Secretion of oxytocin and progesterone by ovine corpora lutea in vitro

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.     

Secretion of oxytocin in pregnant and parturient cows: corpus luteum may contribute to plasma oxytocin at term

Plasma oxytocin (OT) concentrations were determined in 14 late-pregnant and parturient Angus-Hereford cows. Jugular and utero-ovarian veins were cannulated for simultaneous withdrawal of blood samples. Samples were collected at 10-min intervals for 6 h once weekly beginning 60-14 days before the date of expected delivery (group 1), or daily 3-7 days before the due date (group 2). In a third group, samples were collected at 15-min intervals every other day for 12 h beginning 1 wk before calving. Basal levels of OT were low, the overall mean for both veins was 0.46 +/- 0.03 microU/ml until a week before parturition, and then increased to 0.77 +/- 0.1 microU/ml (P < 0.02). Spurts of OT occurred intermittently on all days. Interpeak intervals averaged 71.0 +/- 10.7 min until Day -14, and from Day -14 to Day -1 the intervals were 44.0 +/- 5.3 min (P < 0.05). From Day -60 to Day -25 the amplitudes of OT peaks were low and similar in both veins (mean 1.37 +/- 0.1 microU/ml). From Day -14 to Day -1 the peak amplitudes were 3.6 +/- 0.4 microU/ml on average (P < 0.02). During the last 2 wk the utero-ovarian peak of OT was frequently higher than the peripheral peak. In addition, a number of spurts were observed in the utero-ovarian vein only (solo peaks). On the day of parturition during the first stage of labor, peak amplitudes had increased to 7.3 +/- 2.0 microU/ml, and the interpeak intervals had become shorter than before labor (mean 25.1 +/- 2.6 min). A large surge of OT initiated the expulsive stage of labor. Basal levels rose to 43.1 +/- 16 microU/ml and 38.7 +/- 12.6 microU/ml, and peak levels to 77.4 +/- 19.1 microU/ml and 91.6 +/- 21 microU/ml in the jugular and utero-ovarian veins, respectively. Interpeak intervals had decreased to 17.2 +/- 3.3 min (P < 0.05). Oxytocin levels remained high after delivery of the calf until the placenta was expelled. The posterior pituitary was the source of circulating OT during most of gestation and labor, but the solo peaks observed during late gestation in the utero-ovarian vein were probably of luteal origin or possibly of caruncular origin, because near term, both tissues express OT mRNA. Fetal posterior pituitary is another possible source for these peaks. Our conclusions are that during bovine pregnancy, low amplitude spurts of OT are secreted intermittently; near term, both the frequency and peak amplitude of the spurts increase; and during labor, a dramatic increase in plasma OT precedes the expulsion of the calf. The main source of OT is the posterior pituitary, but near term, a utero-ovarian source secretes additional OT into the systemic circulation.     

Secretion rate of prostaglandin F during induced labor in goats

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 elevated by short-term infusions of PGF_2α 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.     

Secretion of PGF2alpha and oxytocin during hyperthermia in cyclic and pregnant heifers

The effects of acute heat stress (HS) and oxytocin (OT) injection on plasma concentrations of PGF2alpha and OT were examined in cyclic (C; n = 15) and pregnant (P; n = 11) dairy heifers. On Day 17 of synchronized estrous cycles, animals were randomly assigned to either thermoneutral (TN; 20 degrees C, 20% RH) or HS (42 degrees C, 60% RH) chambers. The jugular vein of each heifer was cannulated and blood samples collected hourly for 4 h, then every 15 min for an additional 3 h. Oxytocin (100 IU) was injected (IV) 5 h after the start of blood collection. Plasma samples were assayed subsequently for concentrations of 13,14-dihydro-15-keto PGF2alpha (PGFM) and OT. During the 7-h experiment, body temperature of HS heifers reached 41.2 degrees C as compared to 38.5 degrees C in control heifers. Plasma concentrations of PGFM increased (P<0.05) and peaked 30 min after OT injection in C (890 pg/ml) and P (540 pg/ml) heifers. In C heifers, heat stress failed to alter PGFM concentrations either before or after OT injection. In the P group, PGFM concentrations following OT injection tended to be higher in HS heifers were further TN heifers (peak values of 690 vs. 410 pg/ml). Pregnant TN and HS heifers were further classified as responders or non-responders to OT challenge according to a cutoff value for PGFM of 193 pg/ml (overall mean of C heifers minus 1 SD). Five of six HS and one of five TN pregnant heifers were classified as responders (P<0.06). Oxytocin concentrations in plasma prior to injection of exogenous OT were not affected by HS or pregnancy status. It is concluded that in C heifers, acute HS in vivo does not cause any further rise in PGF2alpha secretion. However, in P heifers, HS appears to antagonize suppressive effects of the embryo on uterine secretion of PGF2alpha, as indicated by the larger proportion of P heifers responding to OT challenge.     

Autocrine/paracrine action of oxytocin in pig endometrium

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F(2alpha) secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF(2alpha). Oxytocin increased (P < 0.01) PGF(2alpha) secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF(2alpha) secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF(2alpha) secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF(2alpha) secretion.     

Secretion rate of prostaglandin F during induced labor in goats.

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.     

Effects of pregnancy and labor on oxytocin levels in human plasma and cerebrospinal fluid

In order to investigate the significance of oxytocin in pregnancy and labor, oxytocin concentrations in plasma and cerebrospinal fluid (CSF) were determined using the specific radioimmunoassay. Plasma and CSF samples were obtained from 23 pregnant women (11 pre labor, 12 in labor), 15 nonpregnant women and 4 men at spinal puncture for anesthesia. In males and nongravidas, CSF levels of oxytocin were significantly higher than plasma levels. Plasma levels in pregnant patients pre or in labor were significantly higher than those in nongravidas. No significant difference between CSF levels in prelabor gravidas (mean +/- SE, 9.7 +/- 1.5 mu u/ml) and nongravidas (10.1 +/- 1.2 mu u/ml) was found. However, CSF levels in gravidas in labor (18.6 +/- 2.3 micromicrons/ml were significantly higher than the levels in prelabor gravidas. These results strongly suggest that oxytocin levels in human plasma and CSF are controlled by different mechanisms and that the increased oxytocin could have some specific central actions.     

Secretion patterns of oxytocin and PGF2alpha-metabolite in response to cervical dilatation in cyclic mares

We conducted the present study to establish a standardized method for cervical stimulation without affecting the endometrium, and to investigate the effect on estrous cycle pattern and concentrations of progesterone, oxytocin and PGF2alpha-metabolite of cervical dilatation in the mare. Six healthy Haflinger mares underwent three different treatments (control, insertion, dilatation) on Days 5 and 7 of the cycles in different orders according to a Latin square design. During dilatation, the balloon of the catheter was inflated stepwise every 30s with warm physiological saline to a maximum of 50 ml. At this stage the size of the balloon was 4.5 cm in diameter and 6 cm length. Estrous cycle length was significantly shortened by dilatation when compared to controls (control: 22.8+/-1.7, insertion: 21.8+/-2.5, dilatation: 20.0+/-1.3 days; P<0.05). Concentrations of progesterone at Days 10, 12 and 14 after ovulation were significantly lower in dilatation cycles. Calculation of the area under the curve (AUC) for progesterone secretion from Day 7 to Day 12 also revealed a significant decrease in progesterone secretion in the dilatation group (dilatation: 34.1+/-7.3, insertion: 35.6+/-7.8, control: 39.1+/-5.9 ng/ml; P<0.05). Cervical insertion and dilatation caused a rapid and pronounced increase in plasma concentrations of oxytocin from basal levels (1.0-6.1 pg/ml) to maximum peaks (insertion: 125.5 pg/ml and dilatation: 305.2 pg/ml). The AUC for oxytocin was significantly higher after insertion (Day 5: 858.4+/-469.9; Day 7: 411.9+/-213 pg/ml/h) and dilatation (Day 5: 1697+/-1725; Day 7: 1078.5+/-764 pg/ml/h) when compared to controls (Day 5: 186+/-98; Day 7: 156+/-23.5 pg/ml/h; P<0.05). Manipulations did not cause considerable changes in plasma PGF2alpha-metabolite concentrations. Because cervical dilatation up to a diameter of 4.5 cm did not cause any immediate PGF2alpha release, the luteolytic pathway is unlikely to be responsible for shortening the length of diestrus and the estrous cycle. The present data suggest an involvement of oxytocin in the shortening of the luteal phase in response to cervical manipulation.     

Novel in vitro system for functional assessment of oxytocin action

One of the classical biological actions mediated by the posterior pituitary hormone oxytocin (OT) is contraction of the uterus at parturition. Moreover, premature activation of the OT system is thought to contribute to preterm labor, a major clinical problem in obstetrical practice. However, the molecular mechanisms linking activation of the OT receptor (OTR) to myometrial contractions are not fully understood. Here, we describe an in vitro system that should serve as a useful tool to study this question at a cellular level. The system consists of a collagen lattice contraction assay and two different human myometrial cell lines: a cell clone from a telomerase-immortalized human myometrial cell population (hTERT-C3) as well as a cell line derived from a primary culture of human myometrial cells (M11). Using this approach, we observed that 1 nM OT promoted an almost maximal effect on cell contraction in both cell lines tested. Furthermore, this dose-dependent, OT-induced contraction was antagonized by the specific OTR antagonist d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)(9)]OVT as well as the clinically used antagonist atosiban. This cell line-based contraction assay enables the application of molecular tools aimed at suppressing or overexpressing specific genes. It is also amenable to high-throughput testing approaches. Therefore, this system represents a powerful and improved experimental model that should facilitate the study of the molecular signal transduction pathways involved in the uterotonic actions of OT.     

Study of oxytocin receptor: II. oxytocin and prostaglandin F2 alpha receptors in human myometria and amnion-decidua complex during pregnancy and labor

Oxytocin receptors (OXT-R) and prostaglandin F2 alpha receptors (PGF2 alpha-R) in human myometrium, amnion and decidua during pregnancy and at parturition were examined in an effort to clarify their role in the initiation and maintenance of uterine contractions. The number of binding sites for OXT in myometria showed an increase as gestation advance (Ist trimester v.s. at term; 205 +/- 90 v.s. 671 +/- 98 fmol/mg protein, N = 5, p less than 0.01), and a rapid decrease following the onset of labor (254 +/- 60 fmol/mg protein, N = 5, p less than 0.02). On the other hand the number of PGF2 alpha-R, remained unchanged throughout pregnancy and in labor. This myometrial PGF2 alpha binding capacity was approximately 1/20 to 1/30 that of the OXT binding, while binding affinity was almost equal. The OXT-R both in amnion and decidua, which was 1/6 to 1/7 that in myometrium, showed no significant changes throughout pregnancy or after the onset of labor. Binding affinity for each tissue was almost the same and appeared to increase towards term but no statistical significance was detected. Present data confirmed the presence of OXT as well as PGF2 alpha receptors in the three functionally distinct entities of pregnant human uterus; myometrium, amnion, and decidua. Among the components, the OXT binding increased only in the myometrium during pregnancy, suggesting this tissue specifically responds to OXT. In contrast, there was a constant binding in myometria for PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of inactivation of oxytocin receptor and inhibition of prostaglandin synthesis on uterine oxytocin receptor and gap junction formation and labor in the rat.

Normal term labor is associated with a surge in myometrial oxytocin receptor formation and gap junction development. We have previously shown that inhibition of prostaglandin synthesis by naproxen sodium, 2.0 mg/day, suppressed oxytocin receptor formation but not gap junction formation and prolonged gestation. In this study, we investigated the effects of a specific oxytocin antagonist on oxytocin receptor formation, gap junction formation, and labor in the rat. [Pen1,Phe(Me)2,Thr4,Orn8]oxytocin, a specific oxytocin antagonist, was infused subcutaneously during the last 3 days of pregnancy at 300 micrograms/day. Measurements of myometrial oxytocin receptor concentrations and gap junction formation on days 21 and 22 and days 22-23 (in labor) pregnant uteri showed no significant differences in the Bmax and Kd values between the control and the treated group. Gestation period was not prolonged by the oxytocin antagonist. However, in a separate group of day 23 pregnant rats, the uterine contractile response to 60 mU of oxytocin i.v. was found completely blocked by 10 micrograms of the oxytocin antagonist. These findings suggest that although functional oxytocin receptors did not appear to be essential for the initiation of labor, oxytocin antagonists may still be effective in the prevention of premature contractions. We also examined the effects of a higher dose of naproxen sodium, 5.0 mg/day, on gap junction formation. At this dose, naproxen sodium suppressed both oxytocin receptor and gap junction formation, prolonged gestation, and delayed parturition by 24 h or longer. Prostaglandin appears to be an important regulator or mediator of oxytocin receptor and gap junction formation and plays a critical role in the initiation of labor.     

Regulation of oxytocin action at the receptor level.

Prior exposure of certain target cells to elevated concentrations of a hormone can cause a reduction in the concentration of receptors for that hormone. Following the initial findings (1–6), many examples of the regulation of receptor concentrations by hormones and drugs have been reported (for a partial list see 7). In most cases a hormone regulates its own receptor concentration. There are examples, however, of hormones that can regulate the concentration of receptors for other hormones. The oxytocin (OT) receptor is a prime example of a system that is regulated by estrogens and progestins. Although changes in receptor concentration can play an important role in modifying the response to a hormone, in most systems studied it has not been possible to relate the changes in receptor concentration to an altered biological response to the hormone. Regulation of the OT receptor system, however, has been shown to have a very important bearing in the initiation of labor and parturition and these aspects of OT action are part of the subject of this review.     

Effect of oxytocin infusion during labor: indocyanin green elimination and serum bilirubin levels

The elimination of indocyanin green (IGG) in selected mature newborn babies (n = 50) was investigated on the first postnatal day. The IGG dose was 2 mg/kg body weight. The half time (t1/2), the elimination constant (K2), the dye distribution volume (ml/kg), as well as the level of serum indirect bilirubin on the third postnatal day were measured and calculated. Healthy, mature newborns from spontaneous labor served as controls (n = 14): the two study groups consisted of either growth-retarded (n = 8) or acidotic (n = 8) neonates. According to the management of deliveries, they were spontaneous, assisted by oxytocin drop infusion (n = 10) or under lumbal peridural anaesthesia + oxytocin drop infusion (n = 8). In the acidotic neonates the elimination constant was significantly lower and the half time significantly longer. In the growth retarded newborn babies the difference was not significant. The increase of the level of indirect bilirubin in serum appearing in the acidotic group on the third postnatal day was significantly greater.