ABSTRACT

The acute antigonadotrophic action of prostaglandin F_2α (PGF_2α) was examined in dispersed luteal cell preparations from immature superluteinized rat ovaries. Cell suspensions prepared by collagenase digestion and purification over a Percoll density gradient were incubated for 1 h in Eagle's minimum essential medium in the presence and/or absence of LH, PGF_2α, N⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP) and forskolin. Medium was assayed for total progesterone and adenosine 3′,5′-cyclic monophosphate (cAMP). Luteal cell preparations showed typical steroidogenic (progesterone) responses to LH, mimicked by both dbcAMP and forskolin. Whilst the threshold LH dose to increase cAMP synthesis was greater than that for progesterone (100 μg/l compared with 1 μg/l), 24 μmol forskolin/l was the threshold dose for both cAMP and progesterone responses. Furthermore, combined doses of LH and forskolin synergistically raised cAMP yet produced less than additive increases in progesterone. Similarly, combinations of dbcAMP plus forskolin produced less than additive progesterone increases. These data suggest that forskolin may not act as a simple mimic of LH.

Prostaglandin F_2α dose-dependently inhibited forskolin-induced cAMP and progesterone synthesis and also inhibited progesterone synthesis induced by dbcAMP. These data suggest that the antigonadotrophic effect of PGF_2α has more than one locus of action, i.e. it both inhibits an adenylate cyclase event associated with cAMP generation and blunts the cellular response to cAMP. The present uncertainty over the exact locus of forskolin's action within the adenylate cyclase complex limits further delineation of the inhibitory action of PGF_2α on LH-responsive adenylate cyclase.

J. Endocr. (1986) 111, 415–423

SUMMARY

1. Sixty crossbred maiden ewes, twelve of which were hysterectomized, and eighteen mature ewes were used during the non-breeding season in two experiments involving injection with 75 mg of progesterone intramuscularly and 1000 i.u. of pregnant mare serum (PMS) subcutaneously, either alone or in combination.

2. Progesterone given as two daily injections of 12·5 mg each for 3 days, followed 2 days later by 1000 i.u. of PMS resulted in oestrus with ovulation in sixteen out of eighteen ewes treated. The two remaining ewes failed to ovulate owing to the presence of active corpora lutea.

3. Three injections of 25 mg progesterone/day, or a single injection of 75 mg progesterone followed by PMS, were decreasingly less effective in inducing oestrus.

4. Progesterone alone in the form of two daily injections of 12·5 mg for 3 days without subsequent PMS, was followed by ovulation in five out of six mature ewes, three of them exhibiting oestrus and producing lambs subsequently.

5. Time relationships were physiologically normal, oestrus commencing 24 to 36 hr after PMS and preceding ovulation by 10–20 hr. The ovaries showed no histological abnormalities.

6. Hysterectomized and intact ewes responded alike, suggesting that the conditioning effect of progesterone is a central phenomenon.

SUMMARY

Three factorial design tests are described, involving 528 anoestrous crossbred ewes, in which were studied the ovarian and behavioural responses to several combinations of dose and types of gonadotrophin, of dose, type and duration and frequency of progestin, and of small doses of oestrogen with gonadotrophin.

Human chorionic gonadotrophin (HCG) increased ovarian weight more than pregnant mares' serum (PMS). Both gonadotrophins were equally effective in inducing ovulation. In one test, but not in the other two, HCG was much less effective than PMS in causing oestrus with ovulation. The most important factors affecting the incidence of oestrus, with ovulation, were the duration and the frequency of injection of progesterone. Injection of 15–45 μg. oestradiol benzoate (ODB) with gonadotrophin increased the proportion of ewes in oestrus, but suppressed ovulation in some cases. HCG tended to increase the incidence of cystic follicles and number of ovulations.

'Proluton-D' (17α-hydroxyprogesterone caproate) was no substitute for progesterone in the ovulation-oestrus phenomenon.

The data suggest that the response to PMS of ewes of different ages and in different years is relatively predictable, whereas that to HCG is highly variable.

SUMMARY

Seven trials were conducted with sixty-nine ovariectomized Suffolk cross-bred ewes to determine the requirements of oestradiol benzoate (ODB), given alone or preceded by 75 mg progesterone (6 × 12·5 mg in oil over 3 days, followed 2 days later by oestrogen), for oestrous behaviour and characteristic vaginal changes.

Progesterone pretreatment resulted in a marked increase of sensitivity to oestrogen. For oestrus, the respective values of the median effective dose (ED 50) for ODB preceded by progesterone and for ODB alone were 22 and 64 μg, the difference being significant (P<0·001). The 99% fiducial limits associated with these estimates were respectively 19 and 26 μg and 52 and 81 μg. For vaginal changes the corresponding values were 10-14-17 and 20-24-28 (P<0·001).

Progesterone pretreatment resulted in an apparently steeper dose-response line for oestrus, and advanced the mean time of onset by about 12 hr. The behaviour pattern following progesterone—ODB appeared to differ from that following ODB alone.

Oestrus in the ewe appears to be under dual hormonal control. Endogenous oestrogen production is insufficient to induce the full psychic and physiological changes associated with normal oestrus, unless the animal has been conditioned previously by progesterone.

ABSTRACT

We previously demonstrated that oxytocin stimulates LH release from rat pituitary cells in vitro and advances follicular development and ovulation in mice in vivo. This study reports an investigation of rat LH levels following in-vivo administration of oxytocin. Injection of oxytocin (10 mIU/g, i.p.) to rats at 07.00, 08.00 and 09.00 h of pro-oestrus or at 09.00, 10.00 and 11.00 h of pro-oestrus advanced the onset of the LH surge (P<0.005) and attainment of peak concentrations of LH (P<0.02) in peripheral blood. On the other hand, the descending phase of the LH surge and the surge amplitude were not altered by oxytocin. Treatment at 05.00, 06.00 and 07.00 h of pro-oestrus or at 11.00, 12.00 and 13.00 h of pro-oestrus had no effect on the LH profile. A higher oxytocin dose (20 mIU/g) inhibited LH release when treatment was begun at 05.00, 07.00 or 09.00 h of pro-oestrus. A lower dose (5 mIU/g) was ineffective in altering LH concentrations. In addition, injections of oxytocin (10 mIU/g) at oestrus, metoestrus or dioestrus had no effect on the release of LH. Thus the efficacy of oxytocin in altering concentrations of LH was dose dependent and also critically affected by the day of the oestrous cycle and the time of pro-oestrus. Removal of endogenous oxytocin activity by the use of an oxytocin receptor antagonist abolished the pro-oestrous LH surge, indicating that oxytocin is a vital physiological component of the LH-releasing mechanism in rats. The study provides unequivocal evidence that oxytocin induces LH release in vivo, but the manifestation of oxytocin activity is dependent upon conditions of exposure.

Journal of Endocrinology (1990) 125, 425–432

SUMMARY

Progesterone levels were determined in the ovarian and jugular vein plasma and the corpus luteum of 68 cyclic Merino ewes (controls) and of 32 ewes which had been treated with intravaginal sponges containing a synthetic progestagen (Cronolone, Searle). The weight and diameter of the corpus luteum and the rate of flow of the ovarian vein blood were also recorded. No corrections for procedural losses were made.

The control ewes showed highly significant (P < 0·001) cyclic changes in the progesterone contents of the ovarian vein plasma and the corpus luteum, and in the mass of the corpus luteum. There were positive correlations (P < 0·001) between ovarian vein plasma progesterone concentration and the weight (r = 0·651), diameter (r = 0·692), total progesterone content (r = 0·775) and progesterone concentration (r = 0·574) of the corpus luteum.

Cyclic changes in the progesterone content of the jugular vein failed to attain significance (0·1 < P < 0·2) but the levels were positively correlated with those in the ovarian vein (r = 0·465, P < 0·001) and with weight (r = 0·432, P < 0·001) and diameter (r = 0·303, P < 0·05) of the corpus luteum.

The Cronolone-treated ewes showed cyclic changes in luteal function similar to those in controls, with the exception of animals treated on the day of oestrus. In ewes in which ovulation was not suppressed, the duration of activity of the corpus luteum, as measured by progesterone concentration in ovarian vein plasma, and concentration and content in the corpus luteum was significantly reduced. By the 12th day of the cycle the corpus luteum had almost completely regressed both morphologically and functionally.

SUMMARY

The levels of free oestrogen (oestrone and oestradiol-17β) in plasma in the ovarian vein were determined in three groups, each of 27 ewes, at nine intervals at about the time of oestrus. One group had a normal oestrus while the other two had been treated for 16 days with intravaginal sponges containing either 10 or 30 mg of a synthetic progestagen (Cronolone, Searle).

In untreated ewes, the mean level (corrected) of oestradiol-17β in plasma from the active ovary rose from 25·3 ng/100 ml at −48 h to a peak of 91·6 ng/100 ml at 0 h (onset of oestrus) and then fell. There was evidence of biphasic production. The mean level of oestrone was relatively high (13·0 ng/100 ml) at −48 h; it fell to 2·0 ng/100 ml between −36 and −24 h and then rose again to 9·4 ng/100 ml at + 12 h. There was no significant change, with time, in the plasma levels of either oestrogen from the non-active ovary. The total amounts of oestradiol-17β and of oestrone produced from both ovaries at an oestrous period were estimated to average 9·7 and 2·4 μg.

In treated ewes, a similar pattern of production of oestradiol-17β was shown by the ewes treated with 30 mg Cronolone. That of ewes treated with 10 mg differed (P < 0·01). Peak level was reached at an earlier stage, relative to the onset of oestrus, and it declined more rapidly, the total amount of oestrogen produced (oestrone + oestradiol-17β) was less (10 mg Cronolone, 8·6 μg; 30 mg Cronolone, 12·1 μg; normal oestrus, 12·1 μg), and there was no biphasic production.

ABSTRACT

Gonadotrophin-releasing activity of oxytocin has previously been demonstrated in vitro and in vivo. This study investigated whether oxytocin is also able to induce LH accumulation in pituitary cells. Following trypsin digestion and mechanical dispersion, pituitary cells from female rats were incubated with oxytocin (100 nmol/l) for 24 h. LH release stimulated by oxytocin increased (P < 0·001) progressively during the incubation indicating a different secretory pattern from the more rapid but less sustained secretion stimulated by gonadotrophin-releasing hormone. Oxytocin also enhanced (P < 0·01) total LH accumulation in the incubation system (released plus cell contents) which was apparent after 7–11 h of stimulation. The release of LH stimulated by oxytocin was reduced by the protein synthesis inhibitor cycloheximide (10 μmol/l). However, cycloheximide did not completely block oxytocin-stimulated LH release; there remained some LH release above that seen in non-stimulated controls (P < 0·01) revealing the presence of a cycloheximide-resistant component in the release mechanism. Furthermore, accumulation of total LH in 24 h incubations was suppressed (P < 0·01) by cycloheximide.

The advancement in LH release which oxytocin has been shown to induce in vivo in pro-oestrous rats was accompanied by an early reduction of pituitary LH stores. However, the fall normally observed in LH content during the surge was markedly attenuated by the oxytocin treatment. Thus, loss of pituitary LH stores was less in oxytocin-treated rats than in saline-treated controls, even though net LH release into plasma was increased. Therefore, oxytocin stimulated the replenishment of LH stores.

Although the mechanism(s) remains to be defined and the relationships between in-vitro and in-vivo results are as yet uncharacterized, the present study demonstrates that oxytocin treatment stimulates LH production in both dispersed cells and intact pituitaries in situ.

Journal of Endocrinology (1992) 132, 277–283

ABSTRACT

The effect of restricting placental growth on maternal glucose, insulin and placental lactogen was investigated in 16 ewes carrying singleton lambs. Uterine caruncles were removed from seven ewes (caruncle ewes) before pregnancy, resulting in reduced placental size and retarded intra-uterine fetal growth. The concentration of insulin in maternal plasma was similar in both control and caruncle ewes. The concentration of glucose was significantly higher in the caruncle than in the control ewes (3·26 ± 0·15 (s.e.m.) mmol/l, number of observations (n) = 9, vs 2·75 ± 0·1, n = 9, P<0·02, and 3·27 ±0·16, n = 7, vs 2·46± 0·11, n = 12, P<0·001, for the carotid artery and utero-ovarian vein respectively). The concentration of ovine placental lactogen (oPL) in the utero-ovarian vein was reduced in the caruncle compared with the control ewes (283± 65 μg/l, n = and 705±106 μg/l, n = 18, P<0·02, respectively). Restriction of placental growth by removal of endometrial caruncles similarly reduced the concentrations of oPL in maternal arterial plasma (231±54 μg/l, n = 9, and 621±96 μg/l, n = 18, P<0·002). Production of oPL by the placenta was also reduced by limiting placental growth to 30±11 μg/min, n = 8, compared with 133±43 μg/min, n = 15, P<0·05, for the controls. Production of oPL per gram of placenta in the caruncle group, although only 34% of the control value, was not reduced significantly. These observations are consistent with the hypothesis that oPL may be involved in the redirection of maternal glucose during pregnancy to maximize the amount available for the fetus.

J. Endocr. (1985) 106, 7–11

ABSTRACT

Neurohypophysial hormones have been implicated in the control of anterior pituitary function, and oxytocin has been shown to stimulate gonadotrophin excretion and ovarian follicular development in certain species. To determine the role of neurohypophysial peptides in the control of gonadotrophin release, their actions on LH and FSH secretion were analysed in rats in vivo and in vitro. In adult female rats, administration of oxytocin during early pro-oestrus advanced the spontaneous LH surge and markedly increased peripheral LH levels at 15.00 h compared with control animals. In cultured pituitary cells from adult female rats, oxytocin and vasopressin elicited dose-related increases in LH and FSH release. Such responses were not affected by a potent gonadotrophin-releasing hormone (GnRH) antagonist that abolished GnRH agonist-induced release of LH and FSH. Oxytocin did not enhance GnRH agonist-stimulated gonadotrophin release to the same extent as it increased basal secretion, but at low concentrations of GnRH agonist the effects were additive. The gonadotrophin responses to oxytocin and vasopressin were inhibited by the specific neurohypophysial hormone antagonists, [d(CH₂)₅ d-Ile²,Ile⁴,Arg⁸]vasopressin and [d(CH₂)₅Tyr (Me),Arg⁸]vasopressin. These results provide direct evidence that neurohypophysial hormones can stimulate gonadotrophin secretion through a receptor system distinct from the GnRH receptor. Such a mechanism could represent a complementary hypothalamic control system for long-term modulation of LH and FSH secretion by exerting a basal or tonic influence on gonadotrophin production.

Journal of Endocrinology (1989) 122, 99–106