Changes in pulsatile LH secretion after ovariectomy in Ile-de-France ewes in two seasons

Two experiments were conducted in Ile-de-France ewes to study changes in pulsatile LH secretion in ewes ovariectomized during anoestrus or during the midluteal phase of the oestrous cycle. In Exp. 1, blood samples were taken every 20 min for 12 h the day before ovariectomy (Day 0). After ovariectomy, samples were taken every 10 min for 6 h (10 ewes per group), on Days 1, 3, 7 and 15. In Exp. 2 samples were taken every 10 min for 6 h (10 ewes per group) on Days 7, 15, 30, 60, 90, 120, 150 and 180 after ovariectomy. Further samples were taken (5 ewes per group) at 9 and 12 months after ovariectomy. There were significant interactions between season and day of sampling for the interval between LH pulses in both experiments. LH pulse frequency increased within 1 day of ovariectomy and the increase was more rapid during the breeding season. There were clear seasonal differences in pulse frequency in Exp. 2. Compared with ewes ovariectomized in anoestrus, pulse frequency was significantly higher for ewes ovariectomized in the breeding season, from Day 7 until Day 120. Once pulse frequency had increased in ewes about the time of the normal breeding season, pulse frequency remained high and subsequent seasonal changes were greatly reduced. Pulse amplitude increased immediately after ovariectomy to reach a maximum on Day 7 and there were no differences between season of ovariectomy in the initial changes in amplitude. In Exp. 2, changes in amplitude followed changes in pulse interval and there was a significant interaction between season and day of sampling. There were no significant effects of season on nadir LH concentrations which increased throughout the duration of the experiments. These results show that, in ovariectomized ewes, LH pulse frequency observed on a given day depends on time after ovariectomy, season at the time of sampling and on previous exposure of ewes to stimulatory effects of season. The direct effects of season on LH pulse frequency and seasonal changes in sensitivity to steroid feedback may contribute to control of the breeding season and their relative contributions to the beginning and end of the breeding season may differ.     

Effects of time after ovariectomy, season and oestradiol on luteinizing hormone and follicle-stimulating hormone secretion in ovariectomized ewes.

During the breeding season, five groups of three ewes were implanted at ovariectomy with 0.36, 0.5, 1.0 and 6.0 cm oestradiol implants or implants containing no steroid. Eleven days after receiving implants, blood samples were taken every 10 min for 6 h; implants were then removed. Treatments were repeated three times during each of two consecutive breeding seasons and four times during the intervening anoestrus. In ovariectomized ewes without steroid treatment, luteinizing hormone (LH) pulse frequency increased from early to mid-breeding season, decreased to a minimum at mid-anoestrus and increased to reach a maximum at the mid-point of the second breeding season, subsequently declining. LH pulse amplitude was inversely related to frequency. Basal serum LH concentrations decreased gradually from the first breeding season to reach a minimum at mid-anoestrus and gradually increased to reach a maximum at the end of the second breeding season. Mean serum LH and follicle-stimulating hormone (FSH) concentrations were higher at the end of the second breeding season compared with the beginning of the first breeding season. All parameters of gonadotrophin secretion were decreased much more by oestradiol during the anoestrus than during the breeding season. LH pulse frequency was decreased during anoestrus and at high oestradiol concentrations during the first breeding season. Apart from LH pulse amplitude, the decreases in all parameters of gonadotrophin secretion were less during the second compared with the first breeding season. The minimum effective dose of oestradiol required to decrease mean and basal serum concentrations of LH during anoestrus was lower than in the breeding season. The minimum effective dose of oestradiol required to decrease mean serum concentrations of FSH was lower in the first compared with the second breeding season. Oestradiol depression of LH pulse amplitude and mean serum concentrations of LH and FSH showed a dose dependency during the breeding season. During anoestrus dose dependency was seen for basal concentrations of LH and mean serum concentrations of LH and FSH. We conclude that significant chronic changes in gonadotrophin secretion occur in the ewe with time after ovariectomy. Sensitivity to oestradiol also changes, and the effects of oestradiol are not always dose dependent. We suggest that the circannual pattern of LH pulse frequency and basal LH secretion are directly linked to the circannual cycle of photoperiod.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year

Overall, significantly more antral follicles greater than or equal to 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 +/- 3 (mean +/- s.e.m.) follicles per ewe, 23 sheep; Day 9-10 of oestrous cycle, 24 +/- 1 follicles per ewe, 22 sheep; P less than 0.01), although the mean numbers of preovulatory-sized follicles (greater than or equal to 5 mm diam.) were similar (anoestrus, 1.3 +/- 0.2 per ewe; oestrous cycle, 1.0 +/- 0.1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9-10 of the oestrous cycle there were significantly more (P less than 0.001) high-amplitude LH peaks (i.e. greater than or equal to 1 ng/ml) in plasma and significantly fewer (P less than 0.001) low amplitude peaks (less than 1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P less than 0.05, prolactin, P less than 0.001). It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.     

Effect of transport on pulsatile LH release in ovariectomized ewes with or without prior steroid exposure at different times of year

The initial aim of the present study was to test whether the stress of transport suppresses LH pulsatile secretion in ewes. In a pilot experiment in the late breeding season, transport resulted in an unexpected response in three out of five transported, ovariectomized ewes pretreated with oestradiol and progesterone. Before transport, seasonal suppression of LH pulses had occurred earlier than anticipated, but LH pulsatility suddenly restarted for the period of transport. This finding was reminiscent of unexplained results obtained in ovariectomized ewes infused centrally with high doses of corticotrophin-releasing hormone after pretreatment with low doses of oestradiol with or without progesterone. Hence, an additional aim of the present study was to examine whether these latter results with corticotrophin-releasing hormone could be reproduced by increasing endogenous corticotrophin-releasing hormone secretion by transport. Subsequent experiments used groups of at least eight ovariectomized ewes at different times of the year with or without prior exposure to steroids to assess whether these unexpected observations were associated with season or the prevailing endocrine milieu. In the mid-breeding season, transport for 4 h in the absence of steroid pretreatment for 8 months reduced LH pulse frequency from 7.5 +/- 0.3 to 6.3 +/- 0.4 pulses per 4 h (P < 0.05) and LH pulse amplitude from 2.6 +/- 0.5 to 1.8 +/- 0.3 ng ml-1 (P < 0.05). Similarly, in the mid-breeding season, 34 h after the cessation of pretreatment with oestradiol and progesterone, transport suppressed LH pulse frequency from 6.1 +/- 0.4 to 5.5 +/- 0.3 pulses per 4 h (P < 0.05) with a tendency of effect on amplitude (6.2 +/- 2.7 to 2.61 +/- 0.6 ng ml-1; P = 0.07; note the large variance in the pretransport data). During mid-anoestrus, evidence of a suppressive effect of transport was only observed on LH pulse amplitude (4.7 +/- 0.6 versus 3.0 +/- 0.5 pulses per 4 h; P < 0.05) in ovariectomized ewes that had not been exposed to ovarian steroids for 4 months. Repetition of the pilot experiment with 12 ewes during the transition into anoestrus resulted in one ewe with LH pulses seasonally suppressed but increased by transport; 11 ewes had a distinct pulsatile LH pattern which was decreased by transport in six ewes. In anoestrus, there was no effect of transport on LH pulse frequency or amplitude in intact ewes, or those ovariectomized 2-3 weeks previously, with or without prior oestradiol and progesterone treatment. However, basal concentrations of cortisol were greater in anoestrus than in the breeding season, and the increment in cortisol during transport was similar in anoestrus and the breeding season but greater during the transition into anoestrus (P < 0.05). Progesterone concentrations increased from 0.31 +/- 0.02 ng ml-1 before transport to 0.48 +/- 0.05 ng ml-1 during the second hour of transport (P < 0.05). In conclusion, transport reduced LH pulse frequency and amplitude in ovariectomized ewes that had not been exposed to exogenous steroids for at least 4 months. In most animals, the previously observed increase in LH pulsatility induced by exogenous CRH was not reproduced by increasing endogenous CRH secretion by transport. However, in four ewes, transport did increase LH pulsatility, but only during the transition into anoestrus in ewes with seasonally suppressed LH profiles after withdrawal of steroid pretreatment.     

Season influences FSH concentration in ovariectomized Ile-de-France ewes

Ile-de-France ewes were ovariectomized during anoestrus or the mid-luteal phase of an oestrous cycle (day of ovariectomy = Day 0). In a short-term study, FSH concentrations were measured in blood samples collected hourly the day before and on Days 1, 3, 7 and 15 after ovariectomy (10 ewes per group). FSH concentrations increased significantly from 6.1 to 16.5 ng/ml within 1 day of ovariectomy and increased further to 47.1 ng/ml by Day 15. Differences between seasons of ovariectomy were not significant. In a long-term study, FSH concentrations were measured in blood samples collected hourly on Days 7, 15, 30, 60, 90, 120, 150 and 180 after ovariectomy in anoestrus or the breeding season (10 ewes per group). Further samples were taken (5 ewes/group) at 240 and 365 days after ovariectomy. The pattern of change in FSH after ovariectomy differed between the two seasons and the interaction between season and sampling day was significant. For ewes ovariectomized during anoestrus, FSH concentrations increased to a maximum by Day 180 and remained high thereafter. In contrast FSH increased more slowly in ewes ovariectomized in the breeding season and differences between the groups were significant from Day 90 to Day 270. However, both groups had similar FSH concentrations at Day 365. These results show that FSH concentrations increase rapidly after ovariectomy. There are seasonal differences in FSH concentrations in the absence of ovarian feedback with increases in FSH concentration around the time of the onset of the breeding season. Once FSH concentrations had reached a maximum, major seasonal changes were no longer apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seasonal changes in pulsatile luteinizing hormone (LH) secretion in the ewe: relationship of frequency of LH pulses to day length and response to estradiol negative feedback

Seasonal changes in pulsatile luteinizing hormone (LH) secretion in ovariectomized ewes were examined over the course of 2 yr in relation to annual changes in environmental photoperiod, shifts in response to estradiol negative feedback control of LH secretion, and timing of the breeding season. Under natural environmental conditions, the frequency of LH pulses in individual ovariectomized ewes changed gradually and in close association with the annual cycle of day length. As days became shorter in late summer and autumn, LH pulse frequency increased; conversely, as day length increased in late winter and spring, frequency declined. Under artificial conditions in which ovariectomized ewes were exposed to different photoperiods, a similar inverse relationship was observed between day length and LH pulse frequency. The seasonal changes in frequency of LH pulses in ovariectomized ewes, although symmetric with the annual photoperiodic cycle, were not temporally coupled to the dramatic shifts in response to estradiol feedback inhibition of LH secretion at the transitions between breeding season and anestrus. The feedback shifts occurred abruptly and at times when LH pulse frequency in ovariectomized ewes was at, or near, the annual maximum or minimum. The tight coupling between LH pulse frequency and photoperiod leads to the conclusion that there is a photoperiodic drive to the LH pulse-generating system of the ewe. The temporal dissociation between changes in this photoperiodic drive and the seasonal shifts in response to estradiol negative feedback support the hypothesis that the neuroendocrine basis for these two phenomena is not one and the same.     

Effects of breed, ovarian steroids and season on the pulsatile secretion of LH in ovariectomized ewes

The effects of season and of oestradiol and progesterone on the tonic secretion of LH were studied in ovariectomized Merino and Suffolk ewes, two breeds which differ markedly in the seasonal pattern of their reproductive activity. In the absence of exogenous steroids, the frequency of LH pulses was lower and the amplitude of the pulses was higher in anoestrus than in the breeding season for Merino and Suffolk ewes 30 days after ovariectomy. In long-term (190 days) ovariectomized ewes, this seasonal change in LH secretion was observed in Suffolk ewes only. During seasonal anoestrus, treatment of ewes with subcutaneous oestradiol-17 beta implants (3, 6 or 12 mm in length) decreased the frequency of LH pulses in a dose-dependent manner, with Suffolk ewes being far more sensitive to the inhibitory effects of oestradiol than Merino ewes. The lowest dose of oestradiol (3 mm) had no effect on the secretion of LH in Merino ewes, but reduced secretion in Suffolk ewes. Treatment of ewes with the highest dose of oestradiol (12 mm) completely abolished LH pulses in Suffolk ewes, whereas infrequent pulses remained evident in Merino ewes. During the breeding season, oestradiol alone had no effect on the pulsatile release of LH in either breed, but in combination with progesterone there was a significant reduction in LH pulse frequency. Progesterone effectively decreased LH secretion in both breeds in both seasons. It was concluded that differences between breeds in the 'depth' of anoestrus could be related to differences in the sensitivity of the hypothalamus to both negative feedback by oestradiol and the direct effects of photoperiod.     

Effects of pentobarbital anesthesia on tonic luteinizing hormone secretion in the ewe: evidence for active inhibition of luteinizing hormone in anestrus

In the ewe, seasonal anestrus appears to result from two effects of inhibitory photoperiod: 1) estradiol gains the capacity to suppress luteinizing hormone (LH) pulse frequency and hence becomes a potent inhibitor of tonic LH secretion and 2) a steroid-independent decrease in LH pulse frequency occurs in ovariectomized ewes. In this study, we have obtained evidence, using pentobarbital anesthesia, that both these actions of photoperiod reflect the activation, in anestrus, of an inhibitory neural system. Administration of pentobarbital to intact anestrous ewes produced a dramatic, 3-fold increase in LH pulse frequency during the 6 h of anesthesia. In contrast, during the breeding season, pentobarbital inhibited LH pulse frequency in luteal phase animals. There was also a seasonal variation in the effects of pentobarbital in ovariectomized ewes. During the breeding season this drug again suppressed LH secretion, inhibiting both LH pulse amplitude and frequency. In anestrus, pentobarbital also suppressed pulse amplitude, but it produced a transitory increase (lasting 3 h) in pulse frequency. To account for the stimulatory actions of pentobarbital, we propose that in anestrus, but not the breeding season, LH pulse frequency is held in check by a set of estradiol-sensitive inhibitory neurons. Further, we suggest that these neurons are activated by inhibitory photoperiod and account for both the steroid-dependent and steroid-independent actions of photoperiod.     

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.     

Plasma LH and FSH in ewes that were either fertile or infertile after long-term grazing of oestrogenic pasture

The levels of plasma LH and FSH were measured in serial blood samples taken at 15-min intervals for 6 h from ewes that had remained fertile after grazing oestrogenic pasture (clover-fertile ewes), from ewes that were permanently affected by clover disease (clover-infertile ewes) and from normal ewes. Two flocks of ewes from different locations were studied. In flock 1, tonic LH secretion (total area under the curve of LH concentration versus time, 1 area unit = 1 ng ml-1 x 1 h) was significantly (P < 0.05) greater in clover-infertile ewes (10.4 area units) during anoestrus than in ewes that had remained fertile after prolonged grazing of oestrogenic clover (5.4 area units). Tonic LH and FSH secretions during the bleeding season and FSH secretion during anoestrus were not significantly different. In flock 2, LH levels during the breeding season were significantly (P < 0.05) elevated in clover-infertile ewes (10.9 area units) compared to normal ewes (5.4 area units) that had never grazed oestrogenic clover. LH secretion in clover-infertile ewes (7.8 area units) was intermediate to that found in infertile and control ewes. Concentrations of FSH, progesterone and ovarian vein oestradiol-17 beta (E2) during the breeding season were similar in the three groups. In another experiment, the positive feedback release of LH following administration of E2 (12.5, 25 or 50 micrograms per ewe) was measured in anoestrous ewes of flock 2. Significantly (P < 0.01) more clover-infertile ewes demonstrated a positive feedback effect than control ewes when given 12.5 micrograms E2 but not when given higher doses. The elevation of LH secretion in permanently affected clover-infertile ewes is inconsistent with the hypothesis that the hypothalamo-pituitary axis of these ewes is less responsive to the negative feedback effect of oestrogen. Furthermore, the patency of the positive feedback loop is consistent with the ability to ovulate.     

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.     

Interactions between inhibin, oestradiol and progesterone in the control of gonadotrophin secretion in the ewe

Experiments were carried out to test the hypothesis that inhibin and oestradiol act synergistically to inhibit the secretion of FSH, to test for effects of progesterone, and to compare the FSH and LH responses to ovarian feedback. In Exp. 1, with 11 ovariectomized and 12 intact Romanov ewes during the anoestrous season, doses of oestradiol (administered by means of subcutaneous implants) that restored normal LH pulse frequencies were insufficient to restore normal concentrations of FSH. In Exp. 2, with 48 ovariectomized Welsh Mountain ewes during the breeding season, a factorial design with 4 ewes per cell was used to assess the responses in LH and FSH to 3 doses of oestradiol (s.c. implants) and 4 doses of bovine follicular fluid ('inhibin', 0.2-1.6 ml s.c. every 8 h). This was done initially in the absence of progesterone and then after 7 days of treatment with progesterone (s.c. implants). Analysis of variance revealed a significant synergistic interaction between oestradiol and inhibin on the plasma concentrations of FSH. Progesterone had little effect. In contrast, there was a significant synergistic interaction between oestradiol and progesterone on the concentrations of LH. 'Inhibin' also inhibited LH secretion but this effect was independent of the two steroids. We conclude that there are basic differences in the way that ovarian feedback acts to control the secretion of LH and FSH in the ewe. FSH secretion appears to be primarily controlled by the synergistic action of oestradiol and inhibin on the anterior pituitary gland, while the secretion of LH is inhibited during the follicular phase by an effect of oestrogen at pituitary level and during the luteal phase by the synergistic action of oestradiol and progesterone at the hypothalamic level. Inhibin, or another non-steroidal factor in follicular fluid, may also play a minor role in the control of LH secretion.     

Separate neural systems mediate the steroid-dependent and steroid-independent suppression of tonic luteinizing hormone secretion in the anestrous ewe

In the ewe, two types of seasonal fluctuations in secretion of tonic luteinizing hormone (LH) have been described: a steroid-dependent change whereby estradiol gains the capacity to suppress LH pulse frequency in anestrus, and a steroid-independent decrease in pulse frequency in ovariectomized animals during anestrus. We have proposed that the former reflects activation, in anestrus, of estradiol-sensitive catecholaminergic neurons that inhibit gonadotropin-releasing hormone (GnRH). Three results reported here support this hypothesis: dopaminergic (pimozide) and alpha-adrenergic (phenoxybenzamine) antagonists increased LH in intact anestrous ewes without altering pituitary responses to GnRH; other dopaminergic (fluphenazine) and alpha-adrenergic (dibenamine) antagonists also increased LH in anestrus; agonists for dopaminergic (apomorphine) and alpha-adrenergic (clonidine) receptors suppressed LH secretion in both seasons, suggesting that the appropriate receptors are present in breeding-season ewes. In contrast, catecholamines do not appear to mediate the steroid-independent suppression of pulse frequency; neither pimozide nor phenoxybenzamine increased LH pulse frequency in ovariectomized ewes during anestrus. When antagonists for 6 other neurotransmitter receptors (muscarinic and nicotinic cholinergic, GABAnergic, serotonergic, opioid, and beta-adrenergic) were tested in anestrus, only cyproheptadine, the serotonergic antagonist, increased pulse frequency in ovariectomized ewes. Cyproheptadine had no effect on frequency during the breeding season. On the basis of these results, we propose that the steroid-dependent and -independent actions of anestrous photoperiod occur via catecholaminergic and serotonergic neurons, respectively.     

Effect of ovariectomy at two periods of the year on LH and FSH basal concentrations and pituitary response to LHRH in the brown hare (Lepus europaeus)

In the brown hare, fertile mating takes place from the beginning of December to September. Seasonal variations of basal concentrations of LH and FSH, and pituitary response to a monthly i.v. injection of LHRH were studied in intact control females and in females ovariectomized during the seasonal anoestrus (OVX1) or during the breeding season (OVX2). In intact females, both basal and LHRH-stimulated LH levels showed an annual variation, with minimal values during anoestrus. During the breeding season, the LH response to LHRH exhibited a biphasic pattern. In contrast, there was no clear seasonal variation in basal and LHRH-stimulated FSH concentrations. After ovariectomy during anoestrus, basal LH remained low for 2 months and began to increase in December. After ovariectomy during the breeding season, LH basal concentrations increased within a few days after the operation. Thereafter, LH values remained high in both groups of females until September, and decreased significantly as in intact females. The pattern of LH release after LHRH remained monophasic in the two groups of ovariectomized females. In OVX1 females, the LH response increased as early as October, was maximum from December to April and decreased progressively until October. IN OVX2 females, the LH response decreased regularly after ovariectomy to a minimum in October. In the 2 groups of ovariectomized females, basal FSH concentrations and pituitary response to LHRH rose rapidly after ovariectomy and did not vary significantly thereafter. These results showed a direct central effect of season on the regulation of basal concentrations of LH, modulated by a negative feed-back of ovarian secretions during the breeding season. In intact hares, the enhanced LH response after LHRH during the breeding season was related to an acute positive effect of ovarian secretions. The regulation of FSH was less dependent on season and remained under a negative control of the ovary throughout the year.     

Opioidergic and nutritional involvement in the control of luteinizing hormone secretion of postpartum Rasa Aragonesa ewes lambing in the mid-breeding season

The role of endogenous opioids and nutrition on the inhibition of luteinizing hormone (LH) secretion during the postpartum period was investigated in a Spanish breed of sheep lambing in the mid-late breeding season. Two groups of adult Rasa Aragonesa ewes housed in individual pens and lambing on 30 December were fed during the suckling period to provide maintenance requirements and the production of 1.1 (M; n=8) or 0.55 (L; n=8) kg of milk per day. On days 10, 20 and 30 after lambing, the effect of a treatment with the opiate receptor antagonist naloxone (1 mg/kg at four hourly intervals) on LH secretion was assessed in half of the ewes of each group, the remaining females receiving four saline injections. After weaning, animals were fed to provide requirements for maintenance of liveweight. Blood samples were collected twice a week from day 20 postpartum until the end of March, and assayed for progesterone and prolactin. Although underfed ewes showed significantly lower mean plasma concentrations during the control period on day 20 postpartum, nutrition did not seem to modify LH secretion before naloxone or saline injections. Moreover, no differences between nutritional groups in the response to naloxone injections on pattern of LH secretion were found. In fact, naloxone treatment induced an increase of mean LH concentrations on days 10, 20 and 30 postpartum (at least, P<0.05), of LH pulse frequency on days 20 and 30 (P<0.05), and of LH pulse amplitude on days 10 and 20 (P<0.05). Underfed ewes during the postpartum period showed a slower decline in plasma prolactin levels, with significant differences on days 29, 36 and 39 after lambing (P<0.05). Only 3 M ewes ovulated before the onset of the seasonal anoestrus period. It is concluded that endogenous opioids are involved in the inhibition of LH secretion during the early suckling period of a reduced seasonality breed of sheep without any influence of nutrition on the response to naloxone treatment; however, ewes underfed before weaning failed to reactivate their cyclicity prior to the onset of the seasonal anoestrus.     

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.     

Seasonal and hormonal control of pulsatile LH secretion in the dairy goat (Capra hircus)

In Exp. 1, the changes in pulsatile LH secretion at the onset of the breeding season were observed in 20 intact, mature Saanen does. Blood was sampled every 20 min for 6 h each week from the beginning of August until the onset of ovulatory activity, as evidenced by cycles in plasma progesterone. The first doe ovulated at the end of August and all were cycling by the end of September. As the first ovulation approached, LH pulse frequency increased by 67% and mean levels of LH increased by 47%. These changes were progressive rather than abrupt. In Exp. 2, seasonal changes in the inhibition of pulsatile LH secretion by ovarian steroids were studied in ovariectomized Saanen does. The animals were untreated (N = 4) or given subcutaneous oestradiol implants (N = 4) and blood was sampled every 10 min for 6 h, twice during the breeding season and twice during the anoestrous season. In each season, the second series of samples was taken after the animals had been treated with progesterone, administered by intravaginal implants. Season did not significantly affect LH secretion in goats not treated with oestradiol, but LH pulse frequency was 54% lower during the anoestrous season than during the breeding season in oestradiol-treated goats. Mean LH concentrations were affected in the same manner as pulse frequency, but pulse amplitude was increased by oestradiol treatment in both seasons. Progesterone had no detectable effect on LH secretion in either season. In Exp. 3, the response to repeated melatonin injections at a set time after dawn was investigated in 11 oestradiol-treated, ovariectomized goats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of follicular development after chronic LHRH immunoneutralization in the ewe

Active immunization of 6 Damline ewes against LHRH during seasonal anoestrus resulted in an inhibition of ovarian cyclicity throughout 2 subsequent breeding seasons. This was associated with a significant suppression of plasma LH and FSH concentrations but no significant effect on prolactin. The ovaries of LHRH-immunized ewes 30 months after primary immunization contained no follicles greater than 2.5 mm in diameter and a greater proportion of follicles between 1 and 2 mm were atretic than in control ewes (N = 8). In-vitro production of testosterone and androstenedione were similar in follicles 1-2 mm in both control and LHRH-immunized ewes (N = 6) and all had little or no ability to secrete oestradiol. However, basal and hCG-stimulated progesterone secretion was suppressed in the follicles from LHRH-immunized ewes. These results show that follicular development beyond 2.5 mm in the ewe is dependent on adequate stimulation by both LH and FSH.     

Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian function in ewes during the transition from breeding season to anoestrus

Transrectal ovarian ultrasonography was conducted in six Western white-faced ewes for 35 days from the last oestrus of the breeding season, to record the number and size of all ovarian follicles > or = 3 mm in diameter and luteal structures. Blood samples were collected once a day for estimation of serum concentrations of follicle-stimulating hormone (FSH), oestradiol and progesterone. Each ewe had five follicular waves (follicles growing from 3 to > or = 5 mm in diameter) over the scanning period. The duration of the growth phase of the largest ovarian follicles did not differ (P > 0.05) between waves, but follicular static and regressing phases decreased significantly (P < 0.05) after the decline in serum progesterone concentrations at the end of the last luteal phase of the breeding season. The intervals between the five follicular waves were: 9.2+/-0.4, 5.2+/-0.7, 8.3+/-0.8 and 5.8+/-0.7 days; the two shorter intervals differed (P < 0.05) from the two longer intervals. Using the cycle-detection program, rhythmic increases in serum FSH concentrations were detected in all ewes; the amplitude, duration and periodicity of FSH fluctuations did not vary (P > 0.05) throughout the period of study. The number of identified FSH peaks (7.8+/-0.5 peaks per ewe, per scanning period) was greater (P < 0.05) than the number of emerging follicular waves. Serum concentrations of oestradiol remained low (< or = 1 pg/ml) on most days, in five out of the six ewes studied, and sporadic elevations in oestradiol secretion above the non-detectable level were not associated with the emergence of follicular waves. The ovulation rate was lower than that seen during the middle portion of the breeding season (November-December) in white-faced ewes but the transitional ewes had larger corpora lutea (CL). Maximal serum concentrations of progesterone appeared to be lower and the plateau phase of progesterone secretion appeared to be shorter during the last luteal phase of the ovulatory season in comparison to the mid-breeding season of Western white-faced ewes. During the transition into anoestrus in ewes, the endogenous rhythm of FSH release is remarkably robust but the pattern of emergence of sequential follicular waves is dissociated from FSH and oestradiol secretion. Luteal progesterone secretion is suppressed because of fewer ovulations and diminished total luteal volume, but it may also result from diminished gonadotropic support. These season-related alterations in the normal pattern of ovine ovarian cycles appear to be due to reduction in ovarian responsiveness to gonadotropins and/or attenuation in secretion of luteinizing hormone (LH) occurring at the onset of the anovulatory season in ewes.