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)     

Increase in concentration of uterine oxytocin receptors and decrease in response to 13,14-dihydro-15-keto prostaglandin F2 alpha in ewes after withdrawal of exogenous progesterone.

Ovariectomized ewes were treated with progesterone and oestradiol to induce oestrus (day of expected oestrus = day 0) and with progesterone on days 1 to 12. The concentrations of endometrial oxytocin receptors and the 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) response induced by oxytocin were measured on days 12, 14, 16 and 18 after the cessation of progesterone treatment on day 12, by a receptor binding assay and direct radioimmunoassay, respectively. During the period of treatment, the concentrations of plasma progesterone were high and remained above 2 ng ml-1 until day 13 when they dropped rapidly to less than 0.5 ng ml-1 by day 14. The concentrations of oxytocin receptors in endometrium of control ewes were high (820.7 +/- 91.7 (SEM) fmol mg-1 protein). Treatment with progesterone significantly (P < 0.01) reduced the concentrations of the receptors on days 12 and 14 (144.1 +/- 65.0 and 200.4 +/- 45.4 fmol mg-1 protein, respectively). The receptor concentrations then increased to relatively high values on day 16 (1021.4 +/- 216.6 fmol mg-1 protein) and remained high until day 18 (677.7 +/- 103.4 fmol mg-1 protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of oxytocin infusion during oestrus and the early luteal phase on progesterone secretion and the establishment of pregnancy in ewes

In Experiment 1, an osmotic minipump containing oxytocin was implanted s.c. in ewes for 12 days beginning on Day 10 of the oestrous cycle, producing approximately 100 pg oxytocin/ml in the plasma. Two days after the start of infusion, all ewes were injected with 100 micrograms cloprostenol and placed with a fertile ram. At slaughter 22 days later, 9 (75%) of the 12 control (saline-infused) ewes were pregnant compared with 1 (11%) of the 9 ewes infused with oxytocin. In the control group, midcycle plasma concentrations of oxytocin were significantly higher in nonpregnant than in pregnant ewes. In Experiment 2, an infertile ram was used throughout to avoid any possible effects of pregnancy and oxytocin infusions were given at different stages of the oestrous cycle. Otherwise the protocol was similar to that in Exp. 1. Oxytocin infusion during luteolysis and the early follicular phase had no effect on the subsequent progesterone secretion pattern, but infusions beginning the day before cloprostenol-induced luteolysis and lasting for 7 or 12 days and infusions beginning on the day of oestrus for 4 days all delayed the subsequent rise in plasma progesterone by approximately 3-4 days. In these animals, the cycle tended to be longer. It was concluded that an appropriate oxytocin secretion pattern may be necessary for the establishment of pregnancy in ewes and that a high circulating oxytocin concentration during the early luteal phase delays the development of the young corpus luteum.     

Temporal relationships between peripheral plasma concentrations of oxytocin, progesterone and 13, 14-dihydro-15-keto-prostaglandin F2α during the oestrous cycle and early pregnancy in the ewe

Peripheral plasma concentrations of oxytocin, 13,14-dihydro-15-keto-prostaglandin F_2α(PGFM), progesterone and LH were determined at 3 hourly intervals during the oesterous cycle (n = 3) and in early pregnancy (n = 4) in sheep. The progesterone and LH concentrations showed that the cycling ewes were samples during the periods of luteal regression (decreasing progesterone concentrations), the preovulatory gonadotrophin surge and the beginning of the next luteal phase (increasing progesterone concentrations). The pregnant ewes had basal LH concentrations and luteal phase concentrations of progesterone (>lng/ml afte day 5 following mating) throughout the whole of the sampling period. Oxytocin concentrations in the non-pregnant ewes decreased around the time of luteal regression to reach low concentrations (mean concentrations of approximately 18pg/ml) during the preovulatory period and then increased after the preovulatory surge. PGFM concentrations exhibited a pulsatile pattern with increasing concentrations as progesterone levels fell. In the pregnant ewes oxytocin concentrations gradually fell until approximately 16 days post-mating (approximately 7–8pg/ml). The magnitude of the pulses in PGFM concentrations were also lower than in the cycling ewes. These results demonstrate that the increased concentrations of PGFM which are found during the period of luteal regression are not caused by increased peripheral concentrations of oxytocin.     

Pulsatile release of oxytocin into the circulation of the ewe during oestrus, mating and the early luteal phase

Two experiments were designed to investigate release patterns of oxytocin into plasma during oestrus and the early luteal phase. In Exp. 1, blood samples were collected from 5 ewes every 30 min for 10 h during 6 days around oestrus and the early luteal phase. During oestrus concentrations of oxytocin were generally low (1.27 +/- 0.54 pg/ml; mean +/- s.d.) but with occasional pulses up to 6 pg/ml. By Day 5 mean basal concentrations had risen to 4.5 +/- 2.1 pg/ml with a fluctuating release pattern. In Exp. 2, a method was developed for continuous blood sampling from conscious, unrestrained ewes. On the predicted day of oestrus following an untreated oestrous cycle, 8-ml blood samples were collected every minute for two 35-min periods (8 ewes: 16 sampling periods). For 6 ewes a ram was introduced to the pen for part of this time, and resulting behaviour was recorded. Additional blood samples were assayed for LH and progesterone to determine the stage of the cycle. Overall mean oxytocin concentrations ranged from 1.5 +/- 0.53 to 6.8 +/- 5.25 pg/ml in different animals. Ewes which were both in oestrus and exposed to the ram showed a pulsatile oxytocin release pattern consisting of low baseline concentrations with short-duration pulses superimposed (duration 1-4 min; amplitude 2.5-31.7 pg/ml; frequency 3.18/h). Coitus was not temporally associated with pulsatile release. However, the importance of the presence of the ram was indicated by total separation of 2 oestrous ewes from the ram until after experimentation. In these animals only 1 pulse of oxytocin was detected in 2.7 h of sampling. It is concluded that, although mean oxytocin concentrations at oestrus were low, short duration pulses were released into the plasma at this time. This effect may be dependent on the presence of a ram.     

Function and lifespan of corpora lutea in ewes treated with exogenous oxytocin

Oxytocin was administered to Dorset and Shropshire ewes in one experiment and to Dorset ewes in a further 4 experiments. In Exp. 1, concentrations of plasma progesterone and lengths of the oestrous cycle in ewes given oxytocin subcutaneously twice a day on Days 0-3, 2-5, 4-7, 6-9, 8-11, 10-13, 12-15 or 14-17 were similar to those of control ewes. In Exp. 2, intraluteal infusions of oxytocin from Day 2 to Day 9 after oestrus had no effect on concentration of progesterone, weight of CL collected on Day 9 or length of the oestrous cycle. In Exp. 3, intraluteal infusions of oxytocin on Days 10-15 after oestrus had no effect on weight of CL collected on Day 15. In Exp. 4, s.c. injections of oxytocin on Days 3-6 after oestrus had no effect on weight of CL collected on Day 9, concentrations of progesterone or length of the oestrous cycle. In Exp. 5, s.c. injections of oxytocin twice a day did not affect the maintenance and outcome of pregnancy in lactating and nonlactating ewes. Exogenous oxytocin, therefore, does not appear to affect luteal function at any stage of the ovine oestrous cycle although oxytocin has been reported by others to alter ovine CL function.     

Control of endometrial oxytocin receptor and uterine response to oxytocin by progesterone and oestradiol in the ewe

The effects of administration of progesterone and oestradiol on ovine endometrial oxytocin receptor concentrations and plasma concentrations of 13,14-dihydro-15-keto prostaglandin F-2 alpha (PGFM) after oxytocin treatment were determined in ovariectomized ewes. Ewes received progestagen pre-treatment, progesterone and/or oestradiol in 11 different treatment schedules. Progestagen pre-treatment decreased oxytocin receptor concentrations in endometrium from ewes treated subsequently with either progesterone for 5 days or progesterone for 5 days plus oestradiol on Days 4 and 5 of progesterone treatment. Oestradiol increased endometrial oxytocin receptor concentrations when administered on Days 4 and 5 of 5 days progesterone treatment. Progestagen pre-treatment followed by progesterone treatment for 12 days caused a large increase in oxytocin receptors and no further increase occurred when ewes were given oestradiol on Days 11 and 12, or when progesterone was withdrawn on Days 11 and 12, or these two treatments were combined. Oxytocin administration caused an increase in plasma PGFM concentrations in ewes which did not receive progestagen pre-treatment, and subsequently received progesterone treatment for 5 days and oestradiol treatment on Days 4 and 5 of progesterone treatment. Similarly treated ewes which received progestagen pre-treatment did not respond to oxytocin. Oxytocin administration also increased plasma PGFM concentrations in ewes which received progestagen pre-treatment followed by progesterone treatment for 12 days, progesterone treatment for 12 days plus oestradiol on Day 11 and 12 of progesterone treatment, progesterone withdrawal on Day 11 and 12, or progesterone withdrawal and oestradiol treatment combined. The results indicate that (1) progesterone pre-treatment affects oxytocin receptor concentrations in the endometrium and uterine responsiveness to oxytocin and (2) progesterone treatment alone for 12 days after a treatment which mimics a previous luteal phase and oestrus is sufficient to induce oxytocin receptors and increase oxytocin-induced PGF release. These results emphasize the importance of progesterone and provide information which can be used to form an hypothesis for control of luteolysis and oestrous cycle length in the ewe.     

Independence of progesterone blockade of the luteinizing hormone surge in ewes from opioid activity at naloxone-sensitive receptors.

Fifteen ovariectomized ewes were treated with implants (s.c.) creating circulating luteal progesterone concentrations of 1.6 +/- 0.1 ng ml-1 serum. Ten days later, progesterone implants were removed from five ewes which were then infused with saline for 64 h (0.154 mol NaCl l-1, 20 ml h-1, i.v.). Ewes with progesterone implants remaining were infused with saline (n = 5) or naloxone (0.5 mg kg-1 h-1, n = 5) in saline for 64 h. At 36 h of infusion, all ewes were injected with oestradiol (20 micrograms in 1 ml groundnut oil, i.m.). During the first 36 h of infusion, serum luteinizing hormone (LH) concentrations were similar in ewes infused with saline after progesterone withdrawal and ewes infused with naloxone, but with progesterone implants remaining (1.23 +/- 0.11 and 1.28 +/- 0.23 ng ml-1 serum, respectively, mean +/- SEM, P greater than 0.05). These values exceeded circulating LH concentrations during the first 36 h of saline infusion of ewes with progesterone implants remaining (0.59 +/- 0.09 ng ml-1 serum, P less than 0.05). The data suggested that progesterone suppression of tonic LH secretion, before oestradiol injection, was completely antagonized by naloxone. After oestradiol injection, circulating LH concentrations decreased for about 10 h in ewes of all groups. A surge in circulating LH concentrations peaked 24 h after oestradiol injection in ewes infused with saline after progesterone withdrawal (8.16 +/- 3.18 ng LH ml-1 serum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic effects of insulin-like growth factor I and gonadotrophins on relaxin and progesterone secretion by ageing corpora lutea of pigs.

Insulin-like growth factor I (IGF-I) is involved in paracrine/autocrine regulation of gonadal steroidogenesis and peptide hormone biosynthesis. This study was designed to determine whether IGF-I alone, or an interaction of IGF-I, is involved in augmenting the actions of luteinizing hormone (LH) and prolactin in controlling relaxin and progesterone secretion from ageing corpora lutea of hysterectomized gilts at days 110, 113 and 116 after oestrus. Luteal tissue slices were incubated for 8 h with IGF-I (0, 50, 300 ng ml-1), LH (0, 100, 1000 ng ml-1), and prolactin (0, 100, 1000 ng ml-1) alone or in combination. Progesterone and relaxin concentrations were determined by radioimmunoassay of spent medium and of homogenates from luteal tissue slices before and after incubation. Porcine luteal tissue from day 110 had a net output of 25 ng progesterone and 26 ng relaxin in the control and of 65 ng progesterone and 2125 ng relaxin in the combined IGF-I, LH and prolactin treatment mg-1 of luteal tissue, respectively. IGF-I, LH and prolactin alone or in combination significantly increased (P < 0.01) progesterone production by luteal tissue from day 110, but they were partially effective at day 113 and ineffective at day 116. By contrast, the same hormone treatments increased relaxin production by luteal tissue from days 110 and 113. Even at day 116, prolactin alone or with LH or IGF-I continued to stimulate relaxin production. In conclusion, IGF-I augments the ability of prolactin and LH to increase relaxin production by ageing corpora lutea; however, a decrease in progesterone secretion and an increase in relaxin secretion at day 113 indicate that different mechanisms control progesterone and relaxin secretion in pigs.     

Suppression of pulsatile luteinizing hormone secretion by gonadotrophin-releasing hormone antagonist does not affect episodic progesterone secretion or corpus luteum function in ewes.

Progesterone secretion has been observed to be episodic in the late luteal phase of the oestrous cycle of ewes and is apparently independent of luteinizing hormone (LH). This study investigated the effects of suppressing the pulsatile release of LH in the early or late luteal phase on the episodic secretion of progesterone. Six Scottish Blackface ewes were treated i.m. with 1 mg kg-1 body weight of a potent gonadotrophin-releasing hormone (GnRH) antagonist on either day 4 or day 11 of the luteal phase. Six ewes received saline at each time and acted as controls. Serial blood samples were collected at 10 or 15 min intervals between 0 and 8 h, 24 and 32 h, and 48 and 56 h after GnRH antagonist treatment and daily from oestrus (day 0) of the treatment cycle for 22 days. Oestrous behaviour was determined using a vasectomized ram present throughout the experiment. Progesterone secretion was episodic in both the early and late luteal phase with a frequency of between 1.6 and 3.2 pulses in 8 h. The GnRH antagonist abolished the pulsatile secretion and suppressed the basal concentrations of LH for at least 3 days after treatment. This suppression of LH, in either the early or late luteal phase, did not affect the episodic release of progesterone. Daily concentrations of progesterone in plasma showed a minimal reduction on days 11 to 14 after GnRH antagonist treatment on day 4, although this was significant (P < 0.05) only on days 11 and 13. There was no effect of treatment on day 11 on daily progesterone concentration, and the timing of luteolysis and the duration of corpus luteum function was unaffected by GnRH antagonist treatment on either day 4 or day 11. These results indicate that the episodic secretion of progesterone during the luteal phase of the oestrous cycle in ewes is independent of LH pulses and normal progesterone secretion by the corpus luteum can be maintained with minimal basal concentrations of LH.     

Uterine oxytocin receptors in cyclic and pregnant cows.

Binding of [3H]oxytocin to uterine subcellular preparations ('oxytocin receptor concentrations') was measured in uterine tissue of heifers and multiparous dairy cows at various stages of the oestrous cycle and during early pregnancy. A method for the assay of ovine uterine oxytocin receptors was optimized for use on bovine tissue. Oxytocin receptor concentrations were increased in cyclic animals around the period of luteolysis and oestrus, rising on Day 15 in endometrium and on Day 17 in myometrium while pregnant animals showed no comparable rise. Receptor concentrations then declined on Day 3 after oestrus in myometrium and on Day 5 in endometrium. Some cyclic animals did not show the expected rise in receptors in the late luteal phase; these animals had abnormally high progesterone concentrations for this stage of the cycle. In animals slaughtered on Day 18 after oestrus and/or insemination which had low oxytocin receptor levels, plasma progesterone concentrations were consistently high; while all animals showing the late luteal phase elevation in receptor values had low progesterone concentrations. Oxytocin receptor and progesterone concentrations were negatively correlated (P less than 0.05). These data support the hypothesis that oxytocin receptor level is a key factor in the process of luteolysis in cattle and that in pregnancy there is suppression of uterine oxytocin receptor at the expected time of luteolysis. We suggest that uterine oxytocin receptor levels are partly controlled by circulating steroid hormones and are suppressed during early pregnancy.     

Effects of oxytocin on cloprostenol-induced luteolysis, follicular growth, ovulation and corpus luteum function in heifers

Twenty-five normally cyclic Holstein heifers were used to examine the effects of oxytocin on cloprostenol-induced luteolysis, subsequent ovulation, and early luteal and follicular development. The heifers were randomly assigned to 1 of 4 treatments: Group SC-SC (n=6), Group SC-OT (n=6), Group OT-SC (n=6) and Group OT-OT (n=7). The SC-SC and SC-OT groups received continuous saline infusion, while Groups OT-SC and OT-OT received continuous oxytocin infusion (1:9 mg/d) on Days 14 to 26 after estrus. All animals received 500 microg, i.m. cloprostenol 2 d after initiation of infusion (Day 16) to induce luteolysis. Groups SC-OT and OT-OT received oxytocin twice daily (12 h apart) (0.33 USP units/kg body weight, s.c.) on Days 3 to 6 of the estrous cycle following cloprostenol-induced luteolysis, while Groups SC-SC and OT-SC received an equivalent volume of saline. Daily plasma progesterone (P4) concentrations prior to cloprostenol-induced luteolysis and rates of decline in P4 following the induced luteolysis did not differ between oxytocin-infused (OT-OT and OT-SC) and saline-infused (SC-SC and SC-OT) groups (P >0.1). Duration of the estrous cycle was shortened in saline-infused heifers receiving oxytocin daily during the first week of the estrous cycle. In contrast, oxytocin injections did not result in premature inhibition of luteal function and return to estrus in heifers that received oxytocin infusion (OT-OT). Day of ovulation, size of ovulating follicle and time of peak LH after cloprostenol administration for oxytocin and saline-treated control heifers did not differ (P >0.1). During the first 3 d of the estrous cycle following luteal regression, fewer (P <0.01) follicles of all classes were observed in the oxytocin-infused animals. Day of emergence of the first follicular wave in heifers treated with oxytocin was delayed (P <0.05). The results show that continuous infusion of oxytocin during the mid-luteal stage of the estrous cycle has no effect on cloprostenol-induced luteal regression, timing of preovulatory LH peak or ovulation. Further, the finding support that an episodic rather than continuous administration of oxytocin during the first week of the estrous cycle results in premature loss of luteal function. The data suggest minor inhibitory effects of oxytocin on follicular growth during the first 3 d of the estrous cycle following cloprostenol-induced luteolysis.     

Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep

Jugular venous concentrations of oxytocin and progesterone changed in parallel during the oestrous cycle in the ewe, falling at luteal regression and rising with formation of the new corpus luteum. These fluctuations in the circulating concentration of oxytocin were not caused by changes in its metabolic clearance rate. On Days 6-9 of the cycle circulating oxytocin concentrations exhibited a diurnal rhythm, peaking at 09:00 h; this rhythm was absent on Days 11-14. Although there was no evidence for increased production of oxytocin at or preceding luteal regression in samples taken daily, more frequent sampling revealed that two thirds of detected surges of uterine secretion of prostaglandin (PG) F-2 alpha were accompanied by raised levels of oxytocin. This oxytocin was not of pituitary origin. Luteal regression induced with cloprostenol on Day 8 after oestrus caused a decrease in circulating progesterone level followed after 24 h by a fall in oxytocin. Measurements of oxytocin in the ovary and other organs before and after treatment with cloprostenol identified the corpora lutea as a major potential source of oxytocin, and suggested that 98% of luteal oxytocin was available for secretion in response to prostaglandin stimulation. The data are consistent with a role for ovarian secretion of oxytocin in response to uterine release of PGF-2 alpha in the control of luteal regression.     

Oestradiol and the preovulatory surges of luteinising hormone and follicle stimulating hormone in ewes during the breeding season and transition into anoestrus

This study was designed to see if giving exogenous oestradiol, during the follicular phase of the oestrous cycle of intact ewes, during the breeding season or transition into anoestrus, would alter the occurrence, timing or magnitude of the preovulatory surge of secretion of luteinising hormone (LH) or follicle stimulating hormone (FSH). During the breeding season and the time of transition, separate groups of ewes were infused (intravenously) with either saline (30 ml h⁻¹; n = 6) or oestradiol in saline (n = 6) for 30 h. Infusion started 12 h after removal of progestin-containing intravaginal sponges that had been in place for 12 days. The initial dose of oestradiol was 0.02 μg h⁻¹; this was doubled every 4 h for 20 h, followed by every 5 h up to 30 h, to reach a maximum of 1.5 μg h⁻¹. Following progestin removal during the breeding season, peak serum concentrations of oestradiol in control ewes were 10.31 ± 1.04 pg ml⁻¹, at 49.60 ± 3.40 h after progestin removal. There was no obvious peak during transition, but at a time after progestin removal equivalent to the time of the oestradiol peak in ewes at mid breeding season, oestradiol concentrations were 6.70 ± 1.14 pg ml⁻¹ in ewes in transition (P < 0.05). In oestradiol treated ewes, peak serum oestradiol concentrations (24.8 ± 2.1 pg ml⁻¹) and time to peak (41.00 ± 0.05 h) did not differ between seasons (P > 0.05). During the breeding season, all six control ewes and four of six ewes given oestradiol showed oestrus with LH and FSH surges. The two ewes not showing oestrus did not respond to oestrus synchronisation and had persistently high serum concentrations of progesterone. During transition, three of six control ewes showed oestrus but only two had LH and FSH surges; all oestradiol treated ewes showed oestrus and gonadotrophin surges (P < 0.05). The timing and magnitude of LH and FSH surges did not vary with treatment or season. In blood samples collected every 12 min for 6 h, from 12 h after the start of oestradiol infusion, mean serum concentrations of LH and LH pulse frequency were lower in control ewes during transition than during mid breeding season (P < 0.05). Oestradiol treatment resulted in lower mean serum concentrations of LH in season and lower LH pulse frequency in transition (P < 0.05). We concluded that enhancing the height of the preovulatory peak in serum concentrations of oestradiol during the breeding season did not alter the timing or the magnitude of the preovulatory surge of LH and FSH secretion and that at transition into anoestrus, oestradiol can induce oestrus and the surge release of LH and FSH as effectively as during the breeding season.     

A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.     

Effect of continuous infusion of oxytocin on length of the oestrous cycle and luteolysis in cattle

In Exp. I oxytocin (60 micrograms/100 kg/day) was infused into the jugular vein of 3 heifers on Days 14-22, 15-18 and 16-19 of the oestrous cycle respectively. In Exp. II 5 heifers were infused with 12 micrograms oxytocin/100 kg/day from Day 15 of the oestrous cycle until clear signs of oestrus. Blood samples were taken from the contralateral jugular vein at 2-h intervals from the start of the infusion. The oestrous cycle before and after treatment served as the controls for each animal. Blood samples were taken less frequently during the control cycles. In Exp. III 3 heifers were infused with 12 micrograms oxytocin/100 kg/day for 50 h before expected oestrus and slaughtered 30-40 min after the end of infusion for determination of oxytocin receptor amounts in the endometrium. Three other heifers slaughtered at the same days of the cycle served as controls. Peripheral concentrations of oxytocin during infusion ranged between 155 and 641 pg/ml in Exp. I and 18 and 25 pg/ml in Exp. II. In 4 our of 8 heifers of Exps I and II, one high pulse of 15-keto-13,14-dihydro-prostaglandin F-2 alpha (PGFM) appeared soon after the start of oxytocin infusion followed by some irregular pulses. The first PGFM pulse was accompanied by a transient (10-14 h) decrease of blood progesterone concentration. High regular pulses of PGFM in all heifers examined were measured between Days 17 and 19 during spontaneous luteolysis. No change in length of the oestrous cycle or secretion patterns of progesterone, PGFM and LH was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Oxytocin is luteolytic in the rhesus monkey (Macaca mulatta)

Oxytocin (10 mi.u./microliter/h) or vehicle (0.5% chlorobutanol in saline, 1 microliter/h) was chronically infused directly into the corpus luteum of normally cyclic rhesus monkeys, by means of an Alzet pump-ovarian cannula system. Infusion of oxytocin (N = 6) or vehicle (N = 5) began 6 days after the preovulatory oestradiol surge, and daily peripheral blood samples were taken. Oxytocin caused a significant (P less than 0.05) decrease in progesterone, beginning 1 day after treatment, and oestradiol after 4 days; progesterone and oestradiol remained significantly depressed until menstruation. However, peripheral LH concentrations remained unchanged. The duration of the luteal phase, menstrual cycle and the onset of menses from the initiation of oxytocin infusion were significantly (P less than 0.01) shorter when compared to those of vehicle-treated controls. These results show that oxytocin can induce functional luteolysis in the primate and supports the hypothesis that oxytocin of luteal origin may play a role in spontaneous luteolysis.     

Effects of prostaglandin E1 or E2 (PGE1; PGE2) on luteal function and binding of luteinizing hormone in nonpregnant ewes

Two studies were conducted to determine the effects of PGE1 or PGE2 on luteal function and binding of luteinizing hormone (LH) to luteal cell membranes in nonpregnant ewes. In Study I, ewes (n=5 per group) received an injection of vehicle (VEH) or 333 micrograms of PGE1 or PGE2 into the tissue surrounding the ovarian vascular pedicle (intrapedicle) on day 7 postestrus. Systemic progesterone concentrations of PGE1-treated ewes were greater (P less than 0.01) than those of VEH-treated ewes at 24 and 48 hr after injection. For PGE2-treated ewes, progesterone concentrations were greater (P less than 0.01) than for VEH-treated ewes only at 24 hr. Neither PGE1 nor PGE2 affected luteal weights or LH binding capacity at 48 hr. Treatment with PGE1, however, increased (P less than 0.10) endogenously bound LH at this time. In Study II, ewes (n=5 per group) received an intrapedicle injection of VEH, or 10 mg of PGE1 or PGE2 on day 8 postestrus. Systemic progesterone concentrations in PGE1-treated ewes were less (P less than 0.01) than for VEH-treated ewes at 24 hr, but by 72 hr were not different from those of VEH-treated ewes. For PGE2-treated ewes, systemic progesterone declined steadily to reach low values by 72 hr. Prostaglandin E2 had no effect on luteal binding of LH at 72 hr, whereas PGE1 increased (P less than 0.05) LH binding capacity and endogenously bound LH. Although PGE2 had no apparent affect on luteal binding of LH in these studies, PGE1 may enhance the function of ovine corpora lutea by stimulating an increase in their binding of LH and capacity to bind LH when the CL receives a luteolytic signal.     

Luteinizing hormone, oestrogen and progesterone levels in peripheral serum of anoestrous and cyclic ewes as determined by radioimmunoassay.

Jugular vein blood was collected daily from four mature ewes throughout anoestrus and the first oestrous cycle of the breeding season until 4 days after the second oestrus. The levels of oestrogen, progesterone and LH were determined by radioimmunoassay. There were fluctuations in the LH level throughout most of the observed anoestrous period with a mean plus or minus S.E. value of 2-3 plus or minus 0-9 ng/ml. High LH values of 20-0, 41-2 and 137-5 ng/ml were observed in three ewes on Day - 24 of anoestrus. A brief minor rise in progesterone level was also observed around this period. Progesterone levels were consistently low (0.11 plus or minus 0-01 ng/ml) before Day - 25 of anoestrus. A major rise occurred on Day - 12 of anoestrous and this was followed by patterns similar to those that have been previously reported for the oestrous cycle of the ewe. Random fluctuations of oestrogens deviating from a mean level of 4-40 plus or minus 0-1 pg/ml were observed during anoestrus and the mean level during the period from the first to the second oestrus was 5-2 plus or minus 0-3 pg/ml. A well-defined peak of 13-3 plus or minus 0-7 pg/ml was seen in all ewes on the day of the second oestrus. Results of the present study suggest that episodic releases of LH occur during anoestrus and periods of low luteal activity. The fluctuations in LH levels, as observed during the period of low luteal activity, i.e. before Day - 25 of anoestrus, were less pronounced during the periods of high luteal activity. The view that luteal activity precedes the first behavioural oestrus of the breeding season is supported.