelectric points between 6.5 and 6.7. These immunoprecipitated polypeptides, defined as bTP-1, constituted the major secretory products of Day 16-25 cow conceptuses. They were larger and more basic than oTP-1 polypeptides (Mr about 18,000; pI 5.4-5.7). Anti-oTP-1 antiserum also recognized the major translation product of Day 17 bovine conceptus mRNA, a polypeptide significantly smaller (Mr approximately 18,000) than the secreted protein. It is suggested that oTP-1 and the homologous bovine protein may play similar roles in the phenomenon of maternal recognition of pregnancy in the two species.