Intact Ewes Research Articles

Quantification of opioid-binding sites in the ewe hypothalamus

Opioid-binding sites were quantified in the ewe hypothalamus using [3H]diprenorphine ([3H]DIP) as the radioligand. [3H]DIP binding to hypothalamic membrane preparations was stereospecific, saturable with respect to [3H]DIP concentration, and linear with hypothalamic membrane protein content. Scatchard analysis revealed a single class of binding sites. There were no significant differences in binding site concentration or binding affinity in hypothalami from intact ewes during the breeding and non-breeding seasons, or from long-term ovariectomized ewes with and without oestradiol treatment during the breeding season. Thus, whilst ovarian steroid hormones are known to modify LH responses to opioids and their antagonists in the ewe in vivo, they do not appear to do this by modulating the numbers of hypothalamic opioid-binding sites.

Effects of ACTH and cortisol on luteolysis in the ewe

Daily administration of 100 IU adrenocorticotrophic hormone (ACTH) on days 12–16 of the oestrous cycle was found to delay oestrus and induce formation of follicular cysts; daily cortisol infusions on days 12–16 did not have these effects. In the ACTH-treated ewes release of 13,14-dihydro-15-keto-prostaglandin F 2α was depressed on days 13–16, and decline in progesterone concentrations was delayed. Intramuscular administration of ACTH (25 IU) significantly raised plasma concentrations of progesterone in ovariectomized ewes, relative to saline-treated controls. Peak value of progesterone were between 0.76 and 1.1. ng.ml −1. In ovariectomized ewes primed with first progesterone (20 mg.day −1 × 6 days) and then oestradiol-17β (500 μg.day −1 × 3 days). ACTH (25 IU) significantly reduced the ability of oxytocin to release 13,14-dihydro-15-keto-prostaglandin F 2α into the peripheral circulation. Both these effects may contribute to the ability of ACTH to delay oestrus in intact ewes.

Role of the corpus luteum of pregnancy in controlling pituitary gonadotrophin secretion during the early post-partum period in the ewe

No difference was found between 5 intact ewes and 5 ewes from which the CL had been excised at Day 70 of pregnancy in the plasma concentration of progesterone at Day 140, and concentrations of progesterone remained below 0.2 ng/ml during the first 20 days post partum. Plasma concentrations of LH, frequency and amplitude of LH pulses were low at Day 140 and increased considerably, particularly in the CL-excised ewes, as early as Day 5 post partum. No significant differences were found between the two groups of ewes in the mean plasma concentrations of FSH for any of the 5 stages examined. Taken together, these results suggest that some factor, other than progesterone, associated with the CL of pregnancy is involved in the inhibition of pulsatile LH secretion during the early post-partum period.

The oestrogen-induced surge of LH requires a 'signal' pattern of gonadotrophin-releasing hormone input to the pituitary gland in the ewe.

Two experiments were conducted with ovariectomized and hypothalamo-pituitary disconnected (HPD) ewes to ascertain the pattern of inputs, to the pituitary gland, of gonadotrophin-releasing hormone (GnRH) necessary for the full expression of an oestrogen-induced LH surge. The standard GnRH replacement to these sheep was to give pulses of 250 ng (i.v.) every 2h; at the onset of experimentation, pulses were given hourly. In experiment 1, groups of sheep (n = 7) were given an i.m. injection of 50 micrograms oestradiol benzoate, and after 10 h the GnRH pulse frequency or pulse amplitude was doubled. Monitoring of plasma LH concentrations showed that a doubling of pulse frequency produced a marked increase in baseline values, whereas a doubling of amplitude had little effect on the LH response. In a second experiment, ovariectomized HPD sheep that had received hourly pulses of GnRH for 16 h after an i.m. injection of oil or 50 micrograms oestradiol benzoate were given either a 'bolus' (2.25 micrograms GnRH) or a 'volley' (500 ng GnRH pulses 10 min apart for 30 min, plus a 500 ng pulse 15 min later). Both groups then received GnRH pulses (250 ng) every 30 min for the next 13 h. Oestrogen enhanced the LH responses to the GnRH treatments, and the amount of LH released was similar in ovariectomized HPD ewes given oestrogen plus bolus or volley GnRH treatments and ovariectomized hypothalamo-pituitary intact ewes given oestrogen. These results suggest that the oestrogen-induced LH surge is initiated by a 'signal' pattern of GnRH secretion from the hypothalamus.

Plasma prolactin concentrations in pinealectomized ewes receiving melatonin treatment and in pineal intact ewes maintained under a non-24-hour photoperiod.

We have investigated the profiles of prolactin secretion in relation to onset of breeding activity in Suffolk-Cross ewes with artificially modified melatonin rhythms. With treatments commencing in midsummer, groups of ewes were 1) subjected to a 8L:16D photoperiod, 2) maintained under a "short" (6L:16D) photoperiod repeated over a 22-h cycle that induces an elevation in plasma melatonin that does not endure for the entire dark phase, 3) pinealectomized (pnx) to abolish plasma melatonin levels, and 4) pinealectomized and treated with a melatonin implant (subcutaneous) to provide a constant (no 24-h rhythm) elevation in plasma melatonin. The onset of breeding activity was significantly advanced in both the 8L:16D and pnx/implant groups compared to the 6L:16D and untreated pnx ewes. The two latter groups displayed a normal timing in seasonal breeding activity. Low and high plasma prolactin levels corresponded with short and long photoperiods during both the 24 and 22-h cycles. There was no clearcut "seasonal" rhythm in plasma prolactin in either of the pnx groups. A clear differentiation was seen between reproductive response and prolactin response, particularly in the case of ewes monitored on 22-h cycles of short photoperiod.

Evidence that the corpus luteum of pregnancy contributes to the control of tonic secretion of LH in the ewe

Concentrations of LH and FSH were measured in blood samples collected from the jugular vein at 20-min intervals for 7 h (09:00-16:00 h) on Days 60, 80, 100 and 120 of pregnancy in 5 intact ewes and 5 from which the CL had been excised on Day 70. In the 5 intact ewes, plasma LH concentrations remained low and unchanged between Days 60 and 120. During this period, pulsatile release of LH occurred irregularly and infrequently. Removal of the CL resulted in an increase in the basal values of LH and in the frequency and amplitude of LH pulses. Concentrations of FSH were relatively constant in all stages of pregnancy examined and were similar in both groups of ewes. These results show that (1) LH concentrations are low during the second half of pregnancy; and (2) LH, but not FSH, increases after CL excision, presumably by removing some luteal factor inhibitor of LH secretion.

Functional organization of the catecholaminergic neural systems inhibiting luteinizing hormone secretion in anestrous ewes.

Both dopaminergic (DA) and noradrenergic (NA) neural systems contribute to the suppression of luteinizing hormone (LH) pulse frequency in intact anestrous ewes. In this study, we explored the functional relationship between these two inhibitory neural systems by determining if DA or NA antagonists could block the inhibitory actions of DA and NA agonists in ovariectomized anestrous ewes. If these systems are linked 'in series' with one (e.g. NA) exerting its effects by stimulating the other (e.g. DA), then one antagonist (e.g. DA) should block the inhibitory actions of both agonists. If they are organized 'in parallel', then each antagonist should only block the actions of the homologous agonist. In the first experiment, the DA agonist, apomorphine, suppressed LH pulse frequency and this action was blocked by the DA antagonist, pimozide, but not by the NA antagonist, phenoxybenzamine. Similarly, in experiment 2, the ability of the NA agonist, clonidine, to suppress LH pulse frequency was blocked by the NA antagonist. However, the DA antagonist also partially blocked the inhibitory effects of this NA agonist. We next repeated experiment 2 using a lower dose of the NA agonist. The lower dose of clonidine again inhibited LH pulse frequency and this effect was completely blocked by the DA antagonist. These results suggest that the NA agonist suppressed LH pulse frequency by stimulating a set of inhibitory DA neurons and are thus compatible with the hypothesis that the catecholaminergic neurons controlling LH secretion in anestrous ewes are organized 'in series' with the NA neurons stimulating a DA neural system that directly suppresses GnRH release.

CONTROL OF THE SURGE RELEASE OF LH AND FSH IN ESTRADIOL- AND PROGESTERONE-TREATED OVARIECTOMIZED EWES

Ovariectomized ewes were treated with silastic rubber implants releasing constant amounts of estradiol. During this time the ewes were treated with progesterone-releasing implants for 8–12 d, followed by a 5-d rest period and a second period of progesterone exposure of approximately 12 d duration. Following the first exposure to progesterone all ewes experienced a transient, but large release of LH and FSH from the pituitary, resembling that seen in intact ewes prior to ovulation (the so called preovulatory LH and FSH surges), but with amplitudes in the lower end of the range for the preovulatory surges in intact ewes. Following the second exposure to progesterone, the amplitudes of LH and FSH surges were reduced in most ewes. Altering mean progesterone levels between 0.9 ± 0.43 ng mL−1 and 2.2 ± 0.26 ng mL−1 in the second exposure did not affect the amplitude of LH or FSH surges, neither did altering estradiol levels from physiological to 4 × physiological. However, altering the temporal pattern of progesterone in both exposures from a sharp increase and decrease at either end of the period, to a more gradual increase and decrease, resembling the normal luteal phase of intact ewes, resulted in an increased amplitude of LH and FSH surges following the second exposure to progesterone. Key words: Ovariectomized ewe, luteinizing hormone, follicle-stimulating hormone, progesterone, estradiol

Effects of an opioid antagonist on pulsatile luteinizing hormone secretion in the ewe vary with changes in steroid negative feedback.

In ewes during the breeding season, estradiol (E) and progesterone (P) synergistically regulate pulsatile luteinizing hormone (LH) secretion. E primarily inhibits LH pulse amplitude and P inhibits LH pulse frequency. To determine if endogenous opioid peptides (EOP) mediate these negative feedback effects, we administered the long-acting opioid antagonist WIN 44,441-3 (WIN) to intact ewes during the luteal and follicular phases of the estrous cycle and to ovariectomized ewes treated with no steroids, E, P, or E plus P. Steroid levels were maintained at levels seen during the estrous cycle by Silastic implants placed shortly after surgery. WIN increased LH pulse frequency, but not amplitude, in luteal phase ewes. In contrast, during the follicular phase, LH pulse amplitude was increased by WIN and pulse frequency was unchanged. Neither LH pulse frequency nor pulse amplitude was affected by WIN in long-term ovariectomized ewes untreated with steroids. In contrast, WIN slightly increased LH pulse frequency in short-term ovariectomized ewes. WIN also increased LH pulse frequency in ovariectomized ewes treated with P or E plus P. WIN did not affect pulse frequency but did increase LH pulse amplitude in E-treated ewes. These results support the hypothesis that EOP participate in the negative feedback effects of E and P on pulsatile LH secretion during the breeding season and that the inhibitory effects of EOP may persist for some time after ovariectomy.

Effects of bovine follicular fluid on gonadotrophin secretion in intact and chronically ovariectomized ewes before and after desensitization of pituitary gonadotrophs to gonadotrophin-releasing hormone.

Intact and chronically ovariectomized ewes were treated for 4 days with charcoal-treated bovine follicular fluid (FF) or charcoal-treated bovine serum during the late-anoestrous period, and the effects on basal and gonadotrophin-releasing hormone (GnRH)-induced secretion of LH and FSH observed. Subsequently, ewes received s.c. implants containing a sustained-release formulation of a potent GnRH agonist D-Ser(But)6-Azgly10-LHRH (ICI 118630) to desensitize pituitary gonadotrophs to hypothalamic stimulation, and the effects of bovine FF and bovine serum were re-assessed 2 weeks later. Chronic exposure (for 2-3 weeks) to ICI 118630 significantly reduced basal levels of LH and FSH in both intact and ovariectomized ewes and completely abolished both spontaneous LH pulses as well as exogenous GnRH-induced acute increases in plasma LH and FSH levels. Treatment with bovine FF significantly reduced plasma FSH levels, but not LH levels, in both intact and ovariectomized ewes before and after chronic exposure to ICI 118630. In intact ewes before exposure to ICI 118630, treatment with bovine FF actually enhanced pulsatile LH secretion and raised mean plasma LH levels by 240% (P less than 0.05). No such stimulatory effect of bovine FF on LH secretion was observed in intact ewes exposed to ICI 118630 or in ovariectomized ewes before or after exposure to ICI 118630, suggesting that the effect probably involved an alteration in ovarian steroid feedback affecting hypothalamic GnRH output. Treatment with bovine FF did not significantly affect the magnitude of GnRH-induced surges of LH or of FSH observed in either intact or ovariectomized ewes before exposure to ICI 118630.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons.

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.

Ovine uterine morphogenesis: histochemical aspects of endometrial development in the fetus and neonate.

Uterine tissues obtained from fetal (d 60 to term; n = 17; d 0 = day of mating) and neonatal (n = 9; d 0 = birth) lambs were subjected to alcian blue-8GX and fluorescein isothiocyanate (FITC)-lectin histochemistry to determine if alcianophilic properties of the epitheliomesenchymal interface (EMI) changed during endometrial morphogenesis and to characterize distribution of binding sites for seven FITC-lectins during uterine development. Neonatal lambs were subjected to bilateral ovariectomy and unilateral hysterectomy (BOHX; n = 3) or unilateral ovariohysterectomy (UOHX; n = 3) on d 0. Remaining tissues were recovered on d 14. Procedures allowed within-animal comparisons of endometrial responses and assessment of the role of the ovary in endometrial morphogenesis. Uteri also were obtained from three intact neonatal lambs by hysterectomy (d 14, d 15 and d 26). Alcianophilic properties of the EMI characteristic of polyanionic glycosaminoglycans (GAG) changed with onset of endometrial remodelling after fetal d 60 and were characterized by loss of EMI alcianophilia at or above .3 M MgCl2 and at low pH. Alcianophilic properties of the neonatal endometrium suggested restabilization of lumenal EMI and destabilization of the EMI in developing endometrial glands. Five of seven FITC-lectins bound to both fetal and neonatal uterine tissue. Tissues from UOHX, BOHX and intact ewes were indistinguishable histochemically. Data provide evidence of a role for GAG in ovine endometrial morphogenesis, ovary-independent initiation of endometrial glandular development, and illustrate potential uses of FITC-lectin conjugates in studies of ungulate uterine tissues.

Changes in LH pulse frequency and serum progesterone concentrations during the transition to breeding season in ewes.

To characterize the changes in LH pulse frequency during the transition to breeding season. LH pulse patterns and serum progesterone profiles were determined in 8 intact ewes from mid-anoestrus to the early breeding season. Overall, 8 increases in LH pulse frequency were observed and these were restricted to 5 ewes. Of the 8 increases, 7 occurred during the 4 weeks before the first cycle, 5 of them within 1 week after a pulse frequency typical of anoestrus (0-2 per 8 h). Six of them occurred less than 1 week before either a full-length luteal phase (n = 2) or a 1-3-day increment in progesterone (n = 4). Seven of these brief progesterone increases were observed in 6 ewes, 5 of them immediately preceding the first full-length luteal phase. These results are consistent with the hypothesis that the seasonal decrease in response to oestradiol negative feedback at the beginning of the breeding season causes an increase in GnRH, and thereby LH pulse frequency. In addition, they demonstrate that the first increase in tonic LH secretion occurs in less than 1 week and, in most ewes, initiates either the first full-length cycle or a transient increase in progesterone, the latter occurring more often.

Effect and Site of Action of Hypothalamic Neuropeptides on Prolactin Release in Sheep

In an attempt to identify a physiological prolactin-releasing factor in the sheep, ovariectomized ewes were given intracarotid injections (10(-8)-10(-7) mol/animal) of thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP), peptide histidine-isoleucine amide (PHI), oxytocin (OT), arginine vasopressin (AVP), substance P (SP), bombesin (BB), neurotensin (NT) and neuropeptide Y (NPY). Administration of TRH, AVP, NT and OT resulted in immediate and significant increases in plasma prolactin concentrations, the greatest stimulatory effect being obtained after TRH; other peptides had no effect in ovariectomized hypothalamo-pituitary intact ewes. AVP, NT and OT failed to release prolactin in ovariectomized ewes. These results suggest that (1) AVP, NT and OT may act via the hypothalamus to regulate prolactin secretion in hypothalamo-pituitary intact ewes; (2) VIP, PHI, SP, BB and NPY appear to have no direct roles at the pituitary level to control prolactin secretion in sheep, and (3) TRH stimulates prolactin secretion in ovariectomized ewes by a direct pituitary action.

Early pituitary follicle stimulating hormone response to gonadotrophin releasing hormone in ewes

The object of our experiments was to characterize the response of plasma follicle stimulating hormone (FSH) within minutes of an i.v. injection of high or low doses of gonadotrophin releasing hormone (GnRH), especially in relation to contemporary changes in luteinizing hormone (LH) concentrations. In the deep anoestrous period (June), three intact ewes and two ovariectomized ewes were injected with 1 μg synthetic GnRH followed 2 h later by a second identical injection. A week later, the same regimen was repeated with the same sheep but with 50 μg GnRH after an interval of 5 h 20 min. Blood samples were collected every 15 sec for 15 min after each injection (early release), then at longer intervals (main release) till the next treatment, followed by sampling for a further 6-h period after the second treatment. FSH was released as soon as the second minute after GnRH injection in all ewes. The mean pituitary FSH response, during this early release, in intact and ovariectomized ewes was similar after either 1 or 50 μg GnRH. However, the main release was less pronounced in the ovariectomized sheep and was not stimulated after the second treatment in all sheep. Three other ewes were injected with 40 μg GnRH and sampled every 15 sec for seven, 6-min periods during the period of release to compare FSH and LH secretion. The profiles reflected a similarity in sensitivity and responsiveness to GnRH, especially soon after GnRH injection. Increases in both hormones were formed by several grouped associated spikes. It is suggested that a readily releasable pool of FSH exists in the ewe. There are probably differences in the mechanisms of synthesis and/or release between pituitary FSH and LH.

Short-term variations of circulating melatonin in the ewe.

Melatonin levels have been studied in venous blood sampled at different frequencies (0.5-, 2-, and 60-min intervals) form intact ewes. All samples were taken during the dark phase of either natural or artificial photoperiods. In one experiment samples were taken simultaneously from both jugular veins to investigate the possible effects of "streaming" on the levels measured. Plasma cortisol was measured to ascertain whether or not the frequent removal of blood activated the ACTH stress axis. Plasma melatonin levels showed considerable variation with peaks of up to 365 pg/ml on a baseline of between 30 and 60 pg/ml. There was consistent evidence of intermittent peaks, the frequency of which increased with an increase in sampling frequency. Plasma cortisol showed no correlation with either the frequency or the amplitude of the melatonin peaks. When plasma samples were taken from both jugular veins a similar melatonin pattern was seen in the samples from both sides, but samples taken from the left jugular vein invariably showed higher levels than those taken from the right vein. This may be due to differential vascular drainage of the pineal to the two sides.

Effect of small doses of bovine follicular fluid on the tonic secretion of gonadotrophins in the ewe.

Previous work has shown that treatment of ewes with steroid-free bovine follicular fluid (bFF), a rich source of inhibin, partially inhibits the increase in mean plasma concentrations of LH induced by ovariectomy. The present experiment was designed to test the hypothesis that this effect was a reflection of reduced LH pulse amplitude which would only be expressed at high (pharmacological) doses of bFF. To do this, we assessed the dose-response to bFF of the secretion of FSH and LH pulses in intact and acutely ovariectomized ewes. In intact ewes, a low dose of bFF (0.2 ml s.c. every 8 h) had no detectable effect on the secretion of FSH, an intermediate dose (0.6 ml s.c. every 8 h) depressed FSH concentrations for about 24 h and a high dose (1.8 ml s.c. every 8 h) reduced FSH concentrations to undetectable levels. In ewes treated with 1.8 ml bFF, FSH concentrations also remained undetectable after ovariectomy and did not increase until treatment was withdrawn. In ewes treated with 0.6 ml bFF, FSH concentrations were maintained at normal intact levels for about 32 h following ovariectomy but then rose to normal ovariectomized levels. In ewes treated with 0.2 ml bFF, FSH concentrations increased immediately after ovariectomy but more slowly than in control ovariectomized ewes. Profiles of LH pulses were recorded after ovariectomy, during and after the withdrawal of bFF treatment. In ewes treated with the highest dose (1.8 ml s.c. every 8 h), mean LH levels and pulse amplitude were lower than in control ewes and increased significantly following withdrawal of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian features contributing to the variability of PMSG-induced ovulation rate in sheep.

In Exp. 1, ovulation rate was measured in three groups of Romanov ewes given two injections of 600 i.u. PMSG 3 weeks apart with the ewes intact (Group I, N = 8), a similar treatment with the ewes intact at the first injection and unilaterally ovariectomized at the second (Group II, N = 8), or unstimulated ewes which were hemispayed at the same time as Group II ewes (Group III, N = 6). In Exp. 2, the follicular population of one ovary was correlated with the number of ovulations induced by 600 i.u. PMSG in the contralateral ovary (10 Romanov ewes). From 8.4 +/- 1.8 (Group I) and 8.2 +/- 3.3 (Group II) CL at the first injection, PMSG-induced ovulation rate at the second injection decreased to 3.9 +/- 1.8 and 3.7 +/- 1.2 in Groups I and II respectively, a value similar for ewes with 1 or 2 ovaries. Furthermore, despite no major changes in the number of antral follicles after the first injection, there was no correlation (r = -0.09) between the response to the two successive injections in intact ewes. Comparison of the ovarian status of the ovary removed before the PMSG injection (Group II ewes of Exp. 1, ewes of Exp. 2) to the number of CL found in the remaining ovary demonstrated that PMSG-induced ovulation rate was not correlated with the overall antral follicle population (r = 0.62 in Exp. 1, r = 0.49 in Exp. 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Pulsatile LH secretion during the preovulatory surge in the ewe: experimental observations and theoretical considerations.

The secretion of LH during the preovulatory surge has been shown to be pulsatile in several species. We confirmed this phenomenon in intact ewes in two experiments in which we studied the LH surge in detail. In the first experiment, the levels of LH were measured in discrete blood samples taken every 5 min around the onset of oestrus in 5 intact Merino ewes. For 2-5 h immediately prior to the onset of the surge, the median pulse interval was 17.5 min (range 10-55) and the median pulse amplitude was 0.70 ng/ml (range 0.28-1.33 ng/ml). During the ascending phase of the surge, the pulse interval did not change significantly (median 18.3 min) but the median pulse amplitude increased 20 fold to 18.0 ng/ml. During the descending phase, the median pulse interval was 22.5 min and the median amplitude decreased to 8.1 ng/ml (P less than 0.005). During the surge, intervals of 10 min and amplitudes over 30 ng/ml were common. In the second experiment with 2 oestrous Romanov x Préalpes-du-Sud ewes, blood was withdrawn continuously and samples were pooled over 4 min periods for 19-27 h. In the resulting profiles, individual pulses were better defined but the pulse frequencies (median interval 15.2 min during ascending phase) and amplitudes (median 35.0 ng/ml) varied in similar fashion to the Merinos. Using a simple mathematical description of pulsatile secretion, and assuming that LH is only released in pulses, we also found that the high concentrations normally achieved during the surge can only be explained by pulse frequencies and amplitudes well above those normally observed in other physiological situations, including the follicular phase or following ovariectomy. These findings strongly support the experimental data and the combined results of the two studies suggest that the initiation and execution of the LH surge is probably accomplished by changes in the amplitude of pulses. A high frequency is also essential and our data suggest that it may be established some time before the onset of the surge proper, but this requires confirmation. Respectively, the changes in amplitude and frequency reflect the pituitary and hypothalamic components of the positive feedback mechanism. The theoretical study suggested that a change in clearance rate could also contribute to the surge and this awaits further investigation.

Progesterone antagonizes the ability of porcine ovarian inhibin to sensitize ovine pituitary cell culture to luteinizing hormone-releasing hormone: dependence on ovaries in vivo.

Progesterone (P4) and a porcine follicular preparation of inhibin (MGRA-IV) have opposite actions on regulation of the ability of LHRH to release LH in ovine pituitary cell culture. Both P4 and inhibin change the response to LHRH. The ability of inhibin to sensitize cultures to LHRH (126-273%) was greatly inhibited (up to 100%) in the presence of P4 (10(-7) M). The inhibitory action of P4 on LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion in ovine pituitary cell culture was dependent on the presence of ovaries in vivo. P4 inhibited 68% of LHRH-stimulated LH secretion in pituitary cultures from intact ewes. However, when cultures were prepared from pituitaries collected on days 9, 21, and 42 after ovariectomy, P4 inhibited LHRH-stimulated LH secretion by only 36%, 13%, and 0%, respectively. Ovariectomy had no effect (P greater than 0.05) on the sensitizing action of inhibin on LHRH-stimulated LH secretion, but ovariectomy did cause a time-dependent decline in the inhibitory action of P4 on inhibin-sensitized LHRH-stimulated LH secretion. Furthermore, when cultures were prepared from pituitaries collected from ewes ovariectomized for 35 days but treated with estradiol implants, both LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion were inhibited as well by P4 as in pituitary cultures from intact ewes. These results suggest that although P4 can completely inhibit the sensitizing action of inhibin on LHRH-stimulated LH secretion, its inhibitory action is dependent on the presence of ovaries or estradiol in vivo.