Characterization of proteins produced in vitro by periattachment bovine conceptuses

Bovine conceptuses from Days 16 (n = 4), 19 (n = 6), 22 (n = 3), and 24 (n = 4), and chorion from Day 69 (estrus/mating = Day 0) were cultured for 24 h in modified minimum essential medium (MEM) in the presence of radioactive L-leucine [( 3H] leucine) to characterize de novo synthesis and release of proteins. Proteins released into MEM were identified by two-dimensional polyacrylamide gel electrophoresis, fluorography, and gel and ion exchange chromatography. Major polypeptides identified in MEM were different from those identified in conceptus and chorionic tissues. Both uptake of [3H] leucine and quality of polypeptides produced de novo and released into MEM were related to stage of conceptus development. Percent retention of [3H] leucine in MEM was lowest (P less than 0.01) in Day 16 cultures (1.2 +/- 4.1%), increased in Days 19 (16.8 +/- 3.7%) and 22 cultures (20.9 +/- 5.8%), and decreased (P less than 0.07) in Day 24 cultures (6.9 +/- 4.1%). Complexity of polypeptides increased after Day 16. Days 16, 19, 22 and 24 conceptus culture MEM was enriched in low-Mr, acidic polypeptides (Mr/isoelectric point ranges: 22K-26K/6.5-5.6, 20K-26K/5.5-5.4, and 16K-20K/5.0-4.5), which were not prominent products of Day 29 and 69 tissues. A high-Mr (Mr +/- SEM; 735K +/- 22K) glycoprotein was produced by all conceptus and chorionic tissues. The transient nature of production of low-Mr polypeptides suggests that they may be required during the periattachment period.     

Platelet-activating factor alters the dynamics of prostaglandin and protein synthesis by endometrial explants from pregnant and cyclic cows at day 17 following estrus

Factors produced by bovine conceptuses alter prostaglandin (PG) and protein secretion by endometrial explants from cyclic cows and induce an intracellular inhibitor of PG synthesis. Endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows at Day 17 following estrus were incubated for 24 h with 0, 0.1, 0.5, 1 and 5 mug platelet-activating factor (PAF)/ml. Cotyledonary microsomes from parturient cows were utilized to determine levels of an intracellular/cytosolic inhibitor of PG synthesis. Endometrial explants from additional cyclic cows (n = 4) were incubated for 24 h with 0 or 5 mug PAF/ml with and without 50 muCi [(3)H]leucine. Endometrial explants (cyclic cows, n = 3) were also incubated for 12 h with each of the following treatments: 1) Control; 2) PAF (1 mug/ml); 3) lyso-PAF (2 to 10 mug/ml); 4) PAF-receptor antagonist (2 to 10 mug/ml); 5) PAF (1 mug/ml) + antagonist (2 to 10 mug/ml); 6) bovine conceptus secretory proteins (bCSP; 25 mug/ml); and 7) bCSP (25 mug/ml) + antagonist (5 mug/ml). Platelet-activating factor had distinct negative and positive dose effects on PGF and PGE-2 secretion, respectively, by explants from cyclic cows, whereas PG secretion was not altered by PAF in the endometrium of pregnant cows. Platelet-activating factor did not alter the level of an intracellular inhibitor of prostaglandin synthesis, whereas, bCSP increased the level of this inhibitor. Platelet-activating factor decreased the incorporation of [(3)H]leucine into tissue and secreted proteins for explants from cyclic cows. Lyso-PAF did not alter endometrial prostaglandin secretion. The effects of PAF but not of bCSP were blocked by the PAF-receptor antagonist. Platelet-activating factor altered PG and protein secretion by the endometrium from cyclic cows, and it may be a potential regulatory factor during early pregnancy if secreted by the bovine conceptus.     

Presence of an intracellular endometrial inhibitor of prostaglandin synthesis during early pregnancy in the cow

Previous studies have detected reduced endometrial secretion of prostaglandins during pregnancy in cattle. The present experiment tested the hypothesis that reduced secretion of prostaglandins is caused by induction of an intracellular endometrial inhibitor of prostaglandin synthesis. The microsomal fraction of parturient bovine cotyledons was utilized as a source of enzymes for prostaglandin synthesis. Endometrial tissues collected at Day 17 of the estrous cycle (n = 12) and pregnancy (n = 12) were homogenized and subjected to differential centrifugation for preparation of microsomes and a high-speed (100,000 x g) cytosolic supernatant. Endometrial intracellular preparations were then examined for the ability to modulate prostaglandin synthesis by cotyledonary microsomes from parturient cows. Endometrial intracellular preparations from cyclic cows decreased (P less than 0.05) PGF synthesis by cotyledonary microsomes to a slight extent (supernatant, 21% reduction; microsomes, 11% reduction), while preparations from pregnant cows markedly decreased (P less than 0.01) PGF synthesis (supernatant, 63% reduction; microsomes, 28% reduction; supernatants vs microsomes, P less than 0.01). Regardless of the amount of arachidonic acid available as substrate (25-400 micrograms) endometrial supernatant from pregnant cows (pooled sample) caused a 50% inhibition (IC50) of prostaglandin synthesis at a tissue equivalent of 270 +/- 9.1 mg. The mechanism of inhibition by endometrial high-speed supernatant from pregnant cows appears to be non-competitive with respect to arachidonic acid. The inhibitor(s) may be proteinaceous (70-75 kDa and 25-35 kDa) and can be precipitated by 20% saturated ammonium sulfate. In conclusion, early pregnancy in cattle appears to be associated with increased amounts of an intracellular endometrial inhibitor of prostaglandin synthesis.     

Identification of bovine trophoblast protein-1, a secretory protein immunologically related to ovine trophoblast protein-1

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.     

Changes in vascular perfusion of the endometrium in association with changes in location of the embryonic vesicle in mares

The equine embryonic vesicle is mobile on Days 12-14 (Day 0 = ovulation), when it is approximately 9-15 mm in diameter. Movement from one uterine horn to another occurs, on average, approximately 0.5 times per hour. Mobility ceases (fixation) on Days 15-17. Transrectal color Doppler ultrasonography was used to study the relationship of embryo mobility (experiment 1) and fixation (experiment 2) to endometrial vascular perfusion. In experiment 1, mares were bred and examined daily from Day 1 to Day 16 and were assigned, retrospectively, to a group in which an embryo was detected (pregnant mares; n = 16) or not detected (n = 8) by Day 12. Endometrial vascularity (scored 1-4, for none to maximal, respectively) did not differ on Days 1-8 between groups or between the sides with and without the corpus luteum. Endometrial vascularity scores were higher (P < 0.05) on Days 12-16 in both horns of pregnant mares compared to mares with no embryo. In pregnant mares, the scores increased (P < 0.05) between Day 10 and Day 12 in the horn with the embryo and were higher (P < 0.05) than scores in the opposite horn on Days 12-15. In experiment 2, 14 pregnant mares were examined from Day 13 to 6 days after fixation. Endometrial vascularity scores and number of colored pixels per cross-section of endometrium were greater (P < 0.05) in the endometrium surrounding the fixed vesicle than in the middle portion of the horn of fixation. Results supported the hypothesis that transient changes in endometrial vascular perfusion accompany the embryonic vesicle as the vesicle changes location during embryo mobility.     

Effect of day of the oestrous cycle, side of the reproductive tract and heat shock on in-vitro protein secretion by bovine endometrium

Endometrial explant cultures were prepared from 16 Brahman x Angus cows killed on Days 0, 2, 5 or 8 after oestrus. Cultures proceeded for 24 h at 39 degrees C (homeothermic) or 43 degrees C (heat shock) in a modified Eagle's minimal essential medium supplemented with 50 microCi L-[4,5(-3)H]leucine. Analysis by two-dimensional polyacrylamide gel electrophoresis of de-novo synthesized proteins secreted into the medium indicated that the major types of secreted polypeptides did not change over Days 0-8. Nevertheless, overall endometrial secretion of protein (incorporation of [3H]leucine into non-dialysable radioactivity in culture supernatants) was greatest at Day 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable material in tissue homogenates was also greatest at Day 0. For tissue cultured at 39 degrees C, several individual polypeptides were secreted at greater rates by endometrium from the horn of the uterus ipsilateral to the corpus luteum, with side differences tending to be greatest at Day 0 or Day 2. Overall, secretion of de-novo synthesized protein by endometrium was significantly elevated by heat shock at Day 0, but not affected thereafter. Nonetheless, heat shock reduced secretion of several individual proteins and exhibited interactions with day of the oestrous cycle and with side of the uterus. Secretion of 7 polypeptides was reduced by heat shock in tissue from the ipsilateral horn of the uterus but not in endometrium from the contralateral horn. We suggest that endometrial protein secretion changes quantitatively during the early oestrous cycle. In addition, there is a local influence of the ovary bearing the corpus luteum on endometrial function that may be disrupted by heat shock.     

Characterization of the uterine environment during early conceptus expansion in the bovine

Fifty-three Angus and Hereford beef cows were utilized to investigate the effect of the conceptus on uterine environment during the period of pregnancy recognition. Blood samples were collected on Days 10, 12, 14, 16 and 18. Cows were randomly assigned to be either mated on the subsequent oestrus or serve as cyclic controls. Blood samples were then collected daily from Day 10 until slaughter on Day 15, 16 or 17 from the initiation of oestrus. Uteri were flushed with physiological saline and flushings analyzed for quantitative and qualitative protein changes, calcium, oestradiol-17β and prostaglandin F content. Endometrial explants of caruncular and intercaruncular tissue, and conceptus tissue recovered from pregnant cows were cultured with [³H]-leucine to determine quantitative and qualitative polypeptide synthesis and release. Plasma progesterone concentrations were similar between pregnant and cyclic cows from Day 10 through 17. Only the uterine content of prostaglandin F significantly increased in the ipsilateral horn of pregnant cows on Days 16 and 17. This increase in prostaglandin content was related to the increase in conceptus length from 25 to 40–80 mm. Conceptus production of bovine trophoblastic protein-1 was also first clearly detectable in fluorographs of medium from conceptuses measuring 25 mm. The complexity of the polypeptides present in the medium increased with conceptus development. Polypeptide synthesis by the endometrium was similar between tissues and days; however, production of two groups of low molecular weight basic polypeptides continued to be intensified on fluorographs from the pregnant horn on Day 17 compared to cyclic cows.     

Synchronization of ovulation in beef cows (Bos indicus) using GnRH, PGF2alpha and estradiol benzoate

The objective of this study was to evaluate protocols for synchronizing ovulation in beef cattle. In Experiment 1, Nelore cows (Bos indicus) at random stages of the estrous cycle were assigned to 1 of the following treatments: Group GP controls (nonlactating, n=7) received GnRH agonist (Day 0) and PGF2alpha (Day 7); while Groups GPG (nonlactating, n=8) and GPG-L (lactating, n=9) cows were given GnRH (Day 0), PGF2alpha (Day 7) and GnRH again (Day 8, 30 h after PGF2alpha). A new follicular wave was observed 1.79+/-0.34 d after GnRH in 19/24 cows. After PGF2alpha, ovulation occurred in 19/24 cows (6/7 GP, 6/8 GPG, 7/9 GPG-L). Most cows (83.3%) exhibited a dominant follicle just before PGF2alpha, and 17/19 ovulatory follicles were from a new follicular wave. There was a more precise synchrony of ovulation (within 12 h) in cows that received a second dose of GnRH (GPG and GPG-L) than controls (GP, ovulation within 48 h; P<0.01). In Experiment 2, lactating Nelore cows with a visible corpus luteum (CL) by ultrasonography were allocated to 2 treatments: Group GPE (n=10) received GnRH agonist (Day 0), PGF2alpha (Day 7) and estradiol benzoate (EB; Day 8, 24 h after PGF2alpha); while Group EPE (n=11), received EB (Day 0), PGF2alpha (Day 9) and EB (Day 10, 24 h after PGF2alpha). Emergence of a new follicular wave was observed 1.6+/-0.31 d after GnRH (Group GPE). After EB injection (Day 8) ovulation was observed at 45.38+/-2.03 h in 7/10 cows within 12 h. In Group EPE the emergence of a new follicular wave was observed later (4.36+/-0.31 d) than in Group GEP (1.6+/-0.31 d; P<0.001). After the second EB injection (Day 10) ovulation was observed at 44.16+/-2.21 h within 12 (7/11 cows) or 18 h (8/11 cows). All 3 treatments were effective in synchronizing ovulation in beef cows. However, GPE and, particularly, EPE treatments offer a promising alternative to the GPG protocol in timed artificial insemination of beef cattle, due to the low cost of EB compared with GnRH agonists.     

Plasma and luteal concentrations of oxytocin in cyclic and early-pregnant cattle.

Concentrations of oxytocin were measured in corpora lutea obtained from heifers throughout the oestrous cycle and first 30 days of pregnancy. Values were low during the first 3 days of the cycle (less than 250 ng/g tissue), increasing to 1312 ng/g by Day 4. Values then further increased up to a maximum of 2344 ng/g on Day 12. Concentrations were similar in cyclic and pregnant animals throughout the midluteal phase and were maintained at approximately 1500 ng/g until the 18th (cyclic cows) or 19th (pregnant cows) day after oestrus, when they were again low. Values subsequently remained less than 250 ng/g in pregnant cattle. Concentrations of oxytocin in jugular venous plasma of cyclic (n = 5) and pregnant (n = 4) cows were measured in samples collected every 15 min for 8 h on Days 14, 16, 18 and 19 after oestrus. There were no significant differences in mean concentrations (range: 2.5-4.7 pg/ml) or in the number, frequency or area under the curve of episodes between either cyclic and pregnant animals, or between days. Mean basal concentrations were higher on Day 16 than on Day 14 (P less than 0.05), values on Days 18 and 19 being intermediate. These findings suggest that the corpus luteum contains a finite amount of releasable oxytocin, which is exhausted by Day 18-19 after oestrus, whether or not pregnancy occurs, and that there is no further accumulation of oxytocin in the animal during early pregnancy. The contribution of luteal oxytocin to jugular venous concentrations appears to be less than in sheep, in which values in the jugular vein closely parallel those within the corpus luteum.     

Concentrations of insulin-like growth factor-I, estradiol and progesterone in follicular fluid of ovarian follicles during early pregnancy in cattle

To determine if the presence of the developing conceptus is associated with changes in intrafollicular concentrations of insulin-like growth factor-I (IGF-I), estradiol (E2) and/or progesterone during early pregnancy in cattle, either pregnant (n=16) or nonpregnant (n=15) cows were slaughtered on Day 10, 15 or 18 postestrus. Ovaries and follicular fluid were collected. Follicles were grouped by diameter: 1.0 to 3.9 mm (small; n=63), 4.0 to 7.9 mm (medium; n=128), and >/= 8.0 mm (large; n=38). The average diameter of large follicles was greater (P<0.05) in pregnant than in nonpregnant cows on Day 10, but on Day 18 it was greater (P<0.05) in nonpregnant than in pregnant cows (11.3 vs 9.7 mm). Status (pregnant vs nonpregnant) did not affect (P>0.10) follicular fluid progesterone nor IGF-I concentrations. In contrast, the status and days postestrus affected (P<0.05) follicular fluid E2 concentrations. Follicular fluid E2 levels in the three follicle size-categories on Day 10 did not differ (P>0.10) between pregnant and nonpregnant cows. However, on Days 15 and 18 postestrus, follicular fluid E2 concentrations in pregnant cows was lower (P<0.05) in large follicles than in nonpregnant cows. We conclude that the presence of a developing conceptus early in pregnancy may alter follicular growth and inhibit follicular E2 production in cattle. These effects appear to be mediated by factors other than IGF-I.     

Early pregnancy associated thrombocytopenia as an initial response to pregnancy in cattle

This study was conducted to determine if early pregnancy-associated thrombocytopenia exists in cattle as has been demonstrated in mice and in humans. Three experiments were designed to compare peripheral platelet counts in pregnant versus nonpregnant animals. In Experiment 1 heifers (n = 25) were artificially inseminated 12 h after the onset of estrus. Peripheral platelet counts in 19 pregnant versus 6 nonpregnant heifers did not reveal any significant differences between groups after insemination. In Experiment 2 embryos were collected nonsurgically from superovulated cows (n =18) on Days 6 to 7 after estrus. Platelet counts were monitored every 12 h after the first insemination until 60 h after the second insemination. Platelet counts and the number of embryos collected nonsurgically from these superovulated donors did not show any significant correlations (P>0.05). Ten recipient heifers synchronized to donor animals received either an unfertilized ovum or a good quality embryo via nonsurgical transfer into the uterus. There were no significant reductions in platelet counts after transfer. In Experiment 3 platelet counts were monitored daily in four pregnant and five nonpregnant recipient heifers between Day 0 and Day 30 after embryo transfer on Day 8 of the cycle. The platelet counts did not reveal any significant differences between the pregnant and nonpregnant groups throughout Days 0 to 30. These results indicate that early pregnancy-associated thrombocytopenia cannot be demonstrated in cattle. Peripheral platelet counts cannot be used as an indicator of early pregnancy in cattle.     

Regulation of heat shock-induced alterations in the release of prostaglandins by the uterine endometrium of cows

Maternal heat stress in cattle may disrupt pregnancy by elevating uterine prostaglandin F(2alpha) (PGF(2alpha)) secretion. The objectives of this study were to determine the effects of elevated temperature (42 degrees C) in vitro upon 1) prostaglandin secretion by endometrial tissue; 2) the actions of extracellular regulators of uterine PGF [conceptus secretory proteins (bCSPs) and platelet-activating factor, (PAF)]; 3) the activity of the cyclooxygenase-endoperoxidase enzyme complex (PG synthetase); and 4) the activity of the endometrial PG synthesis inhibitor present in the endometrium from pregnant cattle. Endometrial explants at Day 17 of the estrous cycle produced more PGF than PGE(2) while elevated temperature caused increased PGF secretion but did not affect PGE(2) secretion. Elevated temperature did not reduce the ability of bCSPs or PAF to suppress release of PGF. The heat shock-induced increase in PGF at Day 17 was not due to the direct effects on PG synthetase, because PGF production from a cell-free cotyledonary microsomal enzyme preparation was reduced at elevated temperature. The activity of the cytosolic inhibitor of cyclooxygenase present in the endometrium of Day-17 pregnant cows could be reduced but not eliminated at 42 degrees C. We conclude that in vitro heat stress induces PGF secretion from the bovine uterine endometrium at Day 17 after estrus. This increase is not accompanied by the loss of regulatory capacity of conceptus products or increased activity of PG synthetase.     

Secretory proteins of the bovine conceptus alter endometrial prostaglandin and protein secretion in vitro

The conceptus is believed to produce factors that regulate endometrial function and prevent luteolysis during early pregnancy. Endometrial tissues were collected from cyclic (n = 8) and pregnant (n = 2) cows at Day 17 post-estrus and cultured for 24 and 48 h with bovine conceptus secretory proteins (bCSP) (0%, 10%, 100%), where the amount of protein produced by a bovine conceptus during 24 h of culture is 100%. Incorporation of [3H]leucine into secreted proteins was determined and examined qualitatively by trichloroacetic acid precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Levels of an intracellular endometrial inhibitor of prostaglandin synthesis were determined with a cotyledonary microsomal test system. Treatment with 10% and 100% bCSP reduced incorporation of [3H]leucine into secreted proteins. However, bCSP selectively induced two secreted proteins (13 and 10 kDa) from endometrium of cyclic cows. Prostaglandin F (PGF) secretion was decreased by bCSP treatment while prostaglandin E2 secretion was unaltered. An intracellular endometrial inhibitor of prostaglandin synthesis was induced by bCSP; synthesis of PGF by the cotyledonary prostaglandin-generating system was decreased when incubated with cytosol of endometrium treated with bCSP, but unaltered by cytosol from control tissues. In conclusion, products produced by the bovine conceptus are capable of regulating endometrial protein and prostaglandin biosynthesis in a fashion that could act to prevent luteolysis in vivo and provide endometrial secretory products for embryonic development.     

Incorporation of a rapid pregnancy-associated glycoprotein ELISA into a CIDR-Ovsynch resynchronization program for a 28 day re-insemination interval

The objective was to compare two resynchronization programs; one that used a blood-based ELISA for pregnancy-associated glycoproteins (PAG) for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 28 d after first TAI, and another that used transrectal ultrasonography for pregnancy diagnosis so that non-pregnant cows were re-inseminated at 35 d after first TAI. The PAG_resynch cows (n = 103) began CIDR-Ovsynch resynchronization on Day 18 after first TAI (Day 0). On Day 25, the CIDR was removed and pregnancy diagnosis with a PAG ELISA was performed. If a cow was not pregnant on Day 25, she was treated with PGF_2α, treated with GnRH 2 d later (Day 27), and TAI on Day 28. Control cows (n = 99) were observed for estrus until Day 25, when they began an identical CIDR-Ovsynch program with pregnancy diagnosis by transrectal ultrasonography on Day 32. If a cow was not pregnant on Day 32, then she was treated with PGF_2α, treated with GnRH 2 d later (Day 34), and TAI on Day 35. There was no difference in pregnancy per AI (P/AI) for either group at first or second insemination. For cows without pregnancy loss, the interval between first and second (P < 0.001) or second and third (P < 0.016) TAI was shorter for PAG_resynch cows compared with Control cows. The interval between first and second or second and third TAI was not different if pregnancy loss cows were included in the analysis. Plasma progesterone concentrations were similar at PGF_2α treatment, and plasma estradiol concentrations increased similarly after PGF_2α treatment for PAG_resynch and Control cows. In conclusion, the 28 d CIDR-Ovsynch resynchronization protocol was comparable to a 35 d CIDR-Ovsynch resynchronization protocol that also included estrus detection. Shortened resynchronization protocols that do not require estrus detection may improve reproductive efficiency in dairy cattle.     

Persistent dominant follicle alters pattern of oviductal secretory proteins from cows at estrus.

The experimental objective was to compare synthesis of oviductal secretory proteins of dairy cows bearing a persistent dominant follicle (PDF) versus a fresh dominant follicle (FDF) at estrus. On Day 7 after synchronized estrus (Day 0), cows received an intravaginal progesterone device and injection of prostaglandin F2alpha (PGF2alpha). On Day 9, cows received an injection of a GnRH agonist (FDF group; n = 3) or received no injection (PDF group, n = 3). On Day 16, all cows received PGF2alpha, and progesterone devices were removed. At slaughter on Day 18 or Day 19, oviducts ipsilateral and contralateral to the dominant follicle were divided into infundibulum, ampulla, and isthmus regions. Explants from oviductal regions were cultured in minimal essential medium supplemented with [3H]leucine for 24 h. Two-dimensional fluorographs of proteins in conditioned media were analyzed by densitometry. Rate of incorporation of [3H]leucine into macromolecules was greater in the infundibulum, ampulla, and isthmus of FDF cows (p < 0.01). Overall, intensities of radiolabeled secretory protein (P) 2 and P13 were greater for FDF than for PDF. In the ampulla, P14 was more intense for FDF while P7 was more intense for PDF. Abundance of P1 in the isthmus was greater for PDF cows. Across regions, P5, P6, P8, P9, and P11 were more intense for PDF than for FDF in the ipsilateral side. In the contralateral side, P19 was more intense for PDF than for FDF, whereas P6, P8, P9, and P11 were more intense for FDF. Differences in biosynthetic activity and in secreted oviductal proteins from cows bearing a PDF may contribute to the decrease in fertility associated with a PDF.     

Bovine trophoblast protein-1 complex alters endometrial protein and prostaglandin secretion and induces an intracellular inhibitor of prostaglandin synthesis in vitro

Evidence exists that conceptuses alter endometrial protein secretion and modulate prostaglandin (PG) synthesis and secretion in cattle. The present study was designed to test the effect of bovine conceptus secretory proteins (bCSP) in general and the bovine trophoblast protein-1 complex (bTP-1) in particular on endometrial function. Endometrial explants from cyclic cows (N = 4) at Day 17 after oestrus were incubated for 24h with 0, 4.8, 24 or 120 micrograms BSA/ml, 1 microgram bTP-1/ml or 12.7 micrograms bCSP/ml. Both bCSP and bTP-1 decreased (P less than 0.005) release of radiolabelled macromolecules into medium and incorporation of radio-labelled precursors into tissue compared to BSA-treated tissues but not tissues treated with medium alone. Secretion of a protein of Mr = 14,900 was enhanced by bCSP treatment as compared to treatment with bTP-1 (P less than 0.025). Both bCSP and bTP-1 decreased PGF secretion of explants (P less than 0.01) compared to BSA. Overall, PGE-2 secretion by bCSP- and bTP-1-treated tissues did not differ from that of BSA cultures, but PGE-2 secretion was greater (P less than 0.025) for bTP-1 than bCSP-treated endometrium. Cotyledonary microsomes from parturient cattle were utilized as a PG-generating system for the detection of inhibitors of PG synthesis. PGF synthesis by the generating system was decreased (P less than 0.05) by 9% by cytosol from BSA-treated explants, whereas cytosol from bCSP- and bTP-1-treated explants decreased (P less than 0.01) PGF synthesis by 42% and 35%, respectively. In summary, both bCSP and bTP-1 inhibit PGF secretion, induce synthesis of an intracellular inhibitor of PGF synthesis, and decrease protein synthesis and secretion. The bTP-1 complex therefore alters PG dynamics by explants in a manner that would function to prevent luteolysis and support the establishment of pregnancy.     

Regulation of endometrial prostaglandin synthesis during early pregnancy in cattle: Effects of phospholipases and calcium In vitro

In experiment 1, endometrial explants from 3 cyclic (Day 17) cows were incubated with arachidonic acid (AA), phospholipase A-2 (PLA-2) and calcium ionophore A23187 (CaI) or control. AA (0.2 mg), PLA-2 (1 U/ml) and Cal (4 μg/ml) increased PGF and PGE secretion. In experiment 2, endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows were incubated +/- Ca⁺⁺ and with either: control, AA, PLA-2, CaI, PLA-2 + CaI, or AA + CaI. PG secretion was higher in cultures with Ca⁺⁺. In presence of Ca⁺⁺, PGF secretion was lower for pregnant than cyclic endometrium. AA with Ca⁺⁺ stimulated PGF and PGE secretion, indicating that AA availability may limit PG secretion. The stimulatory effect of PLA-2 on PGF and PGE secretion was greater in pregnant than cyclic Endometrium. However, CaI inhibited the PLA-2 response of pregnant, but not cyclic endometrium. In experiment 3, endometrium (4 cyclic cows) failed to convert ³H-PGF₂ to PGE₂ or ³H-PGE₂ to PGF₂ Responsiveness of PG secretion to PLA-2, and CaI is altered by reproductive status suggesting that these factors may be involved in the differential regulation of PG production during early pregnancy in cattle.     

Characterization of bovine conceptus proteins produced during the peri- and postattachment periods of early pregnancy

Bovine conceptuses removed from the uterus during the peri- and postattachment periods of placentation (Days 17-24 and 26-38, respectively) were cultured in a modified minimum essential medium in the presence of L-[3H]leucine to characterize in vitro synthesis of proteins released into the medium. Patterns of protein production were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography of dried gels. Four groups of low molecular weight acidic proteins (LMWAP) were observed to be synthesized during the peri- and postattachment periods. The number and relative concentrations of these changed with development. One group (A) consisted of three major and two or more minor isoelectric species (pI approximately equal to 5.8-6.8); these were the major synthesized proteins observed from Days 17-22. The major polypeptides of Group A were present at all time points examined through Day 38 and, in several preparations, appeared as doublets (Mr approximately equal to 22,000 and 24,000) through Day 29 but not thereafter. Group A polypeptides from Day 19 and 36 conceptus cultures were demonstrated by immunoblot analysis to cross-react with antiserum produced against ovine trophoblast protein-1 (oTP-1). A second group of proteins (A1) and a single protein (B) in the 20,000-24,000 Mr range were observed between Days 17 and 22. These were acidic relative to Group A and were not detected after Day 22. A fourth group (C) of LMWAP (Mr approximately equal to 14,000-18,000) was first observed around Day 21 and appeared to increase relative to Group A through Day 29. One protein from this group, C3, was the predominant LMWAP at Day 38.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endometrial suppressor cells in beef cattle

Endometrial cells were recovered post mortem from cyclic and pregnant crossbred beef cattle (n = 5 each) on Days 16 to 18 after estrus, and were evaluated for their ability to suppress lymphocyte responses and release suppressor factor into the culture medium. The suppressor factor was assessed for transforming growth factor-beta (TGF-beta) activity. In addition, Percoll was used to fractionate endometrial cells from Angus cows (n = 4) on Days 16 to 18 of pregnancy to determine the density of the suppressor cells. Endometrial cells from cyclic and pregnant cows suppressed lymphocyte proliferative responses and released suppressor factor into the culture medium. The suppressor factor exhibited TGF-beta activity. Suppressor activities tended to be greatest for fractionated cells with densities of 1.01 and 1.095 g/mL. In conclusion, the bovine endometrium contains low- and high-density suppressor cells capable of releasing suppressor factor. The factor seems to be associated with TGF-beta.     

Fetal survival in the cow after pregnancy diagnosis by palpation per rectum

It is desirable to determine wheter a cow has failed to become pregnant as early as possible, preferably prior to 50 d after insemination. Although palpation per rectum has been the generally accepted method of pregnancy diagnosis in cattle, the procedure may be a significant iatrogenic cause of fetal attrition. In a study conducted at a North Florida dairy from January through June 1982, pregnancy was determined in 192 Holstein-Friesian cows by measuring low milk progesterone (P4) content on day of insemination (Day 0) and elevated P4 on Days 21 and 24. Pregnant cows were randomly assigned to a treatment and a control group. Cows in the treatment group (n = 85) were palpated per rectum twice between Days 42 and 46 after insemination. Cows in the control group (n = 107) were not palpated until both groups were palpated at Day 90. Palpation, done by two experienced clinicians, consisted of palpation of fetal fluid fluctuation, identification of the amniotic vesicle, and slipping of the chorioallantoic membranes. In both groups of cows fetal viability was monitored by milk P4 content. Last milk (5 to 15 ml) was collected from one front quarter on Days 0, 21, and 24 and twice weekly thereafter through Day 63. Milk was defatted by centrifugation and the fat-free milk progesterone content measured by a radioimmunoassay without extraction. The milk P4 test was 80.0% accurate in determining pregnancy in the palpated and nonpalpated cows. In the cows palpated on Days 42 to 46, pregnancy rates declined by 7.5% as determined by palpation at Day 90, or by 11.4% as determined by milk P4 content through Day 63 (both values P < 0.05). Cows that were not palpated on Days 42 to 46 showed a 1.9% increase or a 4.3% decline in pregnancy rates as determined by the same criteria. Before palpation, at Days 42 to 46, pregnancy rates were better in cows that were inseminated in winter (January to March) than in spring (April to June) (82.3% vs 61.6%; P < 0.05); P4 content was higher (winter > spring = 2.13 ng/ml vs 1.38 ng/ml; P < 0.05). First-lactation cows had higher P4 values on Days 21 and 24 than older cows (P < 0.01).