Evidence for oxytocin receptors in cultured bovine luteal cells.

Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Cultured cells from bovine corpora lutea (CL) at different stages (Days 3-5, 8-12, and 15-18 of the estrous cycle) were examined for OT receptors by a radioreceptor assay using the 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2(9)] -vasotocin. Binding specificity was demonstrated in displacement studies with various related peptides. Scatchard analysis revealed the presence of a binding site with an association constant of Ka = 2.6 x 10(9) M-1 and a capacity of 5.9 fmol/micrograms DNA. Additionally, in 50% of the experiments (n = 6) two different binding sites were observed. The Ka of the high-affinity site was 2.6 x 10(10) M-1; its capacity was 0.73 fmol/micrograms DNA. The low-affinity site had an apparent Ka of 4.9 x 10(8) M-1 and a capacity of 8.8 fmol/micrograms DNA. Observation of one versus two binding sites related neither to the assay conditions nor to the state of the individual CL used for the cell culture and therefore appeared to reflect individual variation within the OT receptor population. Significant binding of OT was observed at all luteal stages. OT binding was maximal at the mid-luteal stage (Days 8-12). We conclude that a direct action of OT on the bovine CL is mediated by the OT receptor, supporting the hypothesis that luteal OT plays an important physiological role in the regulation of progesterone release and/or other luteal functions in a paracrine or autocrine fashion.     

Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes

Continuous intravenous infusion of oxytocin (3 micrograms/h) between Days 13 and 21 after oestrus delayed return to oestrus by 7 days (length of cycle 23.3 +/- 0.6 days compared to 16.6 +/- 0.2 days in control ewes). At a lower infusion rate (0.3 micrograms/h) oxytocin delayed luteolysis in only 2 of 5 ewes. Treatment from Day 14, when luteolysis had already begun, was ineffective. Delay of luteal regression by oxytocin had no effect on the length of subsequent cycles. Measurement of circulating progesterone concentrations and luteal weight showed that prolongation of the oestrous cycle was due to prevention of luteal regression. Luteal regression and behavioural oestrus were induced during continuous oxytocin administration begun on Day 13 when cloprostenol was given on Day 15 (mean cycle length, 17.3 +/- 0.21 days). Continuous oxytocin infusion from Day 13 blocked the rise in uterine oxytocin receptor concentrations which normally precedes oestrus. Mean receptor concentrations in caruncular and intercaruncular endometrium and in myometrium were 76, 36 and 9 fmol/mg protein on Day 17 in ewes receiving continuous oxytocin (3 micrograms/h); in control ewes these values were 675, 638 and 130 fmol/mg protein respectively at oestrus. Receptor concentrations on the day of oestrus in ewes receiving oxytocin and cloprostenol were not significantly different from those in control ewes (649, 852, and 109 fmol/mg protein respectively). Since cloprostenol, a PGF-2 alpha analogue, overcame the antiluteolytic action of oxytocin, it is suggested that continuous oxytocin treatment may inhibit uterine production of PGF-2 alpha, possibly by down regulating the uterine oxytocin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local exchange of oxytocin from the ovarian vein to ovarian arteries in sheep

Local transfer of 125I-labeled oxytocin from the ovarian vein to arteries supplying the ovary, the oviduct, and the tip of the uterine born has been investigated. In five sheep, 10 infusions of 125I-oxytocin over a period of 1 h were performed, and the concentration of labeled polypeptide in the peripheral plasma was compared to ovarian arterial plasma. During 2 consecutive infusions into each animal's ovarian vein, blood was collected simultaneously from the following sites: ovarian branch of the ovarian artery (OBOA), tubal branch of the ovarian artery (TBOA), uterine branch of the ovarian artery (UBOA), and from the jugular vein. In all experiments the concentration of 125I-oxytocin in ovarian arterial plasma was higher than in peripheral plasma. The ratio of ovarian artery/jugular vein for 125I-oxytocin was: OBOA 2.8, TBOA 1.8, UBOA 1.6. Based on a 4 ml/min blood flow through ovarian arteries supplying ovary, oviduct, and the tip of the uterine horn, the local transfer of the total amount of oxytocin infused was estimated to be about 1% (range: 0.1-4.4%). Analysis of variance did not reveal significant differences in the exchange ratios between OBOA, TBOA, and OBOA. However, the variances within these groups are significant, presumably because of anatomical variation in the degree of surface contact area between arteries and veins at the ovarian pedicle. It is concluded that polypeptides are locally recirculated to ovaries, oviduct, and the tip of the uterine horn in a higher concentration than is supplied by peripheral blood. This could provide a mechanism for local distribution and concentration of the ovarian peptides that regulate reproductive function.     

Evidence for prostaglandin involvement in early luteal regression of the superovulated nanny goat (Capra hircus)

Feral does of various ages were treated with intravaginal progestagen sponges for 16 days to synchronize oestrus. On Day 2 before sponge removal the goats were given 1200 i.u. PMSG to induce superovulation: 6 of the goats were also injected every 12 h with flunixin meglumine, a prostaglandin (PG) synthetase inhibitor, from Day 3 to 7 of the synchronized oestrous cycle. Jugular blood samples were collected from all females into heparinized syringes at daily intervals over the 2 days before sponge removal, twice daily for the next 2 days, then at hourly intervals from 09:00 to 17:00 h for 2 days and then twice daily for a further 2 days, for measurement of plasma progesterone and the PGF metabolite 13,14-dihydro-15-keto-PGF (PGFM) by radioimmunoassay. Intermittent surges in plasma PGFM concentrations were observed in hourly samples collected from 4/4 untreated females but in only 2/6 of the inhibitor-treated females (P less than 0.05), and the peak plasma PGFM concentrations were reduced in these 2 inhibitor-treated goats compared with the control goats. The corpora lutea (CL) of the inhibitor-treated females appeared to be functional as indicated by the plasma progesterone profile and endoscopic examination of CL. In the control females, however, there was evidence of premature regression of CL. These results suggest that the premature release of PGF-2 alpha may be the cause of premature regression of CL in nanny goats induced to superovulate.     

Seasonal variation of the ovarian follicular dynamics and luteal functions of sheep in the subtropics

This study was undertaken to describe the development of individual follicles and corpora lutea (CL) in Ossimi ewe lambs at different seasons of the year in the subtropics. Seven ewe lambs underwent daily ultrasonographic examination for 20 interovulatory periods (IOP) during spring, winter and autumn. Ovarian follicles >or=2 mm and corpora lutea were counted and measured. Blood samples were taken for progesterone (P(4)) analysis. All ewe lambs included, but one, were cyclic in all seasons studied. Three (65%) and two (35%) follicular waves were detected per estrous cycle. None of the characteristics of the large follicles was affected by season. Follicles >or=2 mm in diameter were significantly higher in winter. The CL developed slowly in autumn. Serum P(4) level was higher in autumn. Double ovulation was observed only in autumn. The data demonstrated that Ossimi sheep in the subtropics were cyclic in most seasons of the year. Season affected mainly the luteal functions with little influences on the follicular characteristics.     

Pulsatile secretion of gonadotrophins, ovarian steroids and ovarian oxytocin during prostaglandin-induced regression of the corpus luteum in the cow

A luteolytic dose (500 micrograms) of cloprostenol was given on Day 12 of the oestrous cycle to 5 heifers. Blood samples were collected simultaneously from the caudal vena cava and jugular vein at 5-20-min intervals from -6 to 0 (control period), 0 to 12 and 24 to 36 h after PG injection. Pulses of LH were secreted concomitantly with pulses of FSH during all sampling periods. However, during the control period separate FSH pulses were detected resulting in a shorter (P less than 0.01) interpulse interval for FSH than LH (93 versus 248 min). LH and FSH pulse frequencies increased (P less than 0.01) beginning 1-3 h after PG to interpulse intervals of 59 and 63 min, respectively, and continued to be maintained 24-36 h after PG. Concomitantly there was a 2-3-fold increase (P less than 0.01) in basal concentrations and pulse amplitude for LH (but not FSH). FSH basal concentrations and pulse amplitudes decreased (P less than 0.05) in 3 heifers 24-36 h after PG. Pulsatile secretion of oestradiol was observed at frequencies similar to LH during the periods 4-12 h (3 heifers) and 24-36 h (2 heifers) after PG, respectively, resulting in higher (P less than 0.05) mean oestradiol concentrations. Progesterone concentrations in the vena cava increased (P less than 0.01) 5-10 min after PG but decreased (P less than 0.01) 67% by 20 min after PG. This decrease was followed by a rise (P less than 0.05) beginning 2-3 h after PG and lasting for an average of 3.3 h.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for luteal cell hyperplasia during pregnancy

The pronounced increase in luteal size and weight that occurs during rat pregnancy has been largely attributed to luteal cell hypertrophy. It is generally believed that hyperplasia does not play a role in luteal growth, since it is thought that luteal cell division in vivo does not occur. Recent data suggest that this may not be the case. Thus, to determine whether luteal cell hyperplasia occurs during rat pregnancy, osmotic minipumps filled with 3H-thymidine were implanted in timed-pregnant rats on Day 6 or Day 11 of pregnancy. These pumps provided a continuous infusion of 10 microCi 3H-thymidine per hour for up to 7 days. Seven days later (Day 13 and Day 18 respectively), rats were killed, and the ovaries were removed and prepared for autoradiography. Labeled cells, which have the morphological characteristics of luteal cells, were clearly observed in autoradiographs of ovaries exposed to 3H-thymidine. The labeling index of these cells from ovaries exposed to 3H-thymidine on Days 6-13 of pregnancy was 6.0% and from ovaries exposed to 3H-thymidine on Days 11-18 of pregnancy was 1.2%. Whether the presence of labeled cells signifies proliferation of luteal cells or whether these cells are derived from another cell type that develops into cells morphologically similar to luteal cells is not known at present. However, regardless of origin, these data clearly demonstrate that the number of parenchymal cells in the corpus luteum does increase during pregnancy.     

Possible role of cyclooxygenase II in the acquisition of ovarian luteal function in rodents

The development of the corpus luteum (CL), which involves angiogenesis, is essential for the establishment of early pregnancy. We investigated the roles of the prostaglandin synthases cyclooxygenase (COX) I and COX-II in angiogenesis and progesterone production in the newly formed CL, using inhibitors of the COX enzymes and the gonadotropin-induced pseudopregnant rat as a model. Injection of indomethacin, a nonselective COX inhibitor, on the day of ovulation and the following day decreased serum levels of progesterone, as did injection of the selective COX-II inhibitor NS-398. In contrast, a selective COX-I inhibitor, SC-560, had no effect on serum progesterone concentrations. None of the inhibitors had any effect on the weight of the superovulated ovaries or on the synthesis of progesterone by cultured luteal cells. To determine whether changes in angiogenesis are responsible for the decrease in progesterone synthesis, we measured hemoglobin and CD34 levels in luteinized ovaries following injection of COX inhibitors and measured the relative frequency of cells positive for platelet-endothelial cell adhesion molecule as a specific marker for endothelial cells. All of these parameters were reduced by the COX-II inhibitors, suggesting that changes in the vasculature are responsible for the decrease in serum progesterone. Histological examination of ovarian corrosion casts indicated that NS-398 inhibited the establishment of luteal capillary vessels following the injection of hCG. The results are consistent with the hypothesis that the activity of COX-II is associated with the formation of functional CL via its stimulation of angiogenesis.     

Evidence for a role of androgens in the growth and maturation of ovarian follicles in rats

Changes in the morphology and steroid content of ovaries were studied after 48 h of intravenous injection of 100 microgram of cyproterone acetate or flutamide to diestrus or estrous rats. Treatment with cyproterone acetate at diestrus caused a decrease in the number of small follicles (less than 200 micrometer), freshly formed corpora lutea and the levels of estradiol-17beta in the ovary, suggesting inhibition of ovulation. Following flutamide administration at diestrus, the number of follicles at all stages of development were reduced with a concomitant decrease in the ovarian levels of the hormones. Thus, flutamide suppressed the growth and maturation of follicles. On administration of these drugs at estrous, the steroid content of ovaries was more pari passu with the increase in the number of mature and medium follicles. The differential effects of the two drugs are discussed in the light of these observations.     

Regression of the corpora lutea in sheep in response to cloprostenol is not affected by loss of luteal oxytocin after hysterectomy

Concentrations of oxytocin in corpora lutea were reduced from 1706 to less than 15 ng/g wet wt after hysterectomy in sheep during the oestrous cycle. Hysterectomy also blocked the appearance of raised levels of oxytocin in ovarian and jugular venous plasma caused by cloprostenol. Administration of cloprostenol to hysterectomized ewes resulted in luteal regression, which occurred as rapidly as in intact animals. Therefore oxytocin in the corpus luteum during the oestrous cycle is unlikely to be involved in intraluteal events mediating prostaglandin-induced luteolysis.     

The role of luteal oxytocin in episodic secretion of prostaglandin F2alpha at luteolysis in the ewe

The role of luteal oxytocin in the generation of luteolytic episodes of prostaglandin F2alpha at luteolysis was investigated. On day 10 of the cycle Dorset ewes underwent either surgical removal of the corpora lutea (lutectomy; n = 4) or sham operation (sham; n = 4). Lutectomised ewes were then administered progesterone by twice daily i.m. injection in corn oil (20 mg/day) until day 14 when treatment was ceased to simulate luteolysis. The concentration of 13, 14 dihydro-15-keto prostaglandin F2alpha (PGFM) was measured in peripheral blood samples collected at 20-min intervals for 8 h on days 12-16 of the cycle. Progesterone and oestradiol concentrations were similar in the two groups over the whole experimental cycle while oxytocin fell dramatically following lutectomy. No prostaglandin F2alpha release episodes were seen on day 12 or 13, while from days 14-16 both groups exhibited a similar episode frequency (lutectomy 0.9/ewe/8 h; sham 0.8/ewe/8 h). Analysis of episode characteristics revealed lower episode amplitude (p<0.05) but longer episode duration (p<0.05) in the lutectomy group. The results demonstrate that a normal frequency of prostaglandin F2alpha release episodes occurs independently of luteal oxytocin secretion. However, luteal oxytocin is involved in regulating the pattern of release, perhaps causing the release of episodes of the magnitude required for the successful completion of luteolysis.     

Role of luteal regression in embryo death in cattle

Inseminated crossbred beef cows and heifers were used in 2 experiments to investigate embryo survival after prostaglandin-induced or spontaneous luteal regression. In Experiment 1, luteal regression was induced by an intramuscular (im) injection of cloprostenol (500 mug) on Day 15 (day of ovulation = Day 0). Progestagen was replaced 24 or 36 h later either by one-and-a-half Syncro-Mate-B (SMB) ear implants (9 mg of norgestomet) and norgestomet solution (2.25 mg, im) containing no estradiol in 1 replicate or by 2 SMB implants (12 mg of norgestomet) and progesterone (100 mg, im) in the second replicate. Combined for both replicates, the Day-24 pregnancy rate in an untreated control group (Group 1, 16/19; 84%) was higher (P < 0.01) than in the 24-h group (Group 2, 9/20; 45%), which also was higher (P < 0.02) than in the 36-h group (Group 3, 3/23; 13%). In Experiment 2, 15 or 16 d after breeding, cattle with a corpus luteum at least 16 x 16 mm were given either 2 SMB implants or no treatment. At 24 to 26 d after breeding, pregnancy rates (48/65, 74% versus 49/68, 72%) were not significantly different, and all but 1 of the pregnant progestagen-supplemented cattle had a functional corpus luteum.     

Prostaglandin F2 alpha-stimulated release of ovarian oxytocin in the sheep in vivo: threshold and dose dependency

To determine the threshold of prostaglandin F2 alpha (PGF2 alpha)-stimulated oxytocin secretion from the ovine corpus luteum, low levels of PGF2 alpha (5-100 pg/min) were infused into the ovarian arterial blood supply of sheep with ovarian autotransplants. PGF2 alpha was infused for six sequential 10-min periods at hourly intervals, 6, 12, or 24 days after estrus (n = 3 for each day). Each cycle day was studied during a separate cycle. Oxytocin and progesterone in ovarian venous and carotid arterial plasma was measured by radioimmunoassay, and secretion rates were determined (venous-arterial concentration x plasma flow). In animals treated on Day 6, 5 pg/min PGF2 alpha caused a significant release of oxytocin (p less than 0.01), whereas in animals treated on Day 12, this threshold was 40 pg/min (p less than 0.05). In animals treated on Day 24, the threshold for oxytocin release was greater than 100 pg/min. PGF2 alpha did not significantly change ovarian blood flow or progesterone secretion rate on any day (p greater than 0.05). To determine residual luteal oxytocin after each threshold experiment, 5 mg PGF2 alpha was given i.m. to all animals. Significantly more oxytocin was released by Day 6 than by Day 12 and Day 24 corpora lutea, and by Day 12 than by Day 24 corpora lutea (1.2 micrograms, 0.7 microgram, and 0.3 microgram, respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of neurophysins during luteal regression in the goat

In non-pregnant goats, ovariectomy on day 12 of oestrous cycle resulted in parallel decrease of oxytocin and progesterone jugular concentration. Similarly, luteolysis, indicated by decreasing progesterone concentration, was accompanied by simultaneous release of oxytocin and oxytocin-associated neurophysins (mean of neurophysin I and II). It is suggested that the neurophysins are secreted concomitantly with oxytocin by the ovary during luteal regressions in the goat.