Characterization of proteins produced in vitro by bovine endometrial explants

Endometrial tissues were obtained from 17 pregnant (P, estrus/mating = Day 0; Day 16, n = 4; Day 19, n = 6; Day 22, n = 3; Day 24, n = 4) and six nonpregnant (NP; Day 16, n = 4; Day 19, n = 2) cattle, as well as from one cyclic (nonbred) cow (Day 4), one later-pregnant cow (Day 69), and both ligated and pregnant uterine horns of three unilaterally pregnant cattle (UP; Day 270). Tissues (approximately equal to 500 mg wet tissue/explant) were cultured for 24 or 48 h in modified minimal essential medium (MEM), in the presence of radioactive amino acid and/or amino sugar substrate(s) (L-[3H] leucine, L-[14C] leucine, and D-[3H] glucosamine), in order to characterize substrate uptake and de novo synthesis and release of proteins and polypeptides in vitro. Endometrial explants from all cattle produced proteins de novo from radiolabeled substrates. Chromatographic (gel filtration, cation, and anion exchange) and two-dimensional polyacrylamide gel electrophoretic analyses revealed complex patterns of primarily acidic radiolabeled polypeptides in dialyzed MEM, which were absent from endometrial tissue homogenates. No qualitative differences were noted in the array of proteins released into MEM associated with either pregnancy status (P vs. NP, UP) or stage of gestation (Days 16, 19, 22, 24, 69, and 270). Medium from all endometrial explants was enriched in polypeptides in four Mr (X 10(3)/pH classes (I, approximately equal to 14/greater than 7.2; II, 19-24/5.4-6.3; III, 28-31/6.9-7.3; and IV, greater than or equal to 150/less than or equal to 5.1).     

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.     

Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares

Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (Mr) 30,000-40,000 (pI values 4.5-5.5 and 6-7), one group of Mr approximately 22,000 (pI 6.5-7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with Mr 20,000 (pI 5-6). The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12-16 of dioestrus or Days 12-18 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Porcine conceptuses secrete an interferon during the preattachment period of early pregnancy

Maternal recognition of pregnancy in sheep and cattle is thought to be initiated by the conceptus secretory proteins ovine trophoblast protein-1 (oTP-1) and bovine trophoblast protein-1 (bTP-1), respectively. Recently, these proteins have been shown to be members of the interferon-alpha (IFN-alpha) family. In this study, we have examined whether pig conceptuses also produce IFN during early pregnancy. conceptuses were collected at Days 8, 10, 11, 12, 13, 15, and 17 of pregnancy and cultured in serum-free medium for 24 h. Day 11 conceptuses secreted a dominant 22,000-24,000 Mr cluster of acidic proteins (pI 5.2-5.4), that appeared to cross-react on immunoblots with antiserum against human IFN-alpha but not against oTP-1. Antiviral activity characteristic of an IFN was present in conceptus culture medium and uterine flushings from Day 11 through Day 17 of pregnancy, but was absent in flushings prior to Day 11 of pregnancy and in flushings from Day 12 nonpregnant gilts. The antiviral activity coeluted with a 22,000-24,000 Mr protein during partial purification through a gel filtration column. The activity was extremely labile, but could be restored by sequential protein denaturation, reduction, and renaturation. We conclude that production of IFN by early conceptuses is not restricted to ruminant species, and may therefore represent a more general phenomenon.     

Characterization of feline conceptus proteins during pregnancy

This study characterized proteins secreted de novo by feline conceptuses collected on Days 10, 12, and 15 (n = 22, preimplantation blastocysts); Days 15, 16, 17, 19, 21, and 25 (n = 6, postimplantation zonary girdle [ZG] i.e. trophoblast and endometrium); and Days 30, 36, 39, and 50 (n = 5, postimplantation ZG and free chorioallantois [CA]) and cultured in Minimal Essential Medium. De novo secretion was shown by incorporation of 3H-leucine into proteins detected in culture media by 2D-PAGE and fluorography. Western blotting, and NH2-terminal amino acid microsequencing. Major radiolabeled proteins identified as they appeared temporally on fluorographs were as follows: feline conceptus protein 1 (fCP1), Mr = 20,000, pI 5.0-5.3; fCP2, Mr = 80,000, pI 6.5-7.2; fCP3a, Mr = 67,000, pI 6.3-6.5; fCP3b, Mr = 67,000, pI 5.9-6.3; fCP4, Mr = 56,000, pI 5.0-6.0; and fCP5, Mr = 29,000, pI 5.0-5.8. The fCP1 was produced by blastocysts on Days 10-15, ZG on Days 16-25, and CA on Day 30; on Days 39-50, CA synthesized 5 proteins, possibly fCP1 isomers. The fCP2, fCP3a and b, and fCP4 were produced by blastocysts on Day 15, ZG on Day 25, and CA on Days 30-50. The fCP5 was made by ZG on Days 16-36 and by CA on Days 30-39. Western blotting identified fCP1 as retinol-binding protein (RBP), fCP2 as alpha fetoprotein, fCP3a as albumin, and fCP3b as transferrin. Amino acid sequence homologies between fCP1 and rabbit and human plasma RBP and porcine conceptus RBP2 were 93, 96, and 100%, respectively, at the first 37 NH2-terminal amino acids. The identities of fCP4 and fCP5 have not been established. Antiviral activity detected in all media was less than 3 units/ml when tested with feline fibroblast cells infected with vesicular stomatitis virus.     

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinol-binding protein: a major secretory product of the pig conceptus

Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.     

Failure of the LH-releasing hormone agonist, deslorelin, to prevent development of a persistent follicle in heifers synchronized with norgestomet

The use of exogenous progestagens for estrus synchronization in cattle can result in a persistent dominant follicle which is associated with reduced fertility. We examined whether the LHRH agonist, deslorelin, would prevent the formation of a persistent follicle in heifers synchronized with norgestomet. The estrous cycles of heifers were synchronized with cloprostenol, and on Day 7 of the ensuing cycle the heifers received one of the following treatments for 10 d: Group C (n = 5), untreated control; Group N (n = 6), injection of a luteolytic dose of cloprostenol on Days 7 and 8 and implant of norgestomet from Day 7 to Day 17 (i.e. typical 10-day norgestomet implant period); Group D (n = 6), injection of cloprostenol on Days 7 and 8 and implants of deslorelin from Day 7 to Day 17; Group ND (n = 6), injections of cloprostenol and both norgestomet and deslorelin implants as above. Follicle growth was monitored using ultrasonography. Group-N heifers showed continued follicle growth and had larger follicles on Day 17 of the cycle than Group-C heifers (16.8 +/- 1.6 and 10.4 +/- 1.6 mm). Follicle growth for Group-D and ND heifers was similar and variable, and seemed to depend on follicle status at the initiation of treatment. Heifers with follicles of 5 to 10 mm (n = 9) in diameter either showed no follicle growth (2 9 ) or developed large follicles (7 9 ), while heifers with follicles approximately 12 mm (n = 3) in diameter showed follicle atresia with no further significant growth. On Day 17, size of the largest follicle was similar for Group-ND (14.3 +/- 2.9) and Group-D (16.8 +/- 1.6) heifers. Heifers in Group N showed estrous behavior 1.8 +/- 0.2 d after treatment, whereas heifers in Groups D and ND did not show estrus for 2 to 4 wk. The results show that combined treatment with progestagen and an LHRH agonist does not consistently prevent the development of a persistent dominant follicle and that return to estrus can be delayed after treatment with an LHRH agonist.     

Estradiol production by preimplantation blastocysts and increased serum progesterone following estradiol treatment in llamas

Estradiol is a potential candidate for the blastocyst signal responsible for maternal recognition of pregnancy in the llama (Lama glama). Two experiments were conducted to determine if the llama blastocyst produces estradiol during the presumed period of maternal recognition of pregnancy and if exogenous estradiol can extend the luteal phase. In Experiment 1, llamas were superovulated with eCG and mated 7 days later (Day 0=day of mating). Blastocysts were collected nonsurgically on Days 7, 9, or 11 or at necropsy on Days 13 and 15 post-mating and cultured for 48h. Conditioned medium was recovered, replaced with fresh medium at 24-h intervals, and assayed for estradiol-17beta. Estradiol production (pg/blastocyst) over the 48-h culture increased (P<0.05) by day of gestation where more estradiol (P<0.05) was produced by Day 11 compared to Day 7 blastocysts, Day 13 compared to Days 7-11 blastocysts, and Day 15 compared to Days 7-13 blastocysts. A dramatic increase was observed between Days 11 and 13 when estradiol production by Day 13 blastocysts increased (P<0.05) more than 50-fold. In Experiment 2, 30 females were induced to ovulate with hCG (Day 0=day of hCG injection). Starting on Day 7 and continuing through Day 15, animals received daily injections i.m. of 0 (n=11), 5 (n=7), or 10mg (n=12) estradiol benzoate (EB) dissolved in isopropylmyristate. Sera were collected immediately prior to each injection and on Days 16, 17, 18, 20, and 22 and analyzed for progesterone. Progesterone concentrations were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas treated with 10mg EB compared to llamas treated with 0mg EB. These results demonstrate that llama blastocysts produce estradiol and exogenous estradiol can enhance and transiently extend luteal progesterone production. Estradiol produced by the preimplantation llama blastocyst may play a role in maternal recognition of pregnancy and early luteal support.     

Effect of delayed metamorphosis on larval competence, and post-larval survival and growth, in the abalone Haliotis iris Gmelin

Marine invertebrate species vary in their ability to delay metamorphosis, and in the degree to which delayed metamorphosis compromises juvenile performance. Abalone (Haliotis iris) larvae were deprived of metamorphosis cues and the effects of delayed metamorphosis on larval competence, and post-larval growth and survival were quantified. Larvae were exposed to a metamorphosis inducer (the coralline alga Phymatolithon repandum (Foslie) Wilks and Woelkerling) on Days 11, 18, 22, 26, 30 and 34 post-fertilisation (temperature 16-17 degrees C). Post-larvae were reared on diatoms (Nitzschia longissima Grunow) for 3-4 weeks post-metamorphosis. Delayed metamorphosis caused progressive negative effects on post-larval performance. Virtually all larvae initiated metamorphosis in response to P. repandum, regardless of larval age. The proportion of post-larvae that developed post-larval shell growth within 2 days of metamorphosis induction dropped only approximately 20% from Day 11 to Day 26 (P>0.05), but was significantly lower by Day 30 and Day 34 (P<0.001). Larvae that metamorphosed on Days 11, 18 and 22 showed high survival (>80%) and growth rates (means of 20-22 μm shell length per day). In contrast, larvae that metamorphosed on Day 26 and Day 30 had poor survival (30-40%) and lower (P<0.05) growth rates (15-16 μm/day). Of the larvae that metamorphosed on Day 34, only 7 (30%) survived their first week post-metamorphosis, and they grew only 2 μm/day on average. Only one of these post-larvae (4%) survived the second week. The visible yolk supply diminished over the life of the larvae and was near zero by Day 34. Nearly all larvae had died by Day 38. H. iris larvae remained competent to metamorphose for at least 3 weeks after they attained competence. Post-larval growth and survival were not reduced if metamorphosis occurred within 3 weeks of fertilisation. This extended period of larval competence implies that H. iris larvae can potentially disperse for up to several weeks before successful metamorphosis.     

Low-molecular-weight polypeptides of vicilin from Vicia faba L. are products of proteolytic breakdown

Vicilin, the main 7-S globulin of Vicia faba L., undergoes cleavage during prolonged treatment at room temperature, which can be inhibited by protease inhibitors such as 1 microM leupeptin. The cleavage products show identical electrophoretic mobilities with the polypeptides normally visible after sodium dodecylsulphate gel electrophoresis of vicilin prepared from mature seeds. N-terminal amino acid analysis of electrophoretically prepared polypeptides reveals serine as common N terminus of the two largest polypeptides of Mr approximately equal to 50000 and 35000. According to serological experiments and peptide mapping the low-molecular-weight polypeptides (Mr approximately equal to 35000; 31000; 19000 and below) have antigenic determinants and amino acid sequences, respectively, that are similar to each other and are all contained within the structure of the large polypeptide of Mr approximately equal to 50000. This leads of the conclusion that polypeptides of Mr approximately equal to 35000 and below are derived by proteolysis from one (or a few closely related) polypeptide(s) of Mr approximately equal to 50000 and that proteolysis starts soon after biosynthesis as a post-translational process within the developing seed. Some experiments indicate the existence of 'nicking' points within the vicilin polypeptides, which are the major cleavage sites during preparation. These observations strongly support the view that 'native' vicilin is a trimeric or tetrameric globulin with polypeptides of Mr greater than or equal to 50000.     

The synchrony of prostaglandin-induced estrus in cows was reduced by pretreatment with hCG

The induction of optimal synchrony of estrus in cows requires synchronization of luteolysis and of the waves of follicular growth (follicular waves). The aim of this study was to determine whether hormonal treatments aimed at synchronizing follicular waves improved the synchrony of prostaglandin (PG)-induced estrus. In Experiment 1, cows were treated on Day 5 of the estrous cycle with saline in Group 1 (n = 25; 16 ml, i.v., 12 h apart), with hCG in Group 2 (n = 27; 3000 IU, i.v.), or with hCG and bovine follicular fluid (bFF) in Group 3 (n = 21; 16 ml, i.v., 12 h apart). On Day 12, all cows were treated with prostaglandin (PG; 500 micrograms cloprostenol, i.m.). In Experiment 2, cows were treated on Day 5 of the estrous cycle with saline (3 ml, i.m.) in Group 1 (n = 22) or with hCG (3000 IU, i.v.) in Group 2 (n = 20) and Group 3 (n = 22). On Day 12, the cows were treated with PG (500 micrograms in Groups 1 and 2; 1000 micrograms in Group 3). Blood samples for progesterone (P4) determination were collected on Day 12 (Experiment 1) or on Days 12 and 14 (Experiment 2). Cows were fitted with heat mount detectors and observed twice a day for signs of estrus. Four cows in Experiment 1 (1 cow each from Groups 1 and 2; 2 cows from Group 3) had plasma P4 concentrations below 1 ng/ml on Day 12 and were excluded from the analyses. In Experiment 1, cows treated with hCG or hCG + bFF had a more variable (P = 0.0007, P = 0.0005) day of occurrence of and a longer interval to estrus (5.9 +/- 0.7 d, P = 0.003 and 6.2 +/- 0.8 d, P = 0.005) than saline-treated cows (3.4 +/- 0.4 d). The plasma P4 concentrations on Day 12 were higher (P < 0.0001) in hCG- and in hCG + bFF-treated cows than in saline-treated cows (9.4 +/- 0.75 and 8.5 +/- 0.75 vs 4.1 +/- 0.27 ng/ml), but there was no correlation (P > 0.05) between plasma P4 concentrations and the interval to estrus. In Experiment 2, cows treated with hCG/500PG and hCG/1000PG had a more variable (P = 0.0007, P = 0.002) day of occurrence of and a longer interval to estrus (4.2 +/- 0.4 d, P = 0.04; 4.1 +/- 0.4 d, P = 0.03) than saline/500PG-treated cows (3.2 +/- 0.1 d). The concentrations of plasma P4 on Days 12 and 14 of both hCG/500PG- and hCG/1000PG-treated cows were higher (P < 0.05) than in saline/500PG-treated cows (7.3 +/- 0.64, 0.7 +/- 0.08 and 7.7 +/- 0.49, 0.7 +/- 0.06 vs 5.3 +/- 0.37, 0.5 +/- 0.03 ng/ml). The concentrations of plasma P4 on Days 12 or 14 and the interval to estrus were not correlated (P > 0.05) in any treatment group. The concentrations of plasma P4 on Days 12 and 14 of hCG/500PG- or hCG/1000PG-treated cows were correlated (r = 0.65, P < 0.05; r = 0.50, P < 0.05). This study indicated that treatment of cows with hCG on Day 5 of the estrous cycle reduced the synchrony of PG-induced estrus and that this reduction was not due to the failure of luteal regression.     

Ovarian activity and plasma concentrations of progesterone and estradiol during pregnancy in jennies

The objectives of this study were to determine ovarian activity (with ultrasound) and plasma concentrations of progesterone and estradiol during pregnancy in jennies. There was considerable ovarian activity during the second month of pregnancy. Secondary corpora lutea (total of 2 to 7 per jenny) were formed (mainly by luteinization) starting on Day 41.8 +/- 1.0 (range Days 38 to 46; ovulation = Day 0). The echogenicity of the primary and secondary corpora lutea gradually decreased during pregnancy. Plasma progesterone concentrations increased between Days 0 and 10 (0.9 and 19.9 ng/mL, respectively), gradually decreased to Day 30 (12.1 ng/mL), increased between Days 30 and 40 (plateau, at approximately 17 ng/mL), gradually declined from Days 110 to 160 (nadir of approximately 6 ng/mL), and remained nearly constant until increasing again just before parturition. Plasma estradiol concentrations increased gradually from Day 65, peaked (1.2 ng/mL) on Day 165 (> or = 1 ng/mL on Days 150 to 210), and decreased thereafter, with very low concentrations during the last 20 d before parturition. Ovarian function and hormone profiles were generally similar to those previously reported during pregnancy in mares.     

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.     

Synchronization of estrus and fertility in beef cattle with two injections of buserelin and prostaglandin

Postpartum beef cows and heifers in Group 1 received 8 mug of buserelin on Day 0 (the beginning of the experiment) and 500 mug of cloprostenol (PGF) on Day 6 (GnRH I, n = 54). In Group 2 (GnRH II, n = 54), the females were injected with buserelin on Day 0 (8 mug) and Day 3 (4 mug), and PGF on Day 6 and Day 9 for females not detected in estrus previously. Animals were bred by AI 12 hours after the onset of estrus. Blood samples were collected on Day -11 and Day 0 to assess cyclicity and on Day 3 and Days 6 to 12 to examine luteal activity. Progesterone levels did not differ between the 2 groups between Days 0 to 9. In both groups, the proportion of spontaneous estruses from Days 0 to 6 was reduced. Precision of estrus was higher (P < 0.005) in the GnRH II group than in the GnRH I group of cows that were detected in estrus between Days 6 and 9. The synchronization rate, interval to estrus, pregnancy and conception rates were similar in GnRH I and GnRH II groups. The conception rate and interval to estrus were similar in cyclic and acyclic cows. Increasing the number of buserelin injections enhanced the precision of estrus, but not the conception rate, without any detrimental effect on luteal activity and induced more estruses in postpartum acyclic beef cattle.     

Changes in adenylate cyclase and phosphodiesterase activities during the growth cycle of adult rat hepatocytes in primary culture

Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.     

Inhibitory effects of adrenocorticotrophin or corticosterone on progesterone secretion during mid-pregnancy in rats

Conceptus number was reduced to one on Day 7 of pregnancy in rats by aspirating all but a single conceptus (Group E) or left at greater than or equal to 8 conceptuses (Group C). In Group E rats, serum progesterone concentrations remained low from Day 12 until autopsy at Day 21. Hypophysectomy on Day 12 significantly increased serum progesterone values after Day 17 of pregnancy, and these increases were blocked by treatment with ACTH (10 U/day, i.p., Days 12-17). Adrenalectomy on Day 12 also induced slight, but statistically significant, increases in serum progesterone concentration after Day 17, and these were overcome by implantation of a 10 mg capsule of corticosterone. In Group C rats, hypophysectomy or adrenalectomy on Day 12 did not change serum progesterone concentrations, but 40 U ACTH/day inhibited progesterone secretion. We conclude from these results that the pituitary-adrenal system exerts inhibitory effects on progesterone secretion during mid-pregnancy in rats.     

The role of endogenous estrogens in the maturation process of the bovine placenta

The plasma estrogen and progesterone concentrations of 19 pregnant cows (average duration of pregnancy 266.0 +/- 2.3 d at the start of the study) were determined daily from Day 6 pre partum to Day 1 post partum. Parturition was induced in all cows by administration of 10 mg i.m. flumethasone. Values were centered around the delivery date (Day 0) following either induced normal calving (n = 3) or surgical delivery (n = 16). In animals showing spontaneous expulsion of the fetal membranes (Group 1, n = 6) the average total estrogen concentration increased significantly from Day 6 until Day 1 before parturition (1329.2 +/- 317.9 to 3719.8 +/- 951.2 pg/ml in total estrogens). A marked decrease was observed on Day 1 post partum (459.4 +/- 344.2 pg/ml). In comparison with Group 1, animals showing either a delayed or partial expulsion of the fetal membranes, or in which the placenta could be withdrawn 16 h after calving (Group 2, n = 5), had consistently lower total estrogen concentrations between Day 6 (595.4 +/- 174.8 pg/ml) and Day 1 (1884.3 +/- 565.1 pg/ml) before parturition. The estrogen values of the cows with retained placenta (Group 3, n = 8) from Days 6 to 0 pre partum were significantly lower than those of Group 2. Total estrogen concentrations of the three groups 1 d post partum did not differ significantly. It is generally recognized that estrogens play an important role in the maturation process of the placentomes. Our investigation demonstrates that not only is the magnitude of the prepartum rise in estrogens of great influence of the maturation process but the duration of this rise is likewise important. These two factors are vital for a normal expulsion of the fetal membranes.