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The primary objectives of this study were to determine whether a delay in the onset of the luteal phase, or high milk urea nitrogen at breeding, or both were associated with failure of pregnancy early in gestation. Milk samples were collected twice daily from cows in a single herd during the week following breeding; single samples were collected on d 14 and 21 postbreeding. Progesterone was measured in all samples, and a total of 156 sample sets was used. The progesterone data combined with results from pregnancy examinations were used to distribute the cows into three groups: 1) pregnant, 2) nonpregnant with a low concentration (< 2 ng/ml) of progesterone on d 21, and 3) nonpregnant with a high concentration (> or = 2 ng/ml) of progesterone on d 21. The interestrous interval for cows in group 3 was longer than that for cows in group 2. Beginning 4.5 d after breeding, pregnant cows had higher concentrations of progesterone than did cows in group 3. Pregnant cows also had higher concentrations of progesterone than did all open cows on d 14 and 21. The onset of the luteal phase was earlier in pregnant cows than it was in cows in group 3. Milk urea nitrogen at breeding was similar in pregnant cows and in cows in group 3, but was higher in cows in group 2. Increased milk urea nitrogen was also statistically associated with decreased fertility. We propose that the cows in group 3 likely had embryos that initiated pregnancy recognition and prolonged luteal function, but these embryos were compromised by suboptimal exposure to progesterone early in development.

Before fertilization, equine spermatozoa adhere to oviduct epithelial cells (OEC) of the mare. The biochemical basis for this adhesion has not been determined. Our objective was to produce an antiserum to block this interaction. Ejaculated spermatozoa were subjected to nitrogen cavitation and spermatozoal plasma membranes enriched by sucrose density gradient centrifugation; membrane enrichment was confirmed by comparative alkaline phosphatase analysis, electron microscopy, and one- and two-dimensional PAGE. Periacrosomal plasma membrane was used as an immunogen for the production of an antiserum, which recognized several components of spermatozoal plasma membrane on Western blots. Antigen-binding fragments (Fab) were isolated by papain digestion from a specific antiserum and from nonimmunized rabbit IgG (control). The periacrosomal regions of epididymal and ejaculated spermatozoa were immunolabeled with antiserum Fab but not control Fab. The immunoneutralizing activity of antiserum Fab was tested in fluorescent cell-binding assays by competitive inhibition of the binding of spermatozoa to OEC monolayers or explants. In both assays, binding of spermatozoa to OEC was reduced as the concentration of specific Fab increased. These results suggest that one or more protein or glycoprotein components of the rostral spermatozoal plasma membrane mediate adhesion between spermatozoa and oviduct epithelium in vitro.

Maturation-promoting factor (MPF) is known to be a key regulator of both mitotic and meiotic cell cycles. MPF is a complex of a B cyclin and the cyclin-dependent kinase cdkl (p34cdc2). Oocyte maturation and its arrest at metaphase of meiosis II (MII) are regulated by changes in MPF activity. In this study, experiments were conducted to examine the dynamics of MPF activity and its constituent proteins during in vitro maturation of bovine oocytes. Bovine oocytes displayed relatively low levels of MPF (histone H1 kinase) activity at the germinal vesicle stage during the first 8 h of maturation. MPF activity increased gradually thereafter, and its first peak of activity occurred at 12-14 h of maturation (presumptive metaphase I), which was followed by an abrupt reduction in activity at 16-18 h, during presumptive anaphase and telophase. MPF activity then increased, reaching a plateau at 20-24 h of maturation (MII stage). This high level of MPF activity was maintained for several hours but decreased gradually after 30 h of maturation and became barely detectable by 48 h of in vitro maturation (IVM) culture. At each time point, there was a significant variation among individual oocytes in histone H1 kinase activity, which was probably due to asynchronous maturation. Abundance of cdk1 increased gradually during the first 8 h and then remained relatively constant except for an apparent reduction at 18-22 h of IVM. The level of cyclin B2 increased quickly during the initial 2 h of culture, and this high level was maintained until 16 h, after which a significant reduction was observed between 18 and 22 h of IVM. The de novo synthesis of cyclin B2, however, exhibited a biphasic oscillation during maturation, with peaks before the onset of MI and of MII. These results have defined the profiles of MPF activity and its individual components during bovine oocyte maturation in vitro. We conclude that active MPF regulates bovine oocyte maturation and that de novo synthesis of cyclin B2 occurs during the process of maturation.

The c-mos proto-oncogene product Mos is believed to be an active component of the cytostatic factor that stabilizes and sustains the activity of maturation-promoting factor. Mos has been found to be responsible for the metaphase arrest of oocytes at the second meiotic division in both Xenopus and the mouse. In this study, we have demonstrated, by Western blot and immunoprecipitation analysis, that an approximately 39-kDa protein, identified as Mos, was present in in vitro-matured (metaphase II stage) bovine oocytes but disappeared in parthenogenetically activated oocytes. The oocytes actively synthesized p39mos at the metaphase II stage (between 22 and 26 h of in vitro maturation [IVM]), whereas little p39mos synthesis was detected during the first 4 h of IVM and it was nondetectable during aging at 44-48 h of IVM, when oocytes lose the capability of normal development after fertilization. Ethanol activation of mature oocytes led to the disappearance of p39mos. beta-Tubulin, but not p34cdc2, was co-precipitated with Mos when extracts of metaphase II-stage bovine oocytes were incubated with Mos antiserum. These results demonstrated that Mos is present and actively synthesized in mature bovine oocytes and that oocytes aged beyond the optimal time for fertilization seem to lose the ability to synthesize the Mos protein. beta-Tubulin was found to be associated with Mos, which suggests a possible role for the cytoskeletal protein in maintaining the meiotic arrest in mature bovine oocytes.

Successful in vitro maturation (IVM) of bovine oocytes requires continual and/or episodic protein synthesis by cumulus-oocyte complexes. This study was designed to expose time-dependent changes in protein synthesis and accumulation by bovine oocytes and cumulus cells during routine IVM. Silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated little if any change in protein species present or their relative contents in oocytes during IVM; one notable exception, however, was the gradual accumulation of a 39-kDa polypeptide between 4-24 hr of maturation culture. Cumulus cells, on the other hand, exhibited no qualitative differences during the period examined, but total protein content did increase during IVM. Metabolic labeling with [35S]-methionine, however, demonstrated changes in protein synthesis, both quantitative and qualitative, by both cell types. Oocytes exhibited a steady or slightly increasing rate of synthesis during the first 12 hr of IVM; thereafter, protein synthesis declined to about 10% of the initial rate by 40 hr in culture. In contrast, protein synthesis in cumulus cells was relatively constant during the first 24 hr. Of greater interest is the demonstration that the synthesis of at least seven oocyte-specific and five cumulus-specific proteins was stage-dependent during maturation. These results indicate that maturation of bovine oocytes is associated with the synthesis of several distinct and temporally expressed proteins which may play roles in the highly ordered sequence of events that culminates in oocyte maturation.

To test the hypothesis that placental lactogen (PL) is a humoral regulator of fetal growth, six singleton sheep fetuses received a continuous intravenous fusion of 1.2 mg/d of purified ovine PL (oPL) for 14 d, beginning on Day 122 of gestation. The plasma concentration of oPL was approximately four-fold higher in infused fetuses than in six control fetuses that received a continuous infusion of saline. The circulating insulin-like growth factor 1 (IGF-I) concentration was also significantly elevated in PL-infused fetuses (43.1 +/- 1.7 vs. 31.9 +/- 4.1 ng/ml; P < 0.05). Animals were slaughtered on Day 136, and the placenta and all major fetal tissues were dissected, weighed, and subsampled for chemical analysis. Fetal weight and crown-rump length were not significantly affected by treatment; however, the aggregate weight of the brain, liver, lungs, and heart tended to be larger (85.3 +/- 2.1 vs. 79.9 +/- 1.5 g/kg fetus; mean +/- SE, P = 0.07) and the thyroid gland was smaller (0.18 +/- 0.1 vs. 0.26 +/- 0.02 g/kg fetus; P < 0.05) in the PL-infused fetuses. The livers of the PL-infused fetuses had also accumulated additional glycogen (13.1 +/- 1.7 vs. 8.4 +/- 0.7 g; P < 0.05). In late gestation, PL within the fetal compartment increases fetal plasma IGF-I concentration and hepatic glycogen deposition and may affect the growth of several vital organs.

Objective: To compare the electrophoretic patterns of proteins synthesized and secreted by oviductal epithelial cell (OEC) explants obtained from young, fertile and aged, subfertile mares., Animals: Young, fertile (n = 5; 2 to 7 years old) and aged, subfertile (n = 5; 17 to 24 years old) mares., Procedure: 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computerized densitometry., Results: Variation in the synthesis and secretion of polypeptides from young, fertile mare OEC (YOEC) and aged, subfertile mare OEC (AOEC) was evidenced by differences in the intensity of radiolabeled polypeptides on fluorograms. Fluorograms for 9 acidic (isoelectric point, 5.09 to 6.50) proteins of low to medium molecular mass (18.2 to 85.0 kd) from YOEC had greater intensity than did those from AOEC. Fluorograms for 7 proteins (10.5 to 45.0 kd; isoelectric point, 5.80 to 6.92) from AOEC had greater intensity., Conclusion: The differences detected in the fluorographic intensities of secreted proteins from YOEC and AOEC may be related to the disparity in embryo development observed between young, fertile and aged, subfertile mares., Clinical Relevance: Failure to maintain pregnancy in aged, subfertile mares may be a result of a suboptimal oviductal environment exerting its effects on the conceptus during early cleavage.

Adhesion of equine spermatozoa to homologous oviduct epithelial cells (OEC) in vitro results in specific changes in spermatozoa and OEC function. To test the hypothesis that adhesion of spermatozoa affects protein synthesis and secretion by OEC, the following treatment groups were established in culture: OEC with culture medium only; control spermatozoa in culture medium only; OEC in coculture with spermatozoa; and OEC and spermatozoa in coculture, but physically separated by a microporous membrane. The experiment was replicated within each of 4 ejaculates from 3 stallions. De novo protein secretion by OEC was measured and compared by incorporation of [35S]methionine, and evaluated, using two-dimensional polyacrylamide gel electrophoresis and fluorography. Monolayers of OEC secreted a large number of proteins of molecular mass ranging from 14 to 205 kd. Adhesion of spermatozoa consistently caused reduced synthesis of 2 OEC secretory proteins and new or increased synthesis of 6 proteins. When spermatozoa and OEC were separated by a microporous membrane, some but not all of these changes were duplicated. Synthesis of 3 OEC secretory proteins, unaffected by binding of spermatozoa, was reduced when spermatozoa were prevented from contact with OEC by a microporous membrane. Adhesion of equine spermatozoa to homologous OEC monolayers and presence of equine spermatozoa resulted in qualitative and quantitative changes in synthesis and secretion of proteins by OEC. These changes have implications for storage, longevity, and maturation of spermatozoa.

The time of activation of the embryonic genome (maternal-embryonic transition) in equine embryos was investigated by assessing incorporation of 3H-uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with 3H-uridine (560 microCi/ml) for 10 hr, while eight-cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 microCi/ml for 0.5-1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counterstained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight-cell, and five-cell stages incorporated 3H-uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four- to six-cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five- to six-cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight-cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of 3H-uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four- to eight-cell stage in equine embryos.

The effects of testosterone on the epiphyseal growth plate of metacarpal bones of growing sheep were evaluated in 20 rams, 20 wethers, and 20 wethers receiving subcutaneous testosterone replacement therapy. Two animals from each testosterone treatment group were slaughtered at 14-d intervals from 49 to 133 d, and then at 28-d intervals until 217 d, for a total of 10 slaughter ages. Immediately after slaughter, the cannon bones were dissected of extraneous tissue, weighed, and their lengths measured. Growth plates from the metacarpal bones were isolated and explants were cultured for 24 h in medium containing [3H]thymidine. After autoradiography, labeling index was calculated as the ratio of labeled to total nuclei in the resting and proliferative zones of the growth plate. Testosterone increased (P < .03) weight and length of the metacarpal bone. Increased bone length due to testosterone was associated, in part, with a higher (P < .05) labeling index in chondrocytes of the proliferative zone of the growth plate. Labeling indices in the resting zone chondrocytes of rams were higher near the time of puberty. Accelerated growth followed by cessation of growth occurs concurrently with puberty in males of several species and is accompanied by an increase in the blood concentration of testosterone. Testosterone may mediate this accelerated growth by first increasing bone growth and then depleting the source of stem cells in the cartilage growth plate, the site where growth in length of long bones occurs.

Polypeptides secreted by uterine tube epithelial cells (UTEC) may facilitate sperm cell capacitation in vivo. This experiment evaluated the effect of sperm-UTEC co-culture on de novo protein synthesis by epithelial cells of the tubal isthmus. Comparisons of the patterns of proteins secreted into medium were made between four culture groups incubated for 24 h in the presence of 35S-methionine: group 1, sperm cells alone; group 2, control UTEC monolayers; group 3, UTEC co-cultured with sperm cells; and group 4, UTEC partitioned by a diffusible membrane from sperm cells during culture. Two-dimensional PAGE followed by fluorography was used to analyze conditioned medium containing secreted proteins from each group. The experiment was replicated four times. Sperm cells alone secreted no detectable proteins, whereas control UTEC monolayers produced a wide array of polypeptides. Sperm cells attached to UTEC in co-culture within minutes, and the resultant protein profile for these UTEC differed markedly from that of the control UTEC. Several new proteins were seen only from co-cultured cells, whereas other protein groups that were present with UTEC alone were absent in the co-culture medium of group 3. The protein pattern expressed by UTEC partitioned from sperm cells (group 4) was intermediate between that of the group 2 controls and that of co-cultured UTEC (group 3). In summary, the attachment of sperm cells to the UTEC during co-culture changed the types and quantities of proteins secreted into the conditioned medium as compared to those of control UTEC monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Chimeric sheep-goat pregnancies were established in 24 ewes and 29 does by transferring 251 embryos, prepared by the blastomere-aggregation technique, to 52 ewes and 61 does. Fifteen does experienced early pregnancy failure; however, term offspring were delivered by 24 ewes (17 lambs, 3 kids, 6 chimeras) and 14 does (6 lambs, 9 kids, 6 chimeras). (Fetal classifications were based on phenotype, red blood cell isozymes, and lymphocyte antigen expression). RIAs for ovine and caprine placental lactogen detected chimerism in the binucleate cell population of the trophoblast throughout the pregnancies of 2 ewes and 7 does; these pregnancies resulted in the birth of 12 healthy offspring. Histological examinations of intact placentomes from 2 of these recipients revealed a continuous cellular trophoblast apposed to a syncytium as in normal placentas. Chimerism was detected electrophoretically in the membranes of the placentas with binucleate cell chimerism and in 17/28 of the other placentas. Data collected on placental lactogen production, chimerism in the conceptus, and placental morphometry were examined with respect to the stages of the blastomeres aggregated to form the chimeric embryo and with respect to fetal status at delivery. For comparison, analogous data were collected on sheep-goat concepti that developed from embryos prepared by inner cell mass transplantation.

Developmentally competent bovine blastocysts were produced by adding transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.

Two-cell bovine embryos produced in vitro were cultured in serum-free medium containing the soluble glycoprotein fibronectin (50 micrograms ml-1) to study the function of the extracellular matrix in early development. Some of the embryos (48/164, 29.3%), developed beyond the 16-cell stage compared with none of the 179 controls. Fibronectin at lower (5 micrograms ml-1) or higher (300 micrograms ml-1) concentrations did not promote embryo development (0/89 and 0/82, respectively). Indirect immunofluorescence demonstrated the presence of both fibronectin and its receptor on the surface of eight-cell embryo blastomeres, and biotinylated fibronectin demonstrated that exogenous fibronectin could cross the zona pellucida. These results, demonstrating the successful culture of bovine embryos in serum-free medium, support the hypothesis that the extracellular matrix, specifically fibronectin, plays a role in early development of bovine embryos.

In vitro produced, 2-cell bovine embryos were cultured in serum-free medium supplemented with various combinations of growth factors to test the hypothesis that these polypeptide factors are able to signal preimplantation development. The developmental arrest that occurs during the 8-cell stage with typical culture methods might be relieved by a growth factor-dependent mechanism that would stimulate expression of the embryonic genome, thereby mimicking events that occur in vivo in the oviduct during the fourth cell cycle (8- to 16-cell stage). Subsequently, other growth factors might promote compaction and blastulation, processes which normally occur in the uterus. The effects of growth factors on early embryos were evaluated using phase contrast microscopy to monitor progression to the 8-cell stage, completion and duration of the fourth cell cycle, and blastocyst formation. Platelet derived growth factor (PDGF) promoted development beyond the 16-cell stage in 39.1% of the 2-cell embryos examined in all experiments. The duration of the fourth cell cycle among these embryos was approximately 26 hours. During development after the 16-cell stage, PDGF reduced the proportion of embryos bastulating from 12.7% to 5.8%; in contrast, transforming growth factor alpha (TGF alpha), acting during the same developmental time period, increased the proportion of embryos blastulating from 8.6% to 40.6%. These results, using serum-free medium, indicated that PDGF signalled completion of the fourth cell cycle. TGF alpha, and perhaps basic fibroblast growth factor (bFGF), promoted blastulation of 16-cell embryos during subsequent culture.

Placental lactogen (PL) is found in fetal plasma throughout gestation, and PL receptors occur on many types of fetal cells. In this study, the entry rate of PL into the fetal circulation was estimated by injection of 125I-labelled ovine PL into two mid- and four late-gestation fetuses. At both ages, PL appears to be distributed into two body pools. One pool has a rapid half-life (approximately 9 min) and a volume of distribution approximately 8% of body weight, while the second pool has a longer half-life (approximately 45 min) and a distribution volume only 4% of body weight. The first pool is presumably blood plasma, but the physiological identity of the second pool is unknown. The effective half-life of PL is approximately 15 min, and the liver is suggested as a probable major site of degradation. These estimates were confirmed in late gestation by measuring fetal plasma concentrations of PL in response to a continuous infusion of unlabelled PL. The kinetic parameters estimated in this study can be used to determine the quantity of exogenous hormone required to alter PL concentration in fetal plasma in a predictable manner.

Research has indicated that trophoblastic vesicles (TV) formed from Day-14 equine conceptuses would prolong luteal maintenance in mares after surgical transfer to the uterus at Day 10 after ovulation. The current study assesses TV as a further model for maternal recognition of pregnancy in mares. The objectives of the study were to determine the ability of TV to prolong luteal maintenance in mares, their effect on endometrial production of prostaglandin F (PGF) in vitro, and their ability to secrete polypeptides in vitro. In contrast to our previous study (Ball et al., 1989b), transfer of TV from Day-12 or -14 equine conceptuses to recipient pony mares at Day 10 or 12 post ovulation did not significantly prolong luteal maintenance compared to sham-operated control mares. Prolonged luteal maintenance was noted in 1/10 control mares and 1/15 mares that received TV. Trophoblastic vesicles from Day-14 conceptuses significantly reduced production of PGF by Day-14 pregnant endometrium in vitro. However, intact Day-14 conceptuses failed to reduce PGF secretion in the same culture system. TV secreted an array of polypeptides that were similar in molecular weight range to those produced by intact conceptuses or conceptus fragments at Day 12 or 14. Although this study failed to confirm our earlier finding that TV prolong luteal maintenance in recipient mares, this study does indicate that TV may be a useful model for evaluating maternal recognition of pregnancy in mares.

Placental lactogen (PL) was isolated from goat cotyledonary tissue by a combination of mild alkaline extraction, anion and cation exchange chromatography, chromatofocussing and molecular filtration. The product, enriched 15,000-fold from the initial extract, was homogeneous when examined by SDS-gel electrophoresis (Mr 22,500) and isoelectricfocussing indicated a pI of 8.35 with a trace contaminant of pI 8.0. When assessed by relative binding activity in radioreceptor assays (RRA), goat PL exhibited somatotropic activity equivalent to 2.2 units/mg dry weight and lactogenic activity equivalent to 28.5 units/mg. A radioimmunoassay (RIA) for goat PL is described that is highly sensitive (190 pg/tube) and has acceptable repeatability within and between assays (6 and 13%, respectively). The assay is not affected by goat pituitary extracts or partly purified goat growth hormone and prolactin. Despite the marked increase in sensitivity of the RIA over that previously available when goat PL was measured by RRA, the hormone was not detected in jugular plasma of goats before Day 44 of pregnancy; concentrations increased thereafter and highest levels were measured during the last third of pregnancy in animals bearing triplets. Measurements by RIA are in general agreement with those obtained earlier in several studies in which RRAs were used. The hormone was detected in amniotic fluid. Maternal concentrations of goat PL declined before parturition and were undetectable by 18 h post partum.

The hypothesis that placental secretion of progesterone (P4) and ovine placental lactogen (oPL) are controlled through different mechanisms was tested. Placental tissue was obtained at days 133-138 of pregnancy, and explant incubations were established using 200 mg tissue per flask in 5 ml O2-saturated DMEM containing 24 mM HEPES and lacking phenol red (pH 7.4). Following a 30-min preincubation, and a 15-min control period, test substances were added and incubations continued, with periodic gassing, for 4 h at 37 degrees C in a shaking water bath. Dopamine (DA), norepinephrine (NE) and epinephrine significantly stimulated P4 production (P less than 0.05). The enhancement of placental P4 production was mimicked by the addition of 8-bromo-cyclic adenosine monophosphate and forskolin (P less than 0.05). The response to catecholamines was abolished by the addition of propranolol (P less than 0.05) but not by phentolamine (P greater than 0.05). Inclusion of a membrane-permeant substrate for P4 synthesis, 25-hydroxycholesterol, increased basal (P less than 0.05) but did not enhance agonist-induced P4 production (P greater than 0.05). High performance liquid chromatographic analysis of placental tissue demonstrated the presence of DA (80.8 +/- 7.07 pg/mg) and NE (48.8 +/- 5.77 pg/mg), as well as catecholamine metabolites. Addition of 1,2-dioctanoyl-sn-glycerol (DAG) or phorbol 12-myristate-13-acetate (PMA) enhanced oPL secretion (P less than 0.05) without affecting P4 production. The response to DAG and PMA, representing the release of considerably more oPL than can be detected by extracting the tissue, was not influenced by treatment with cycloheximide (P greater than 0.05) indicating that secretion of preformed oPL is regulated by the protein kinase C pathway. These results support the hypothesis that the secretion of oPL and the production of P4 are controlled by different mechanisms.

The effects of N-acetylimidazole on specific binding of oxytocin to microsomal fractions of bovine mammary gland were studied. N-acetylimidazole suppressed oxytocin binding, with time and concentration dependence. Decreased oxytocin binding activity appeared to be due to decreased affinity of the hormone for its receptor. Acetylation of oxytocin, rather than of oxytocin receptors, seemed to be responsible for the decreased binding.

Relationships between foetal corticosteroid concentrations, utero-ovarian prostaglandin F (PGF) and maternal peripheral progesterone have been examined in detail in goats shortly before spontaneous parturition at term. Foetal corticosteroids increased during the last 13-11 days of gestation and particularly sharply during the last 3 days and even during advanced labour. About 24 h before parturition, acute releases of PGF were evident in the vein draining the pregnant uterine horn, and these corresponded closely to the time of luteal regression. Further release of PGF occured when progesterone declined to low levels, probably reflecting in the course of labour. The changes observed before premature parturition, induced by infusing ACTH into foetal goats, were similar except for the more rapid increase in foetal corticosteoid concentrations. Immature neonates born after ACTH treatment were viable, placental delivery was normal and lactogenesis occurred in the mothers indicating that the treatment promoted full expression of the critical perinatal events. The early, acute releases of PGF were ipsilateral to the ACTH-infused foetus and were luteolytic provided the corpora lutea were also on that side. Luteolysis failed or was abnormally delayed if the corpora lutea were contralateral and prolonged ACTH treatment of the foetuses in such cases caused foetal death probably because of premature failure of the placenta. Similar findings were noted if ACTH infusion of the foetus was accompanied by simultaneous progesterone treatment of the mothers in order block the induction of labour. It was suggested that placental changes occurring during foetal hypercortisolism might be caused by increased placental oestrogen synthesis and the effect of this on the foeto-maternal junction along with a stimulatory action on PG synthesis in the maternal placenta. Experimental disruption of the normal sequence of events, when labour was blocked by progesterone, proved to be lethal to the foetus if the loss of placental integrity progressed sufficiently. The chain of regulatory signals linking increased activity of the foetal adrenal with parturition thus appears to involve stimulation of oestrogen biosynthesis, PGF release from the maternal placenta and the start of physical changes at the placental junction. Provided the foetus and corpora lutea are ipsilateral, the early releases of PGF effect luteolysis and a withdrawal of progesterone from the maternal circulation. When progesterone concentrations are sufficiently low, labour is initiated and its progress reflected by further release of PGF. The control mechanisms, which also provide for the final maturation of the foetus, clearly enable a close synchronization of the various perinatal events which are essential for the transition from foetal to postnatal life.

A complete set of electromyographic recordings, plasma samples and behavioural observations were collected from 2 mares beginning 7 days pre partum, through parturition and into the early post-partum period. During the week pre partum, EMG activity was elevated, occurring 26-73% of the time. Activity was least during the day and greatest at night with no significant difference for the hours of the day or between days pre partum. During the 24 h before delivery, EMG activity was increased for 7-13 h (55-80%) during the daylight hours. EMG activity decreased 2-4 h immediately preceding delivery of the foal, with an abrupt increase at rupture of the chorioallantois. At delivery, EMG activity consisted of events containing a series of 10-13 discrete bursts of increasing amplitude occurring in rapid succession. After fetal delivery there was a reduction in activity until placental delivery followed by very long (2-22 min) trains of potentials.

Jugular plasma concentrations of oestradiol-17 beta, prolactin, progesterone and 13,14-dihydro-15-keto-prostaglandin F-2 alpha (PGFM) were measured at 2-h intervals during the last 4 days of pregnancy in 6 goats. During advanced labour and delivery, samples were obtained more frequently and assayed for oxytocin. The animals were housed in a barn with continuous dim lighting. A distinct pattern of oscillation in prolactin concentrations, with peaks during the late afternoon, was apparent during the last 3 days. Geometric means of peak concentrations doubled each day and became of longer duration; night-time nadir values remained low except during the last night before parturition. A progressive increase in oestradiol-17 beta, with mean levels doubling every 36 h, was apparent during the last 3 days. There was no sharp pre-partum increase in oestradiol-17 beta. Correlated (r = 0.83) with the increase in oestradiol-17 beta was a gradual increase in PGFM and when the latter reached approximately 1000 pg/ml, the non-reversible decline in progesterone reflecting pre-partum luteolysis occurred. Subsequent changes in PGFM related closely to an approximately 20-fold increase in the ratio of oestradiol-17 beta to progesterone until maximal PGFM levels of 26.5 +/- 4.2 ng/ml were reached at delivery. Basal concentrations of oxytocin (8-15 microU/ml) were measured before the last 60 min and markedly higher, though erratic, concentrations were detected at various times before appearance of the allantochorion. Maximal oxytocin values (range 180-1570 microU/ml) occurred within minutes before or after delivery of the first fetus. The results suggest that increased pre-partum production of oestradiol-17 beta, in addition to provoking sufficient release of prostaglandins to cause luteolysis, may modulate either the sensitivity or set-points for an endogenous rhythm in prolactin secretion at the end of pregnancy. The nature of the oxytocin changes suggest that, after labour has evolved sufficiently, delivery is precipitated by an abrupt increase in oxytocin secretion.

Recordings of uterine electrical activity were made from 5 pregnant pony mares from Day 141 to 320 of pregnancy. Three types of activity were identified. Short, medium and long bursts were quantified as the percentage of time each occurred during the hour analysed and further categorized according to frequency, amplitude and duration. The uterus was most active during the early stages recorded and became increasingly quiescent after Day 240. Short-burst activity was greatest when the uterus was most quiescent. Long bursts showed the greatest percentage of activity until Day 220 and then decreased. Medium-burst activity was present throughout the period studied and high amplitude synchronous medium bursts peaked at Day 221-240.

Progesterone concentrations measured in plasma samples from 280 bats captured during pregnancy or early lactation were related to fetal attributes indicative of stage of pregnancy. Fetal weight increased exponentially from 40 mg at crown-rump length of 6 mm to 2000 mg at 23 mm (term). Fetal weights at term accounted for up to 35% of the weight of intact pregnant animals. Progesterone concentrations increased from less than 5 ng/ml at 2 mm estimated crown-rump length to plateau values of approximately 65 ng/ml (geometric means) from 16 mm crown-rump length until the most advanced stages of pregnancy. Mean concentration in 8 post-partum bats, most of which were actively lactating, was 8.4 ng/ml; 11.6 ng/ml was measured in one animal that was carrying a wet neonate when sampled yet was still pregnant when captured 5 h earlier. Placental concentrations of progesterone ranged from 43 to 964 ng/g wet weight of tissue and mean values increased in a similar fashion though were about 4-fold greater than changes in plasma concentrations of the steroid. The concentrations in placental tissue were at least 15- to 20-fold higher than could be expected from blood contamination, indicating that placental steroidogenesis is likely to occur in this species.

Cytosolic and nuclear progesterone (P) and estrogen (E) receptors (PR and ER, respectively) were measured in uterine tissues of rabbits at the end of pregnancy (days 25, 27, 29, 30, and 31 and 0-10 h postpartum) and pseudopregnancy (days 13 and 18). At both times, plasma P concentrations fell dramatically after a period of prolonged elevation, effecting changes in uterine function. Plasma concentrations of P remained elevated until day 29 of pregnancy and then fell continuously until postpartum, whereas plasma estradiol levels did not change. The numbers of nuclear ER and PR were constant from days 25-29. Concomitant with the fall in plasma P on day 30, levels of nuclear ER and PR doubled and remained elevated on day 31 and postpartum. In all cases, the numbers of nuclear receptors were negatively correlated with P concentrations. The number of cystosolic ER in the myometrium increased on day 30 of pregnancy and remained elevated on day 31 and postpartum. Although levels of cytosolic ER in the endometrium appeared to rise similarly, the change was not significant. The number of cytosolic PR in endometrium and myometrium did not change significantly. Between days 13 and 18 of pseudopregnancy, plasma P declined while plasma estradiol remained constant, as during pregnancy. At this time, cytosolic PR increased 4-fold, and nuclear PR doubled. Nuclear ER also increased between days 13 and 18, but the apparent increase in cytosolic ER was not significant. These data suggest that receptors for both steroids increase as the ratio of plasma estradiol to P increases at the end of pregnancy and pseudopregnancy. Surprisingly, nuclear PR levels are highest at term, when the influence of P in suppressing myometrial activity and preventing the onset of labor is removed. Thus, in the rabbit, the level of PR does not directly reflect the physiological response to P.

Pregnant ewes were chronically exposed to thermoneutral (TN; 20 degrees C, 30% relative humidity) or hot (H; 40 degrees C 9 h/d, 30 degrees C 15 h/d, 40% relative humidity) environments between d 64 and 136 to 141 of pregnancy. They were sampled for blood at 14-d intervals during this period for measurement of plasma metabolites and hormones, then slaughtered and dissected to measure conceptus weights, dimensions and fetal organ weights. Rectal temperatures of H ewes were elevated .3 to 1.0 C degrees above those of TN ewes throughout the experiment. Voluntary feed intakes were not altered by heat exposure except after 120 d of pregnancy, when feed intake was about 25% lower (P less than .10) by H than by TN ewes. Blood 3-hydroxybutyrate concentrations were not affected by heat, but plasma glucose concentrations were greater in H than in TN animals after 120 d (P less than .05). Placental weight, reduced by 54% (P less than .001) by heat exposure of ewes, was correlated positively with fetal weight and correlated negatively with fetal/placental weight ratio, fetal brain/liver weight ratio and fetal relative heart weight. Late in pregnancy, plasma concentrations of progesterone, cortisol and placental lactogen were reduced (P less than .01) in H ewes, whereas triiodothyronine levels were markedly lower (P less than .03) at all stages of pregnancy. Plasma concentrations of prolactin were elevated dramatically (P less than .01) and a modest increase (P less than .03) in somatotropin levels was recorded in H ewes. These results are consistent with our hypothesis that heat-induced fetal growth retardation is secondary to a primary reduction in placental growth; this could be mediated partly by reduced peripheral activity of thyroid hormones. Heat-induced reductions in secretion of progesterone and ovine placental lactogen more likely were a consequence than a cause of placental stunting.

Premature parturition was induced in five pregnant goats infused intravenously with 4.65-8.4 mg estradiol-17beta but not in one treated with 5.85 mg estradiol-17alpha. A single intramuscular injection of 12 mg estradiol benzoate (8.8 mg estradiol-17beta equivalents) was also effective. These doses were estimated to provide plasma concentrations of estradiol-17beta in the physiological range for animals at sponteneous parturition. Circulating plasma concentrations of progesterone decreased and lactogenesis occurred before all instances of induced parturition but no such changes resulted from infusion of estradiol-17alpha. Placental delivery was normal in all animals but neonates of more than 10 days prematurity were non-viable.

Concentrations of progesterone and of 13,14-dihydro-15-keto-prostaglandin F-2 alpha (PGFM) were measured in plasma collected from 6 bitches every 3 h starting 2.8-4.6 days before parturition (birth of first pup) and continuing until 0.4-0.8 days post partum, and in additional samples collected less frequently. Progesterone concentrations at 48, 24, 12 and 3 h pre partum averaged 2.8 +/- 0.3, 2.2 +/- 0.4, 1.0 +/- 0.3 and 0.7 +/- 0.2 ng/ml. At those times PGFM values averaged 380 +/- 80, 800 +/- 220, 1450 +/- 450 and 1930 +/- 580 pg/ml, respectively. Mean concentrations of PGFM increased about 2.5-fold between 48 and 15 h pre partum in association with the onset of luteolysis, and then increased another 2.5 times before parturition as progesterone fell to nadir values. Peak levels of PGFM ranged from 1060 to 7150 pg/ml (2100 +/- 600 pg/ml) and occurred within 1-9 h after the birth of the first pup and before the birth of the last pup. These results suggest that prepartum luteolysis in dogs is initiated by increases in maternal concentrations of PGF, and that progesterone withdrawal causes a further increase in PGF which completes luteolysis and provides a major portion of the uterotonic activity causing expulsion of pups.

To understand the factors responsible for the increased pre-partum output of cortisol from the adrenal glands of the goat foetus, we measured changes in responsiveness to ACTH in vitro of dispersed adrenal cells from foetal goats of various gestational ages and we determined the output of cortisol (F) and corticosterone (B) in the presence of exogenous progesterone (P4) and 17 alpha-hydroxyprogesterone (17 alpha-OHP4). The increment in F output after ACTH increased 5-fold between day 100 and days 147 or 154 (term). This increase was not associated with changes in the ED50 for ACTH stimulation of F output. The basal and ACTH-stimulated output of F was similar to that of B at day 100 but 5-7 times greater than that of B at day 154. There was a significant effect of ACTH on P4 output on days 77, 100 and 147 but net utilization of P4 by the cells at term. The ratio of exogenous P4: 17 alpha-OHP4 converted to F varied with gestation and increased from 0.11 at day 100 to 0.99 at day 154. Exogenous P4 was converted to B at all stages of pregnancy. We conclude that in the goat there is an increase in the responsiveness of foetal adrenal cells to ACTH between day 100 of gestation and term. One factor associated with this change in adrenal responsiveness may be an increase in the activity of the enzyme 17 alpha-hydroxylase.

Relationships between plasma flow and plasma concentrations of prostaglandin F were examined in the utero-ovarian veins of three pregnant goats. Plasma flow, measured by veno-arterial dilution of para-Aminohippurate in two goats, was unchanged or increased slightly when PGF concentrations were elvated by short-term infusions of PGF2alpha into a uterine vein. Utero-ovarian plasma flow was measured during labor in two goats. Flow doubled during advanced labor and then decreased sharply to very low rates during the terminal expulsive phase of stage II labor. A total of 8.3 and 9.5 mg PGF was released into the utero-ovarian vein of two goats during the last 6 hours before fetal delivery and maximal release rates of approximately 100 ug. min-1 were obtained some 5-10 minutes before delivery was completed. The highest plasma concentrations of PGF were detected immediately after completion of fetal delivery when utero-ovarian plasma flows were lowest.

Imidazole, at concentrations between 10(-3) and 10(-2) M, exerts a profound stimulatory effect on rabbit uterine strips obtained during pregnancy and studied isometrically in vitro. The action is not duplicated by N-alkylimidazoles which have greater potency as inhibitors of thromboxane synthetase but the effect of imidazole was antagonized by isoproterenol or theophylline. Biochemical analysis indicated that imidazole at concentrations greater than 5 x 10(-4) M stimulated both high and low affinity forms of cyclic AMP phosphodiesterase. The uterus of pregnant rabbits is profoundly refractory to any kind of pharmacological stimulation and the effects of imidazole, acting to stimulate phosphodiesterase, suggest that the integrity of the adenyl cyclase-cyclic AMP-protein kinase system is a necessary requirement for this organ to remain quiescent during pregnancy.

The growth process exemplifies changes in the priorities of different tissues for available nutrients according to a developmental program. We propose that a higher order of endocrine regulation over and above that provided by homeostatic mechanisms directs the flow of nutrients to support the physiological or developmental process of highest prevailing priority. The term homeorhesis is applied to this regulatory phenomenon and is distinguished from the more familiar concept of homeostasis. The documented actions of growth hormone and prolactin as somatotrophic agents are discussed and their candidacy as likely homeorhetic vectors is proposed. Certain shortcomings in the quality of available hormone preparations and inconsistencies between potencies in various tests performed in vitro and in vivo are noted. We question the appropriateness of the use of experimental routines suited to exploring acute metabolic phenomena in acquiring a deeper understanding of the long-term process of growth. Despite the varied nature of supportive data, growth hormone, prolactin, and the closely related placental somatomammotropin exhibit the desired properties of homeorhetic hormones--they direct the flow of nutrients to the process of highest priority, partly by coordinating nutrient utilization by competing tissues.

Radioreceptor assays for prolactin-like (lactogenic) activity and growth hormone (GH)-like activity have been used to study concentrations of caprine placental lactogen (PL) in the circulation during pregnancy. Both lactogenic and GH-like activities from less than 100 ng/ml (ovine prolactin- and human GH-equivalents) about 60 days after mating to reach peak levels (400-1600 ng/ml) between days 110 and 130 of pregnancy. The levels of both activities increased in essentially the same fashion but during the last 15 days of pregnancy, lactogenic activity declined less than GH-like activity. This divergence was most pronounced at parturition when levels of lactogenic activity increased (approximately 700 ng/ml) despite very low (less than 200 ng/ml) levels of GH-like activity being measured and this probably reflected increased secretion of pituitary prolactin near parturition. When serum from a pregnant goat or a simple alkaline extract of placental cotyledons was fractioned on a column packed with Sephadex G-100, lactogenic and GH-like activities eluted together with distribution coefficients of approximately 0-5-0-6. The possibility that caprine PL serves physiologically as a luteotrophin and/or mammotrophin during pregnancy in goats is discussed.

Control of metabolism during pregnancy and lactation involves two types of regulation-homeostasis and homeorhesis. Homeostasis control involves maintenance of physiological equilibrium or constancy of environmental conditions within the animal. Homeorhesis is the orchestrated or coordinated control in metabolism of body tissues necessary to support a physiological state. Regulation of nutrient partitioning during pregnancy involves homeorhetic controls arising from the conceptus. This assures growth of the conceptus (fetus and fetal membranes) and gravid uterus as well as development of the mammary gland. With the onset of lactation many--perhaps even most--maternal tissues undergo further adaptations to support rates of lipogenesis and lipolysis in adipose tissue are examples of important homeorhetic controls of nutrient partitioning that are necessary to supply mammary needs for milk synthesis. The interactions between homeorhesis and homeostasis during pregnancy and lactation and possible endocrine control are discussed. While not definitively established, roles for placental lactogen and prolactin are attractive possibilities in homeorhetic regulation of maternal tissues to support pregnancy and the initiation of lactaion, respectively.

The effects of excess dietary calcium on bone growth were quantified in 44 ewes fed ad libitum diets that contained one of four levels of dietary calcium (0.37, 0.56, 1.13 and 1.80%) and 0.42% phosphorus. Animals were slaughtered at one of six ages, circulating concentrations of hormones and minerals were measured and bone morphometry was evaluated. None of the diets impaired normal bone growth of the metacarpal as evaluated by overall length, cortical index and growth plate width. Circulating plasma concentrations of calcium and gastrin increased as dietary calcium levels increased, but all values were within the normal range for sheep. Plasma phosphorus was unaffected by the level of dietary calcium. Diet had no affect on T3, T4 or estradiol-17 beta. Age significantly (P less than 0.05) affected the metacarpal length, growth plate width and circulating calcium and phosphorus. The metacarpal in these ewes was 95% of mature length by 172 d of age. Attainment of mature length preceded growth plate closure by slightly over 300 d. These results demonstrate that elevated dietary calcium levels do not adversely influence bone growth and that the presence of an intact growth plate does not infer that further bone growth will occur.

Concentrations of plasma progesterone were similar to values reported in the literature except that a significant decrease in progesterone during the last day, but before parturition, was detected by systematic, high-intensity blood sampling. Mean concentrations of oestradiol-17 beta increased sharply and significantly, plateaued for 132.8 +/- 1.5 days (mean +/- s.e.m., N = 9), then declined sharply in each mare. There was obvious variation between the mares in when these increases and decreases in oestradiol-17 beta occurred, with the events being related closely to ambient photoperiod conditions rather than to the stage of pregnancy. Concentration of 13,14-dihydro-15-keto-prostaglandin F-2 alpha (PGFM) remained at low levels (less than 400 pg/ml) until Day 200 then increased to peak pregnancy levels (greater than 2000 pg/ml) by Day 300 and remained at this value until parturition. The concentrations of oxytocin remained basal (less than 15 microU/ml) throughout pregnancy and increased only at the beginning of the expulsive stage of labour. There was an increase, although not statistically significant, in the relative concentrations of oestradiol-17 beta to progesterone beginning 3 days before parturition, with the highest value of the ratio occurring at fetal delivery. Far more striking were acute changes in PGFM and oxytocin during parturition. Maximal concentrations of PGFM (approximately 30 ng/ml) and oxytocin (greater than 200 microU/ml) were measured between rupture of the chorioallantois and the completion of delivery. Closely timed samples from one animal showed that oxytocin increased (more than 10 standard deviations of the mean levels during late pregnancy for this animal) before any change in PGFM. In another dystocic mare, both oxytocin and PGFM peaked in the initial stages of delivery but only oxytocin remained elevated until the dystocia was remedied. The results suggest that an abrupt increase in oxytocin secretion precipitates the expulsive phase of parturition in mares.

Ovarian tissues were obtained from cyclic goats during the early, mid and late breeding season. Immunoreactive oxytocin was measured by RIA in tissue extracts after chromatography on octadecylsilica cartridges. Luteal oxytocin concentrations were significantly greater during the early breeding season than during the mid or late breeding season. Oxytocin is luteolytic in goats. High concentrations of luteal oxytocin may be related to the high incidence of short estrus at the onset of the breeding season.

Treatment of pregnant rats with antiestradiol (A-E2) serum twice a day, starting at 2100 hours on day 19, sharply increased circulating total estradiol (E2) above control values and drastically reduced the biologically active E2 unbound (E2U) to A-E2 in plasma and uterine tissue. This A-E2-induced and--sustained E2U deficiency was not accompanied by similar changes in plasma and tissue progesterone (P), since P decreased similarly in the control and A-E2--treated rats in preparation for parturition. However, in the A-E2 rats the reduction in E2U was accompanied by a small, though significant, decrease in uterine vein prostaglandin F (PGF) during labor. This A-E2--provoked regulatory imbalance significantly altered normal parturition. In comparison with controls, labor in the A-E2 rats was delayed and prolonged by extended intervals between deliveries of individual fetuses of the same litter. The delay in the onset of labor significantly increased the birth weights of the newborn rats. Whether E2U deficiency is directly responsible for the asynchronic myometrial activity that delays and prolongs labor or whether it is mediated by reduced PGF release remains to be determined.

The objective was to study control of mature size by characterizing metacarpal growth plate closure in relation to relevant bone growth-regulating hormones in two breeds exhibiting distinct differences in mature frame size. Thirty-four Suffolk and 34 Dorset ram lambs were slaughtered in pairs within breed at birth, weaning and monthly intervals until 420 d and then bimonthly to 600 d. Plasma growth hormone was depressed to undetectable levels due to the high-energy, ad libitum-fed diet. Plasma insulin-like growth factor-I (IGF-I) rose over the growth period from 116 ng/ml (newborn Suffolk) to a high of 451 ng/ml (420-d Dorset); it appeared to peak at approximately 400 d and then declined to a stable level. Dorsets consistently exhibited higher IGF-I levels. The thyroid hormones exhibited no apparent age association. An age-associated rise was detected for testosterone, but not for estradiol. Mature metacarpal lengths were estimated to be 147.2 and 127.4 mm for Suffolks and Dorsets, respectively. Ninety-five percent of mature length was attained in Suffolks by 226 d and in Dorsets by 165 d. Growth plates, however, did not begin to appear closed until 390 d and closure was not complete in all animals until 480 d, suggesting that metacarpal growth rate was dissociated temporally from growth plate closure. Although growth plate closure likely is controlled by the endocrine system, there were no apparent relationships between circulating hormones and growth plate width, age at closure or zonal divisions within the growth plate, suggesting that the growth plate experiences a very different hormonal environment than what can be measured in the circulating blood.