Reduced fertility associated with low progesterone postbreeding and increased milk urea nitrogen in lactating cows.

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.

Antibody directed against plasma membrane components of equine spermatozoa inhibits adhesion of spermatozoa to oviduct epithelial cells in vitro.

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.

Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of bovine oocytes.

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.

Expression of Mos proto-oncoprotein in bovine oocytes during maturation in vitro.

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.

Temporal distinctions in the synthesis and accumulation of proteins by oocytes and cumulus cells during maturation in vitro of 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.

Effect of chronic infusion of placental lactogen on ovine fetal growth in late gestation.

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.

Characterization of polypeptides synthesized and secreted by oviductal epithelial cell explants obtained from young, fertile and aged, subfertile mares.

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.

Effect of coculture with stallion spermatozoa on de novo protein synthesis and secretion by equine oviduct epithelial cells.

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.

Initiation of transcription and nucleologenesis in equine embryos.

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.

Effects of testosterone on skeletal growth in lambs as assessed by labeling index of chondrocytes in the metacarpal bone growth plate.

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.

Effects of chimerism in sheep-goat concepti that developed from blastomere-aggregation embryos.

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.

De novo protein synthesis by bovine uterine tube (oviduct) epithelial cells changes during co-culture with bull spermatozoa.

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)

Transforming growth factor beta and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.

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.

Effect of fibronectin on early embryo development in cows.

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.

Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle.

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.

Kinetics of placental lactogen in mid- and late-gestation ovine fetuses.

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.

Trophoblastic vesicles and maternal recognition of pregnancy in mares.

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.

Purification, partial characterization, and development of a specific radioimmunoassay for goat placental lactogen.

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.

Differential control of placental lactogen release and progesterone production by ovine placental tissue in vitro.

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.