Localization of prostaglandin H synthase, prostaglandin dehydrogenase, corticotropin releasing hormone and glucocorticoid receptor in rhesus monkey fetal membranes with labor and in the presence of infection.

Prostaglandins (PGs) play a central role in primate parturition by their actions on uterine contractility and on cervical ripening. Rhesus monkey placentation is hemochorial and the endocrine events surrounding parturition are qualitatively similar to human pregnancy. Although there is an increase in PG production before the onset of labor, little is known about the cellular localization of the PGH synthase (PGHS) or the 15-hydroxy PG dehydrogenase (PGDH) in the fetal membranes of nonhuman primates and whether it changes at term in spontaneous labor or during preterm labor associated with infection. Placental corticotropin releasing hormone (CRH) and the glucocorticoid receptor (GR) have also been implicated as mediators in parturition by virtue of their roles in PG production. We utilized immunohistochemical methods to localize the inducible isoform PGHS-2, PGDH, GR and CRH in rhesus monkey amnion, chorion and attached decidua. Tissues were obtained at cesarean section during late pregnancy, in spontaneous labor at term and in premature labor induced by Group B streptococcal intraamniotic infection. Specific staining for immunoreactive (ir)-PGHS-2 was observed in amnion epithelial and mesenchymal cells and to a lesser extent in chorion and decidua. In contrast, ir-PGDH was localized primarily to the extravillous trophoblast layer of chorion. GR was localized to both the cytoplasm and nucleus of amnion epithelial cells, subepithelial fibroblasts, chorion trophoblasts and in decidua. Immunostaining for CRH was found in amnion and in scattered decidual cells but was most intense in the chorion trophoblast layer. There was no demonstrable change in this overall pattern of immunostaining in association with the onset of labor at term except for a decrease in staining for ir-PGDH in chorion. Experimental Group B streptococcal chorioamnionitis resulted in preterm labor and extensive necrosis of extravillous trophoblast cells with subsequent loss of chorionic ir-PGDH and relative sparing of ir-PGHS-2 in amnion epithelium which favors the net production of PGs. The expression pattern of these effectors in the rhesus monkey fetal membranes points to a functional role of PGs and glucocorticoids in the process of term and preterm parturition which is similar to that in human pregnancy.

Progesterone receptor localization and isoforms in myometrium, decidua, and fetal membranes from rhesus macaques: evidence for functional progesterone withdrawal at parturition.

OBJECTIVE: It is not known whether withdrawal of progesterone (P) action is a prerequisite for parturition in women or in nonhuman primates because concentrations of circulating progesterone or progesterone receptors (PR) in myometrium and decidua do not decrease before delivery. To examine this potentially important regulatory mechanism, we determined PR isoforms, PR localization, and mRNA in myometrium, decidua, and fetal membranes from rhesus monkeys during pregnancy and in spontaneous labor at term. METHODS: Gestational tissues were obtained midpregnancy (day 80-100), late pregnancy (day 130-145), and during spontaneous labor at term (day 161-167). Samples of rhesus monkey myometrium, decidua, chorion-decidua, and amnion were collected and analyzed for total nuclear and cytosolic PR by competitive binding assay. Progesterone receptor isoforms were identified and quantified by Western blot analysis, and PR mRNA was determined by a specific ribonuclease protection assay. Nuclear PR was localized by immunohistochemistry with monoclonal anti-PR (JZB39) after microwave stabilization. RESULTS: Myometrium and decidua showed no change in total PR during pregnancy and labor. Nuclear PR was not detected in fetal membranes by binding assay but was localized in amnion epithelial and mesenchymal cells and in chorion laeve cytotrophoblasts by immunohistochemistry. Staining for PR was substantially less by serial antibody dilution in fetal membranes than in decidua. Message for PR was confirmed in all tissues analyzed. A significant (P <.05) shift in the ratio of PR isoforms (from PR-B dominance at midpregnancy to PR-A dominance in labor) was observed in myometrium but not in decidua. Both PR-A and PR-B isoforms and PR nuclear staining were nearly undetectable in amnion obtained during labor. CONCLUSION: A shift to PR-A dominance in myometrium at term together with a loss of PR in fetal membranes provides evidence for a functional progesterone withdrawal mechanism, which may facilitate the initiation of parturition in primates.

Maternal or fetal origin of rhesus monkey (Macaca mulatta) amniotic fluid leukocytes can be identified by polymerase chain reaction using the zinc finger Y gene.

Leukocytes can be found in substantial numbers within the intrauterine tissues and amniotic fluid of women, and play a central role in the pathophysiology of infection-related preterm labor by their production of proinflammatory mediators. It remains unclear whether these leukocytes represent a fetal immune response, a maternal response, or a combination of the two. The objective of this study was to develop a test in the rhesus monkey (Macaca mulatta) suitable for determining the percentage of male fetal cells present in a population of leukocytes recovered from blood or amniotic fluid. We found inadequate specificity for rhesus monkey cells using commercial human Y-chromosome paint kits (fluorescence in situ hybridization (FISH)). Human-specific primers for the repetitive Y chromosome DYZ-1 locus employed in the polymerase chain reaction (PCR) produced an unacceptable percentage of false positives. However, we successfully developed a PCR-based test using rhesus-specific primers for the zinc finger Y (ZFY) locus. Densitometry of PCR products from known ratios of male and female adult peripheral leukocytes generated a linear standard curve which provided quantitative results and required only 400 cells per sample. The rhesus beta globin (RBG) gene served as an internal control. The PCR test correctly discriminated the sex of peripheral leukocytes in 20 adult males, 20 adult females, two male fetuses, and one female fetus. Serial samples of amniotic fluid from four chronically catheterized rhesus monkeys bearing male fetuses were used to confirm the utility of this assay for quantifying fetal cells in amniotic fluid. In conclusion, we have developed a PCR test which is suitable for distinguishing male from female cells in adult and fetal blood and in amniotic fluid, which lends itself to a variety of diagnostic and biologic applications in the rhesus monkey and potentially in other nonhuman primates.

Indomethacin blocks interleukin 1beta-induced myometrial contractions in pregnant rhesus monkeys.

OBJECTIVE: We sought to determine whether blockade of prostaglandin synthesis with indomethacin prevents interleukin 1beta-induced increases in uterine contractions in a nonhuman primate model. STUDY DESIGN: Maternal and fetal vascular catheters, intra-amniotic fluid pressure catheters, and fetal electrocardiographic and myometrial electromyographic electrodes were implanted in 11 rhesus monkeys at 124 +/- 2 days' gestation (term, 167 days). After postsurgical stabilization (136 +/- 2 days) indomethacin 50 mg was administered orally twice daily for 5 days (n = 6). On day 3 human recombinant interleukin 1beta 10 microg was infused into the amniotic cavity over 2 hours. Five days after the last indomethacin dose the study was repeated without indomethacin treatment. Uterine activity was continuously monitored and quantified as the hourly contraction area (millimeters of mercury. seconds per hour) in the experimental group and a control group (n = 5) that received interleukin 1beta alone. At timed intervals amniotic fluid was sampled for leukocyte counts and assayed for prostaglandin E(2) and F(2alpha), the inflammatory cytokines interleukin 1beta, interleukin 6, interleukin 8, tumor necrosis factor alpha, and interleukin 1 receptor antagonist by specific assays. RESULTS: Uterine activity was increased severalfold from baseline after interleukin 1beta infusion alone and in the absence of indomethacin treatment (P <.05). There was no increase in uterine contractility when interleukin 1beta was infused concurrently with indomethacin treatment. Concentrations of amniotic fluid leukocytes and cytokines increased significantly after interleukin 1beta infusion in both the presence and absence of indomethacin. Amniotic fluid prostaglandins E(2) and F(2alpha) were suppressed during indomethacin treatment but rose significantly after interleukin 1beta infusion in the absence of indomethacin. Except for higher interleukin 6, cytokine levels were unaltered by indomethacin. CONCLUSIONS: After interleukin 1beta infusion, indomethacin blocked the development of uterine activity. Amniotic fluid prostaglandins were suppressed by indomethacin treatment, but cytokines and leukocytes were not. These results suggest that prostaglandins or possibly other indomethacin-suppressible compounds stimulate uterine activity after interleukin 1beta infusion in late-gestation rhesus monkeys or that indomethacin has direct tocolytic effects.

Amniotic fluid prolactin is decreased by experimental intrauterine infection or interleukin-1beta infusion but not via prostaglandins in pregnant rhesus macaques.

Amniotic fluid contains a high concentration of prolactin produced and secreted by the decidua. In vitro models have suggested that bacterial products inhibit prolactin secretion by decidual cells. To further examine this potentially important regulatory mechanism in the whole animal, chronically instrumented pregnant rhesus monkeys were prepared. Experimental infection was induced by intraamniotic or choriodecidual inoculation of 10(3)-10(6) group B streptococcus. Alternatively, interleukin (IL)-1beta was infused into the amniotic cavity. Finally, indomethacin was coadministered with IL-1beta to block the production of prostaglandins (PGs). The average prolactin level prior to inoculation (0 h) equaled 34.0 +/- 6.4 microg/ml. There was a 40% decrease in prolactin by 37 h postinfection (n = 6) and a 71% decrease between 61 and 72 h postinfection (n = 3, p < 0.01 vs. before infection). Infusion of IL-1beta also caused a decrease in amniotic fluid prolactin. There was a 42% decrease in prolactin between 0 and 24 h postinfusion (p < 0.05) and a 66% decrease between 25 and 72 h after IL-1beta infusion (p < 0.05; n = 6). Coadministration of indomethacin with IL-1beta prevented the accompanying increase in PGs but did not prevent the decrease in prolactin (n = 5). Amniotic fluid prolactin levels in untreated monkeys were stable and without a prepartum decline during the sampling period from 130 to 166 days of gestation. In summary, intrauterine bacterial infection decreases amniotic fluid prolactin, and IL-1beta mimics this effect. The effect of IL-1beta on amniotic fluid prolactin does not appear to be mediated by PGs and may involve a direct effect of IL-1beta on decidual cells.

The ratio of progesterone receptor isoforms changes in the monkey corpus luteum during the luteal phase of the menstrual cycle.

Recent evidence suggests that progesterone is required for ovulation, luteinization, and the maintenance of luteal structure and function in primates. Progesterone action is mediated by intracellular progesterone receptors (PRs), and PRs are detectable by immunocytochemistry in the monkey corpus luteum. However, changes in total luteal PR and PR isoform expression have not been quantitated in the corpus luteum during its life span in the menstrual cycle. This study was initiated to identify and quantify PR isoforms in the macaque corpus luteum throughout the luteal phase of the natural cycle by means of Western blotting. Several antibodies generated against the human PR recognized two bands of consistent molecular weights in monkey tissues, and these bands comigrated with PR-A and PR-B from human T47D cells. Taken together, these data suggest that the two proteins identified were macaque PR-A and PR-B. The estimated molecular weights of monkey PR-A and PR-B were approximately 90,000 and 120,000, respectively. PRs were detected in a variety of macaque tissues, including the endometrium, whole ovary, and decidua, but not in spleen, which is PR-negative by other techniques. Whereas PR-A was the predominant isoform observed in endometrium and decidua, PR-B predominated in the ovary without a dominant follicle or corpus luteum as well as in the corpus luteum. In luteal tissue, PR-A levels decreased (p < 0.05) over the course of the luteal phase, while PR-B levels were unchanged. Hence the ratio of PR-B to PR-A (PR-B:PR-A) increased (p < 0.05) from early to very late luteal phase. Since PR-B:PR-A can alter gene expression in response to progestins and antiprogestins in vitro, the temporal changes in PR-B:PR-A in the monkey corpus luteum may contribute to functional differences in luteal responses to progesterone and other steroids in vivo.

Inhibition and augmentation of progesterone production during pregnancy: effects on parturition in rhesus monkeys.

OBJECTIVES: Uterine quiescence during mammalian pregnancy is attributed to progesterone. However. systemic progesterone levels remain elevated in primates before parturition. Epostane, a selective 3beta-hydroxysteroid dehydrogenase inhibitor, and progesterone (with or without epostane) were administered to late pregnant rhesus monkeys to clarify the role of progesterone in primate parturition. STUDY DESIGN: On days 122 to 132 of gestation (term 167 days), 11 rhesus monkeys (Macaca mulatta) with timed pregnancies were divided into three treatment groups: (1) epostane alone (10 mg/kg subcutaneously), (2) epostane with progesterone subcutaneously in Silastic silicone rubber capsules, and (3) progesterone implants only with no surgical instrumentation. Maternal and fetal blood and amniotic fluid were sampled for progesterone, estrone, estradiol, cortisol, testosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, and amniotic fluid was sampled for prostaglandins E2 and F2alpha. Uterine activity was monitored continuously by electromyography and intraamniotic pressure. Cervical status was assessed by a modified Bishop's score. Production of prostaglandins E2 and F2alpha by amnion was determined by tissue superfusion. The group of three noninstrumented monkeys, which received only progesterone Silastic silicone rubber implants subcutaneously at 146 to 148 days, were observed until spontaneous vaginal delivery. RESULTS: Epostane reduced maternal and fetal progesterone levels by 75% and 50%, respectively, followed by increased uterine activity and cervical ripening within 24 hours and vaginal delivery within 48 hours. Amniotic fluid progesterone decreased to undetectable levels. Progesterone implants prevented the epostane-induced decrease in maternal and fetal progesterone levels and the associated myometrial and cervical changes until the implants were removed. Alterations in other steroid hormones were consistent with inhibition of 3beta-hydroxysteroid dehydrogenase. Amniotic prostaglandin E2 production was increased sixfold by epostane (p < 0.05) but did not reach the high levels normally seen at spontaneous parturition. Animals that received progesterone implants alone had markedly elevated circulating progesterone concentrations yet were delivered spontaneously at term (range 163 to 167 days). CONCLUSIONS: Progesterone withdrawal induces preterm labor and delivery (which can be blocked by progesterone substitution) but exogenous progesterone, even in substantial quantities, does not prevent parturition at term.

Fetal and maternal endocrine responses to experimental intrauterine infection in rhesus monkeys.

OBJECTIVE: Our purpose was to describe the temporal and quantitative relationships among intrauterine infection, fetal-placental steroid biosynthesis, and preterm labor in a nonhuman primate model. STUDY DESIGN: On approximately day 130 of gestation (term 167 days) chronically instrumented rhesus monkeys (Macaca mulatta) were infected with 10(6) colony-forming units of group B streptococci either by intraamniotic (n = 4) or choriodecidual (n = 2) inoculation. As controls, four additionally chronically instrumented noninfected monkeys were followed up to spontaneous parturition. Amniotic fluid and maternal and fetal arterial blood were serially sampled in all monkeys (both before and after infection) for progesterone, estrone, estradiol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, and cortisol by specific radioimmunoassays, and uterine activity was continuously recorded. RESULTS: Spontaneous parturition was preceded by gradual and significant increases in the plasma concentrations of fetal dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione and fetal and maternal levels of estrone, estradiol, and progesterone but not by changes in cortisol. In contrast, infection-associated parturition (either intraamniotic or choriodecidual) was characterized by abrupt increases in fetal dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, progesterone, and cortisol but not by increases in maternal or fetal estrone or estradiol. Infection-associated steroid changes occurred concurrently with or after increases in uterine activity. CONCLUSION: Infection-associated preterm parturition is associated with dramatic increases in fetal adrenal steroid biosynthesis but not by corresponding increases in placental estrogen biosynthesis. This suggests that fetal stress in accompanied by placental dysfunction and that infection-associated parturition is not dependent on the increased estrogen biosynthesis observed in spontaneous parturition.

Interleukin-1 beta intra-amniotic infusion induces tumor necrosis factor-alpha, prostaglandin production, and preterm contractions in pregnant rhesus monkeys.

OBJECTIVE: To describe the temporal and quantitative consequences of intra-amniotic interleukin-1 beta infusion in a nonhuman primate model. METHODS: On days 128-138 of gestation (term 167 days), four chronically instrumented rhesus monkeys (Macaca mulatta) underwent serial intra-amniotic infusions of 2, 5, and 10-20 micrograms recombinant human interleukin-1 beta. Each infusion was for 2 hours, and subsequent infusions were at least 48 hours later. Amniotic fluid was sampled serially both before and after infusion for interleukin-1 beta, tumor necrosis factor-alpha (TFN-alpha), and prostaglandin (PG) E2 and F2 alpha by specific assays, and uterine activity in each monkey was recorded continuously. RESULTS: Intra-amniotic concentrations of interleukin-1 beta rose dramatically after infusion. This rise was rapidly followed by the appearance of TNF-alpha in the amniotic cavities of all animals, with maximal levels reached 5 hours after the initiation of the infusion. Both interleukin-1 beta and TNF-alpha were rapidly cleared from the amniotic fluid and returned to baseline levels by 24-48 hours. Increases in PGE2 and F2 alpha paralleled those of the two cytokines but remained elevated for the duration of the experiments. The stimulation of uterine contractility from a pre-infusion level of 200 mmHg. seconds/hour to 6000 mmHg. seconds/hour occurred an average of 6-10 hours after interleukin-1 beta infusion. These stimulations were transient, usually abating by 22 hours after infusion, and did not result in frank labor. CONCLUSION: In the rhesus monkey, intra-amniotic infusion of interleukin-1 beta rapidly induces production of intra-amniotic TNF-alpha as well as PGE2 and F2 alpha, followed by uterine contractility. Uterine activity diminishes as cytokine levels return to pre-infusion levels, even in the presence of elevated intraamniotic PG levels. Tumor necrosis factor-alpha may act synergistically with interleukin-1 beta in the pathophysiology of cytokine-related preterm labor.

An experimental model for intraamniotic infection and preterm labor in rhesus monkeys.

OBJECTIVE: Our purpose was to describe the temporal and quantitative relationship between intraamniotic infection and preterm labor in a nonhuman primate model. STUDY DESIGN: On day 130 of gestation (term 167 days) four chronically instrumented rhesus monkeys (Macaca mulatta) were infected with an intraamniotic inoculation of 10(6) colony-forming units of group B streptococci. Four additional noninfected monkeys were followed up to spontaneous parturition as controls. Amniotic fluid was serially sampled in all monkeys both before and after inoculation for bacterial growth, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, prostaglandin E2, and prostaglandin F2 alpha, and uterine activity was continuously recorded. RESULTS: Increases in uterine contractility occurred 28 hours (range 14 to 40 hours) after inoculation and were preceded by increases in amniotic fluid cytokines and prostaglandins. Intraamniotic concentrations of tumor necrosis factor-alpha, interleukin-6, and interleukin-1 beta all rose dramatically 9, 15, and 18 hours after infection and 10 to 20 hours before increases in uterine contractility. In spontaneous parturition only interleukin-6 concentrations rose moderately (from 0.1 to 1.2 ng/ml). Increases in prostaglandin E2 and prostaglandin F2 alpha paralleled those of the cytokines. Peak prostaglandin concentrations in intraamniotic infection exceeded by severalfold concentrations seen in spontaneous parturition (16,046 pg/ml vs 2765 pg/ml for prostaglandin E2, p < 0.05; and 5547 pg/ml vs 708 pg/ml for prostaglandin F2 alpha, p < 0.05). In spite of intraamniotic none of the monkeys were febrile or had peripheral leukocytosis at the onset of labor. CONCLUSION: In the rhesus monkey, after intraamniotic infection, there is a predictable and sequential increase in amniotic fluid tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, followed by increases in prostaglandin E2 and prostaglandin F2 alpha. These increases all occur before an increase in uterine contractility and before clinical signs of infection. Our data provide evidence for a cause-and-effect relationship between intraamniotic infection and preterm labor and support the utility of measuring interleukin-6 or other cytokines in the diagnosis of intraamniotic infection.

Induction of interleukin-1 receptor antagonist in rhesus monkeys after intraamniotic infection with group B streptococci or interleukin-1 infusion.

OBJECTIVE: Interleukin-1 receptor antagonist is a natural inhibitor of interleukin-1, a cytokine implicated in the initiation of preterm labor after intraamniotic infection. The effects of intraamniotic infection and interleukin-1 infusion on the appearance of interleukin-1 receptor antagonist in amniotic fluid and fetal and maternal plasma were assessed with a monkey model. STUDY DESIGN: On day 130 of pregnancy four chronically catheterized rhesus macaques received intraamniotic inoculations of group B streptococci, three monkeys received intraamniotic infusions of recombinant human interleukin-1 beta, and three monkeys received buffered saline solution infusions. At timed intervals samples of amniotic fluid, fetal plasma, and maternal plasma were assayed for interleukin-1 beta and interleukin-1 receptor antagonist by immunoassays. Uterine activity was continuously monitored by intraamniotic pressure catheters and by electromyographic activity. RESULTS: Interleukin-1 receptor antagonist, but not interleukin-1 beta, was present in the amniotic fluids of all monkeys before intervention. Infection induced the appearance of interleukin-1 beta and an increase in interleukin-1 receptor antagonist in the amniotic fluid. Interleukin-1 beta infusion resulted in a similar increase in the intraamniotic concentration of interleukin-1 receptor antagonist. Both infection and interleukin-1 beta infusion were followed by the transient appearance of interleukin-1 receptor antagonist in the plasma of all fetuses. The subsequent decrease in plasma levels was paralleled by increased amniotic fluid levels of interleukin-1 receptor antagonist. Interleukin-1 beta and interleukin-1 receptor antagonist were not detected in maternal plasma. Both infection and interleukin-1 infusion induced preterm labor in all treated animals. CONCLUSIONS: Interleukin-1 receptor antagonist is a normal component of monkey amniotic fluid. Intraamniotic infection or the appearance of interleukin-1 beta in the amniotic fluid results in increased production of interleukin-1 receptor antagonist. Under physiologic conditions interleukin-1 receptor antagonist in amniotic fluid may inhibit interleukin-1-induced preterm labor.

Prostaglandin production during spontaneous labor and after treatment with RU486 in pregnant rhesus macaques.

The production of prostaglandins from amnion, chorion, decidua, and myometrium was studied in a superfusion system to determine the level of prostaglandin production during late pregnancy, during spontaneous labor, and after in vivo treatment with RU486. Tissues were divided into three groups: those from pregnant control animals, those from animals receiving RU486 (20 mg/kg/day for 3 days) in vivo, and those from animals in spontaneous term labor. Tissues were collected at cesarean section and placed in the superfusion system. After a 30-min equilibration period, fractions of media were collected after passing through the tissue chambers and were assayed for prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2). The results showed a significant increase in PGF2 alpha from decidua of animals treated with RU486 compared to pregnant controls (pregnant: 3.01 +/- 0.45 ng/g/min; RU486: 4.45 +/- 0.28 ng/g/min; p < 0.05). There was a dramatic increase in PGE2 production by amnion from animals in spontaneous labor but not after RU486 treatment (pregnant: 0.21 +/- 0.06 ng/g/min; RU486: 0.56 +/- 0.09 ng/g/min; spontaneous labor: 5.83 +/- 0.43 ng/g/min; p < 0.01). We conclude that while progesterone is important for maintaining uterine quiescence during pregnancy, progesterone receptor blockade by RU486 does not lead to an increase in PGE2 production by amnion as is shown during normal spontaneous labor.

Plasma oxytocin and nocturnal uterine activity: maternal but not fetal concentrations increase progressively during late pregnancy and delivery in rhesus monkeys.

OBJECTIVE: The purpose of the study was to determine whether rising maternal or fetal plasma oxytocin concentrations could be responsible for the increasing levels of nocturnal uterine activity on the nights preceding delivery. STUDY DESIGN: Chronically catheterized pregnant rhesus monkeys were exposed to a 16-hour light, 8-hour dark photoperiod (dark 11 PM to 7 AM). Uterine activity and maternal arterial plasma oxytocin concentrations were measured concurrently at weekly intervals in late gestation, on the night preceding term delivery (158 to 167 days, n = 4), and during delivery (149 to 170 days, n = 6). Fetal carotid arterial plasma oxytocin levels were measured during episodes of nocturnal uterine activity in six animals. The effect of oxytocin infusions into the fetus (30 to 480 ng/kg/hr) on uterine activity and on maternal and fetal plasma oxytocin levels was also determined (n = 3). RESULTS: Maximal nocturnal oxytocin concentrations in the maternal plasma rose progressively during late gestation from 9.9 +/- 3.5 pg/ml at 130 to 139 days to 28.7 +/- 9.8 pg/ml on the night preceding term delivery (p < 0.005); a significant increase in nocturnal uterine activity accompanied this rise (p < 0.001). Maternal oxytocin concentrations were elevated during labor and increased further at delivery (62.5 +/- 5.5 pg/ml, p < 0.05). There was no increase in fetal plasma oxytocin during nocturnal uterine activity (3.1 +/- 0.2 pg/ml) or during labor. Fetal oxytocin infusions raised fetal plasma oxytocin concentrations sixtyfold but had no effect on maternal plasma oxytocin concentrations or on uterine activity. CONCLUSIONS: Elevated maternal plasma oxytocin concentrations are responsible, at least in part, for the increasing magnitude of nocturnal uterine activity episodes as term approaches and for the elevated uterine activity before delivery at night. Fetal plasma oxytocin does not contribute to nocturnal uterine activity or to maternal plasma oxytocin concentrations.

Breed differences in circulating equine relaxin.

Equine relaxin has been previously determined in a small number of pregnant Thoroughbred mares. To better define the normal pregnancy pattern of relaxin, the current study reports on a much larger number of mares. It also was designed to determine if all equids have the same gestational pattern of relaxin secretion. Plasma samples were collected weekly in 24 Standardbred mares, every 7-10 days in 10 pony mares, and daily in late pregnancy from 16 burros. Standardbreds had higher concentrations of relaxin than that reported for Thoroughbreds during most of gestation and did not exhibit the midpregnancy nadir in relaxin concentrations observed in Thoroughbreds. Relaxin concentrations in Standardbreds showed a small but steady decline from Day 150 until delivery. Pony mares had lower relaxin concentrations throughout pregnancy than other mares and had continuously increasing concentrations during gestation. Burros had relaxin concentrations intermediate to ponies and other mares in late gestation. Burros induced to foal with oxytocin showed a sharp increase in relaxin concentrations. No effect of the sex of the offspring was observed in relaxin profiles in Standardbred mares. Each of three Standardbreds with abnormal termination of pregnancy exhibited abnormally low relaxin concentrations at some point in the gestation prior to termination of the pregnancy. Thus, relaxin may be an indicator of placental functioning and used to assess at-risk pregnancies in mares.

Comparison of plasma oxytocin and catecholamine concentrations with uterine activity in pregnant rhesus monkeys.

Pregnant rhesus monkeys exhibit diurnal changes in uterine activity (UA), with episodes of increased UA during the early hours of darkness. The estrogenic environment during late pregnancy serves a permissive role in the maintenance of nocturnal UA episodes and may involve myometrial interactions with oxytocin (OT) and/or alpha-adrenergic stimuli. In the present study we have used chronically catheterized pregnant rhesus monkeys to measure diurnal changes in maternal plasma OT, epinephrine, norepinephrine, and dopamine. We also determined the effects of infusing an OT antagonist (ORF 22164) and the alpha-adrenergic antagonist phentolamine on nocturnal UA episodes. Animals were exposed to a 16-h light, 8-h dark photo-period, with the hours of darkness between 2300-0700 h. Maternal plasma samples were collected at 3-h intervals for 36 h and analyzed by RIA for OT and by high performance liquid chromatography for catecholamines. Plasma OT was correlated with UA in animals that displayed nocturnal UA episodes (r = 0.76; P less than 0.01). Maximal OT concentrations occurred at 2400 h in these animals; plasma OT was higher during the hours of darkness compared to levels during the light phase (10.4 +/- 1.9 and 3.0 +/- 0.3 pmol/L, respectively; n = 4). Some animals did not display nocturnal episodes of increased UA and showed no increase in OT concentrations during the hours of darkness. Maternal plasma catecholamine concentrations were not correlated with nocturnal UA and were maximal during the light phase. Nocturnal UA was abolished within 30 min of infusion of the OT antagonist, but phentolamine infusions had no effect on nocturnal UA. We conclude that 1) changes in maternal plasma catecholamine concentrations are not involved in the generation of nocturnal UA; 2) the presence of episodes of increased UA at night results from increased maternal plasma OT concentrations; and 3) the absence of nocturnal UA in some animals can be explained by a reduced level of OT secretion.

Antagonism of oxytocin in rats and pregnant rhesus monkeys by the novel cyclic hexapeptides, L-366,682 and L-366,948.

Two cyclic hexapeptides unrelated in chemical structure to oxytocin (OT) were shown in vivo to be antagonists of the contractile action of OT on the uterus. In anesthetized rats challenged with OT (1 micrograms/kg) administered as an i.v. bolus, L-366,682 [cyclo-(L-Pro-D-Trp-L-Ile-D-pipecolic acid-L-pipecolic acid-D-His)] and L-366,948 (D-2-naphthyl-alanine in place of D-Trp) were equipotent with AD50 values of about 100 micrograms/kg i.v. At doses of L-366,682 or L-366,948 causing approximately 90 to 95% block (approximately the AD95 dose) of OT, the duration of action of the antagonists exceeded 145 min. Both compounds exhibited selectivity in the rat, as a dose of either at 300 micrograms/kg i.v. shifted the dose-response for OT-induced uterine contraction to the right by approximately 5-fold but did not affect the dose-response to prostaglandin F2 alpha. Furthermore, neither compound, at a dose of 3 mg/kg i.v., antagonized the action of arginine vasopressin acting at V-1 (pressor effect in pithed rats) or V-2 (antidiuretic) receptors. In conscious, freely moving, pregnant rhesus monkeys, L-366,948 or L-366,682 given i.v. or s.c. were effective antagonists of uterine contractions elicited by an infusion of OT. OT- or arginine vasopressin-like agonist activity was not observed in any of the in vivo models. It is concluded that L-366,682 and L-366,948 act in vivo as reasonably potent, long-acting and selective antagonists at OT receptors in the rat and rhesus uterus.

Amniotic fluid lipoxygenase metabolites during spontaneous labor and after RU486 treatment during late pregnancy in rhesus macaques.

To determine changes in amniotic fluid (AF) lipoxygenase metabolites prior to spontaneous labor and after RU486 administration, we implanted AF and vascular catheters and myometrial electromyographic (EMG) electrodes in 8 rhesus macaques at 120-130 days of pregnancy (term = 167 days). Four animals had AF samples taken serially until they delivered their infants normally at term. The other four animals received RU486 (20 mg/kg/day) for 3 days. AF samples were collected every 2-3 days and at 12 hour intervals for 72 hours before and after treatment with RU486. Uterine activity was monitored continuously. LTB4, 5-HETE and 15-HETE were measured by radioimmunoassay. In untreated animals, LTB4 and 5-HETE concentrations in AF increased significantly (P less than 0.05) 4 days before delivery with no change in 15-HETE. After RU486, mean levels of LTB4 and 5-HETE were increased although the difference was not statistically significant. No change in 15-HETE levels was observed. In conclusion, LTB4 and 5-HETE increase in AF before the onset of spontaneous labor. Progesterone receptor blockade by RU486 does not reproduce the changes in AF lipoxygenase metabolites observed during normal parturition.

Uterine estrogen receptors are increased by RU486 in late pregnant rhesus macaques but not after spontaneous labor.

Progesterone withdrawal as a mechanism for parturition in primates is controversial. The progesterone antagonist RU486, given in late pregnancy to rhesus monkeys at a dose of 47 mmol/kg.day (20 mg/kg.day), causes an increase in uterine activity, but not the expected increase in amniotic fluid prostaglandins or cervical dilatation. We, therefore, studied the effect of RU486 on estrogen receptor (ER) localization and concentration in reproductive tract tissues in rhesus monkeys during late gestation and after spontaneous labor at term. Distribution of ER in pregnant uterine tissues was studied by immunocytochemical techniques and quantified by a biochemical assay, both of which employed a monoclonal antibody specific for ER. ER was not present in amnion and chorion by immunocytochemical investigation; however, a significant increase in receptor staining was seen in decidua and myometrium after RU486 treatment compared to that in both pregnant control tissues and parturient tissues. Sucrose gradient assay of nuclear (n) and cytosolic (c) ER revealed a low level of ER (expressed as fmol of estradiol bound/mg of DNA) in pregnant and parturient decidua (pregnant: nER = 7.3 +/- 2.4, cER = 17.1 +/- 6.4; parturient, nER = 7.7 +/- 3.1, cER = 16.4 +/- 8.8) and myometrium (pregnant: nER = 21.7 +/- 4.1, cER = 20.8 +/- 5.3; parturient: nER = 30.0 +/- 2.8, cER = 10.7 +/- 6.7). In contrast, tissues collected from RU486-treated animals contained high levels of ER in decidua (nER = 52.3 +/- 16.8, cER = 240.5 +/- 145.3) and myometrium (nER = 77.0 +/- 19.2; cER = 66.5 +/- 31.6). We conclude that 1) the increase in ER in decidua and myometrium after RU486 treatment is the result of a decrease in the inhibitory action of progesterone on ER and documents the progesterone receptor antagonism by RU486 during induced myometrial contractility in late pregnant rhesus monkeys; 2) the absence of ER from amnion and chorion indicates that the normally observed increase in prostaglandin production by rhesus fetal membranes during labor is not mediated by ER; and 3) the absence of a change in the concentration of ER in decidua and myometrium from pregnant control monkeys and those in spontaneous labor indicates that an increase in ER (and, by inference, a withdrawal of receptor-mediated progesterone inhibition) is not part of the normal events in preparation for parturition in primates.

The effect of relaxin infusion on prolactin and growth hormone secretion in monkeys.

To test the hypothesis that relaxin may affect pituitary hormone secretion, synthetic human relaxin was infused into cycling and pregnant rhesus monkeys. Doses ranging from 0.154-1540 ng/kg.min were calculated to achieve circulating relaxin concentrations of 1 pM to 10 nM. Low (0.154 and 1.54 ng/kg.min), intermediate (15.4 and 154 ng/kg.min), and high (1540 ng/kg.min) doses of relaxin were infused for 15 min each hour into ovulating monkeys at the midluteal phase of the menstrual cycle in two separate experiments. Serum GH and PRL were measured by RIA, and serum relaxin was determined by enzyme-linked immunosorbent assay. Relaxin was undetectable in peripheral plasma during the control saline infusion and during infusion of the lowest dose of relaxin. Serum relaxin levels reached 0.011, 0.119, 0.965, and 13.0 nM with 1.54, 15.4, 154, and 1540 ng/kg.min, respectively. Serum GH was significantly elevated over basal levels upon infusion of relaxin from 1.54-1540 ng/kg.min; however, a plateau was observed with the intermediate doses, and a decrease in the magnitude of the response was observed at the highest dose. Serum PRL increased over basal levels with 15.4 and 154 ng/kg.min, but there was no difference in the magnitude of the increase between these doses. PRL levels during infusion of the highest dose of relaxin were similar to control levels. These data suggest that relaxin can stimulate secretion of GH and PRL in cycling monkeys within a defined dose range, but that a decrease in pituitary responsiveness occurs at higher doses. One high dose of relaxin (2600 ng/kg.min) was infused for 1 h into the maternal and then the fetal circulations of chronically catheterized and tethered pregnant monkeys between 120-140 days gestation. Upon infusion of relaxin into the maternal circulation, there was a significant elevation of PRL in the mother but not the fetus. Upon infusion of relaxin into the fetus, there was no consistent change in PRL secretion in either the mother or the fetus. In conclusion, relaxin may have a heretofore undescribed role in pituitary physiology during the menstrual cycle and in pregnancy.