Steroid secretion rates and plasma binding activity in androstenedione-immune ewes with an autotransplanted ovary

Mature Merino ewes in which the left ovary and its vascular pedicle had been autotransplanted to the neck were divided into control (N = 5) and immunized groups (N = 6). The immunized ewes were treated (2 ml s.c.) with Fecundin 1 and 4 weeks before the start of blood sampling. Ovarian and jugular venous blood was collected every 10 min at two stages of the follicular phase (21-27 h and 38-42 h after i.m. injection of 125 micrograms of a prostaglandin (PG) analogue) and during the mid-luteal phase (8 h at 15-min intervals). The ewes were monitored regularly for luteal function and preovulatory LH surges. Hormone concentrations and anti-androstenedione titres were assayed by RIA and ovarian secretion rates of oestradiol-17 beta, progesterone and androstenedione were determined. After the booster immunization, progesterone increased simultaneously with titre in immunized ewes, reaching 30 ng/ml at the time of PG injection when median titre was 1:10,000. All ewes responded to PG with LH surges 42-72 h later: 2 of the immunized ewes then had a second LH surge within 3-4 days at a time when peripheral progesterone values were 2-3 ng/ml. The frequency of steroid and LH pulses was greater in immunized ewes (P less than 0.05) during the luteal phase but not the follicular phase. The secretion rate of androstenedione was 6-10 times greater (19-37 ng/min; P less than 0.001) in immunized ewes at all sampling stages. Progesterone secretion rates were 3 times greater (16 micrograms/min; P less than 0.001) during the luteal phase in immunized ewes. The amplitude of oestradiol pulses was significantly reduced in immunized ewes (4.8 vs 2.1 ng/min at +24 h and 6.5 vs 2.8 ng/min at +40 h in control and immunized ewes, respectively: P less than 0.05) during the follicular phase. However, the mean secretion rate of oestradiol at each phase of the cycle was not significantly different between treatment groups. Analysis of bound and free steroid using polyethylene glycol showed that greater than 98% of peripheral and ovarian venous androstenedione and 86% of peripheral progesterone was bound in immunized ewes but there was no appreciable binding (less than 0.1%) in control ewes. Similarly, 50% of ovarian venous oestradiol was bound in immunized ewes compared to 15% in control ewes. We conclude that immunization against androstenedione increases the secretion rate of androstenedione and progesterone but not of oestradiol.(ABSTRACT TRUNCATED AT 400 WORDS)     

FACTORS AFFECTING THE SECRETION OF LUTEINIZING HORMONE IN THE EWE

(1) Luteinizing hormone (LH) is secreted as discrete pulses throughout all stages of the reproductive cycle of the ewe, including pre-pubertal, seasonal and lactational anoestrus, and the luteal and follicular phases of the oestrous cycle. Secretion is probably also pulsatile during the preovulatory surge of LH. (2) The secretion of LH is affected by the ovarian steroids, oestradiol and progesterone, both of which act principally to reduce the frequency of the pulses. During the luteal phase the two steroids act synergistically to exert this effect, and during anoestrus oestradiol acts independently of progesterone. Androstenedione secreted by the ovary apparently has no role in the control of LH secretion. (3) The amplitude of the pulses may also be affected by the steroids but there are conflicting reports on these effects, some showing that amplitude is lowered by the presence of oestrogen and others showing increases in amplitude in the presence of oestrogen and progesterone. (4) The secretion of LH pulses is affected by photoperiod, social environment and nutrition. Under the influence of decreasing day-length, oestradiol alone cannot reduce the frequency of pulses and the ewe experiences oestrous cycles. When day-length is increasing, the hypothalamus becomes more responsive to oestradiol which reduces the frequency of the pulses. (5) A hypothetical pheromone secreted by rams can increase the frequency of the LH pulses in anoestrous ewes and thereby induce ovulation, possibly by inhibiting the negative feedback exerted by oestradiol. (6) The relationships between nutrition and reproduction are poorly understood, but it seems likely that the effects of nutrition are mediated partly through the hypothalamus and its control of the secretion of LH pulses. (7) The pulses of LH secreted by the anterior pituitary gland are evoked by pulses of GnRH secreted by the hypothalamus. The location of the centre controlling the GnRH pulses and the neurotransmitter involved are not known.     

Effects of medroxyprogesterone acetate (MAP) on ovarian antral follicle development, gonadotrophin secretion and response to ovulation induction with gonadotrophin-releasing hormone (GnRH) in seasonally anoestrous ewes

When ovulation is induced with gonadotrophin-releasing hormone (GnRH) in anoestrous ewes, a proportion of animals fail to form normal (full-lifespan) corpora lutea (CL). Progesterone treatment before GnRH prevents luteal inadequacy. It remains uncertain whether a similar effect, achieved with medroxyprogesterone acetate (MAP) from intravaginal sponges, is mediated by influences on growing ovarian follicles and/or secretion of gonadotrophic hormones, before and after GnRH treatment. Two experiments were performed, on 13 and 11 anoestrous Western white-faced ewes, respectively. Seven and six ewes, respectively, received MAP-containing sponges (60 mg) for 14 days; the remaining ewes served as untreated controls. To test the effect of timing of GnRH administration after pre-treatment with MAP-releasing sponges, GnRH injections (250 ng every 2h for 24h followed by a bolus injection of 125 microg of GnRH i.v.) were given either immediately (Experiment 1) or 24h after sponge removal in the treated ewes (Experiment 2). Ovarian follicular dynamics (follicles reaching >or=5mm in size) and development of luteal structures were monitored using transrectal ultrasonography. In Experiment 1, the mean ovulation rate (0.7+/-0.3 and 1.0+/-0.4) and proportion of ovulating ewes (57 and 67%, respectively) did not vary (P>0.05) between MAP-treated and control ewes. Normal (full-lifespan) CL were detected in 29% of treated and 67% of control ewes (P>0.05). In Experiment 2, the mean ovulation rate (2.3+/-0.2 and 1.2+/-0.6; P<0.05) and percentage of ewes with normal (full-lifespan) CL (100 and 40%, respectively; P<0.10) were greater in the treated compared to control ewes. In Experiment 1, the mean peak concentration of the GnRH-induced LH surge was lower (P<0.05) in MAP-treated than in control ewes. There were no significant differences between MAP-treated and control ewes in the characteristics of follicular waves, mean daily serum FSH concentrations, and secretory parameters of LH/FSH, based on intensive blood sampling conducted 1 day before sponging and 1 day before sponge removal. It is concluded that treatment with MAP has no effect on the tonic secretion of LH/FSH or follicular wave development in anoestrous ewes. However, the GnRH-stimulated LH discharge was attenuated in the ewes that received MAP-impregnated sponges for 14 days and were treated with GnRH immediately after sponge withdrawal. Ovulatory response and CL formation were increased when GnRH was administered 24 h after sponge removal.     

Ovarian function in nutritionally induced anoestrous cows: effect of exogenous gonadotrophin-releasing hormone in vivo and effect of insulin and insulin-like growth factor I in vitro

Ovarian function of nutritionally induced anoestrus cows was evaluated in vivo (Expt 1) and in vitro (Expt 2). In Expt 1, 32 nutritionally induced anoestrous beef cows were divided into four treatment groups receiving: (1) saline infusions at one pulse every 4 h for 13 days (control); (2) 2 micrograms GnRH at one pulse every 4 h (2 micrograms infused in 1.8 ml saline over 5 min) for 13 days (GnRH-4); (3) 2 micrograms GnRH at one pulse every 1 h for 13 days (GnRH-1); and (4) continuous infusion of 2 micrograms GnRH (a total of 2 micrograms in 34 ml h-1) for 13 days (GnRH-C). On the last day of treatment, cows were killed, ovaries were removed and follicular fluid samples (n = 149) were collected. The percentage of cows with luteal activity on day 13 was significantly different (P < 0.01) among treatments (0, 25, 75 and 25% for control, GnRH-4, GnRH-1 and GnRH-C cows, respectively). Owing to the large percentage of ovulatory cows in the GnRH-1 group (n = 6), anovulatory cows (n = 2) were removed from this treatment group for statistical analysis, as were cows with luteal tissue from the GnRH-4 (n = 2) and GnRH-C (n = 2) groups. The numbers of small (1.0-4.9 mm) and medium plus large (> or = 5 mm) follicles were not affected (P > 0.10) by treatment. However, GnRH-4 cows (n = 6) had greater (P < 0.05) concentrations of oestradiol in follicular fluid than did control (n = 8) but not GnRH-1 (n = 6) or GnRH-C (n = 6) cows. Concentrations of insulin-like growth factor I were greater (P < 0.05) in the follicular fluid of GnRH-1 cows than in all other treatment groups. Concentrations of androstenedione and progesterone in follicular fluid were not affected (P > 0.10) by treatment or follicle size. The binding activity of insulin-like growth factor binding proteins was not affected by GnRH treatment. However, the binding activity of insulin-like growth factor binding protein 2, 29-32 kDa and 22 kDa insulin-like growth factor binding proteins were greater (P < 0.05) in small versus medium plus large follicles. In Expt 2, granulosa cells were collected from nutritionally anoestrous cows to determine whether ovarian cells from anoestrous cows have the capacity to respond to insulin-like growth factor I or insulin in vitro. Both insulin-like growth factor I (20 and 200 ng ml-1) and insulin (10, 100 and 1000 ng ml-1) increased (P < 0.05) granulosa cell proliferation and progesterone production. In conclusion, pulsatile infusion of 2 micrograms GnRH (every 1 or 4 h) for 13 days into nutritionally induced anoestrous cows results in increased intrafollicular oestradiol and insulin-like growth factor I concentrations and can stimulate ovulation without markedly affecting concentrations of androstenedione or progesterone, or the binding activity of insulin-like growth factor binding proteins, in follicular fluid. In addition, granulosa cells from nutritionally induced anoestrous cows have the capacity to respond to insulin-like growth factor I and insulin in vitro, indicating that the decrease in trophic factors observed with restricted feeding does not reduce the response of the ovary to insulin-like growth factor I and insulin.     

Melatonin and the pituitary response to GnRH in prepubertal and adult ewes

GnRH-induced LH-release was studied in female lambs from 5 weeks of age until puberty and in adult anoestrous ewes. In pre-pubertal animals LH was released rapidly after GnRH treatment but after puberty the responses became slower and more sustained although the peak concentration did not change. In neither pre-pubertal nor adult sheep did prior treatment with melatonin influence LH release after GnRH treatment.     

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.     

Direct effect of prolactin, induced by TRH injection, on ovarian oestradiol secretion in the ewe

The effect of sustained high plasma levels of prolactin, induced by repeated 2-h i.v. injections of thyrotrophin-releasing hormone (TRH; 20 micrograms), on ovarian oestradiol secretion and plasma levels of LH and FSH was investigated during the preovulatory period in the ewe. Plasma levels of progesterone declined at the same rate after prostaglandin-induced luteal regression in control and TRH-treated ewes. However, TRH treatment resulted in a significant increase in plasma levels of LH and FSH compared to controls from 12 h after luteal regression until 5 to 6 h before the start of the preovulatory surge of LH. In spite of this, and a similar increase in pulse frequency of LH in control and TRH-treated ewes, ovarian oestradiol secretion was significantly suppressed in TRH-treated ewes compared to that in control ewes. The preovulatory surge of LH and FSH, the second FSH peak and subsequent luteal function in terms of plasma levels of progesterone were not significantly different between control and TRH-treated ewes. These results show that TRH treatment, presumably by maintaining elevated plasma levels of prolactin, results in suppression of oestradiol secretion by a direct effect on the ovary in the ewe.     

Effect of oestradiol treatment during GnRH-induced ovulation on subsequent PGF2alpha release and luteal life span in anoestrous ewes

In sheep, induction of ovulation during anoestrus is accompanied by a high incidence of short luteal phases, though pre-treatment with progesterone can overcome this problem. We have investigated the effects of supplementing oestradiol during GnRH-induced ovulation on subsequent PGF2alpha release and luteal life span. Thirty anoestrous crossbred ewes received 250 ng GnRH i.v. at 2 h intervals for 48 h to induce ovulation either alone (group 1; n=10) or in association with either an i.m. injection of 20 mg progesterone 3 days earlier (group 2; n=10) or 3 i.m. injections of 10 microg oestradiol at 8 h intervals on the second day of GnRH treatment (group 3; n=10). Laparoscopy, performed 3 days following GnRH to confirm ovulation and 8 days later, coupled with plasma progesterone analysis were used to determine luteal life span. On day 4 following GnRH, plasma samples were collected at 20 min intervals for 8 h to monitor PGF2alpha release. One ewe from group 1 failed to ovulate and was excluded from further analysis. All groups showed an increase (P<0.01) in plasma oestradiol during GnRH treatment, with group 3 showing a marked (P<0.001) increase over that seen in the other two groups. In group 1 there were 1.4+/-0.2 PGF2alpha episodes/ewe/8 h. In group 2, pre-treatment with progesterone caused the complete inhibition of PGF2alpha episodes (0 episodes/ewe/8 h) while in group 3, treatment with oestradiol resulted in a significant reduction (0.3+/-0.1 episodes/ewe/8 h) compared with group 1 (P<0.01). In group 1, 9/9 ewes exhibited short cycles compared with 2/10 ewes in group 2 (P<0.01). In group 3 the proportion of ewes showing short cycles 7/10 ewes was not significantly different from the other groups. While treatment with oestradiol caused a significant attenuation of PGF2alpha release, this was associated with only a partial reduction in the incidence of short cycles.     

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.     

Pulsatile GnRH administration stimulates the number of pituitary GnRH receptors in seasonally anoestrous ewes

Twenty seasonally anoestrous ewes were pretreated with progesterone for 4 days and divided into four equal groups. Ewes in Group 1 received no GnRH treatment and were slaughtered immediately after progesterone removal. Ewes in Groups 2, 3 and 4 received i.v. injections of 250 ng GnRH every 2 h for 36 h starting at the time of progesterone removal. Ewes in Group 2 were slaughtered immediately after the 36 h GnRH pulsing, while ewes in Groups 3 and 4 were given a bolus injection of 125 micrograms GnRH at this time and were slaughtered 2 and 10 h after the bolus injection, respectively. Blood samples were collected every 30 min from ewes in Group 4 only, from 4 h before the start of GnRH treatment until 10 h after the bolus injection. Pulsing with GnRH resulted in episodic release of LH, and the bolus injection of GnRH was immediately followed by a preovulatory type LH surge in those ewes in which an endogenous surge had no already begun. The pituitary GnRH receptor numbers were significantly higher for the ewes in Group 2 than for any of the other treatment groups, while there was no significant difference in the receptor numbers between Groups 1, 3 and 4. The results suggest an up-regulation of GnRH receptors resulting from pulsatile GnRH therapy.     

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 mug 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 mug 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 mug 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 mug 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.     

Time series analysis of plasma LH and FSH concentrations as a method of assessing episodic secretion

Time series analysis was used to detect LH and FSH episodes in untreated seasonally anoestrous ewes and prepubertal heifers, and in these animals when treated with low doses of GnRH. For comparison, these profiles were also assessed for episodic secretion by subjective, visual appraisal methods and by cycle detection-an objective threshold method. In untreated animals, time series analysis detected recurring events in the LH and FSH profiles, the period lengths of which varied between individual animals. When GnRH was injected at 2-h intervals, cycles in LH secretion with period lengths of 120 min were recorded in all animals, of 60 min in all ewes and 11/12 heifers, and of 40.5 min in 22/24 ewes and 10/12 heifers. The cycles with period lengths of 60 and 40.5 min are probably artefacts of this method of analysis. No consistent cycles in FSH release were detected in GnRH-injected anoestrous ewes, but 120-min cycles were recorded in 8/12 GnRH-injected heifers. When GnRH was administered to seasonally anoestrous ewes by continuous infusion, recurring cycles in both LH and FSH secretion were evident. However, there was no consistency in their period lengths and the mean number and frequency of cycles were similar to pretreatment values. The number of episodes detected by visual appraisal was influenced by the choice of episode definition. Both methods identified LH, but not FSH, episodes in response to each injection in all GnRH-injected animals. Cycle detection, which does not identify individual episodes, recorded LH and FSH episode frequencies similar to those detected by the more stringent method of visual appraisal. Time series analysis detected an FSH response to GnRH injections in prepubertal heifers that was not identified by the other methods of analysis. However, because of the asymmetric nature of LH episodes, it also detected cycles in LH profiles that were probably spurious. Subjective decisions influenced the frequencies of LH and FSH episodes recorded by visual appraisal, and the variation in episode amplitude in these profiles made cycle detection inappropriate. Each of these methods can contribute to the interpretation of hormone profiles, but their constraints and limitations must be recognized.     

Ovarian antral follicular dynamics and their associations with peripheral concentrations of gonadotropins and ovarian steroids in anoestrous Finnish Landrace ewes

Daily transrectal ultrasonography of ovaries was done in seven Finn ewes during three 17-day periods from May to July. Blood samples were collected each day for estimation of the serum follicle-stimulating hormone (FSH), oestradiol and progesterone concentrations, and also every 15 min for 6 h, halfway through each period of ultrasonographic examination, to determine the patterns of gonadotropic hormone secretion. Four ewes ceased cycling from March to mid-April (ewes entering anoestrus early) and three in May (ewes entering anoestrus late). In all ewes cyclicity resumed during the period from mid-August to mid-September. The growth of ovarian antral follicles to periovulatory sizes of >/=5 mm in diameter was seen at all stages of anoestrus. An average of four waves of follicular development (follicles growing from 3 to >/=5 mm in diameter before regression) with a periodicity of 4 days were recorded during each of the three scanning periods. There was a close temporal relationship between days of follicular wave emergence and peaks of successive FSH fluctuations. Ewes entering anoestrus late exceeded ewes that became anoestrus early in numbers of large (>/=5 mm in diameter) ovarian antral follicles and maximum follicle diameter. Peak concentrations of transient FSH increases were higher (P<0.05) in ewes entering anoestrus late than in ewes entering anoestrus early. The secretion of luteinising hormone, (LH; mean and basal level, and LH pulse frequency, but not amplitude) was lowest during the month of June in all ewes. Oestradiol production was markedly suppressed throughout anoestrus. Peaks of progesterone secretion appeared to occur at regular intervals and were associated with the end of the growth phase of the largest follicles of sequential waves. In conclusion, the growth of ovarian follicles to ostensibly ovulatory diameters is maintained throughout anoestrus in Finn ewes and periodic emergence of follicular waves is correlated with an endogenous rhythm of FSH secretion. The present study also provides evidence for the inverse relationship between the time of the onset of seasonal anoestrus and the number and size of antral follicles developing throughout anoestrus in Finn ewes, and indicates that differences exist in both the secretion of and ovarian responsiveness to gonadotropic hormones among early and late anoestrous ewes.     

Effect of the introduction of rams during the anoestrous season on the pulsatile secretion of LH in ovariectomized ewes

Introduction of rams to ovariectomized ewes treated with oestradiol implants (N = 10) increased the frequency of LH pulses from 4 X 8 to 10 X 6 pulses per 12 h. This effect was reflected by increases in mean levels of LH and the basal levels upon which the pulses were superimposed. In ewes that had not been treated with oestradiol (N = 5), there was no significant increase in pulse frequency but mean and basal levels of LH increased slightly after the introduction of rams. In a second experiment, similar effects of the introduction of rams were seen in ovariectomized ewes treated with oestradiol or oestradiol + androstenedione (N = 16), but no significant effects of the rams were observed in untreated ewes (N = 8) or ewes treated only with androstenedione (N = 7). No preovulatory surges of LH were observed in the 30-h period after the introduction of rams. It was concluded that the ram stimulus probably evokes the increase in pulse frequency by inhibiting the negative feedback action of oestradiol, and that the surge normally observed in entire ewes is dependent on the ovarian response to these pulses. However, the observation of responses in some ewes not treated with oestradiol also raises the possibility that the ram stimulus can act directly on the hypothalamic neurones that control the secretion of LH, and that this effect is enhanced in the presence of oestrogen.     

Ovarian capillary blood flow in seasonally anoestrous ewes induced to ovulate by treatment with GnRH

The microsphere technique was used to obtain estimates of ovarian capillary blood flow near ovulation, in 8 seasonally anoestrous ewes, which were induced to ovulate by GnRH therapy. Plasma progesterone concentrations were monitored in jugular blood sampled between Days 4 and 7 after the onset of the preovulatory LH surge. The ewes were then slaughtered. Three of the ewes were treated with a single injection of 20 mg progesterone before GnRH therapy. In these ewes and 1 other, plasma progesterone values increased after ovulation and reached 1.0 ng/ml on Day 7 following the preovulatory LH surge (normal, functional CL), whilst in the other 4 ewes progesterone concentrations increased initially then declined to 0.5 ng/ml by Day 7 (abnormal CL). In the ewes exhibiting normal luteal function, the mean ovarian capillary blood flow was significantly greater (P less than 0.01) than that for ewes having abnormal luteal function. Irrespective of the type of CL produced, capillary blood flow was significantly greater (P less than 0.05) in ovulatory ovaries than in non-ovulatory ovaries. These findings indicate that the rate of capillary blood flow in ovaries near ovulation may be a critical factor in normal development and maturation of preovulatory follicles and function of subsequently formed CL.     

Effect of stress-like concentrations of cortisol on follicular development and the preovulatory surge of LH in sheep

Stress-like concentrations of cortisol increase the negative feedback potency of oestradiol in castrated male sheep. A similar cortisol-dependent response in female sheep might be expected to suppress gonadotrophin secretion and impair follicular development and ovulation. The oestrous activity of 21 female sheep was synchronized using progestogen-treated vaginal pessaries to test this hypothesis. Stress-like concentrations of cortisol (60-70 ng ml-1) were established by continuous infusion of cortisol (80 micrograms kg-1 h-1; n = 13) beginning 5 days before, and continuing for 5 days after, pessary removal. Control animals (n = 8) received a comparable volume of vehicle (50% ethanol-saline) over the 10 day infusion period. Serum concentrations of oestradiol increased progressively in control sheep during the 48 h immediately after pessary removal. This increase in serum oestradiol was blocked or significantly attenuated in sheep receiving stress-like concentrations of cortisol. Preovulatory surge-like secretion of LH was apparent in control animals 58.5 +/- 2.1 h after pessary removal. In contrast, surge-like secretion of LH was not observed during the 5 days after pessary removal in 54% (7 of 13) of sheep receiving cortisol. Moreover, the onset of the surge was significantly delayed in the cortisol-treated ewes that showed surge-like secretion of LH during the infusion period. The ability of episodic pulses of exogenous GnRH to override the anti-gonadal effect of cortisol was examined in a second study. Oestrous activity of 12 ewes was synchronized using progestogen-containing pessaries as described above. Ewes were randomly assigned to one of three treatment groups (n = 4 ewes per group). Animals received cortisol (100 micrograms kg-1 h-1; groups 1 and 2) or a comparable volume of vehicle (group 3) beginning 5 days before, and continuing for 2 days after, pessary removal. Pulses of GnRH (4 ng kg-1 h-1, i.v.; group 1) or saline (groups 2 and 3) at 1 h intervals were initiated at pessary removal and continued for 48 h. Serum concentrations of oestradiol were not significantly increased after pessary removal in sheep receiving cortisol alone. Conversely, serum concentrations of oestradiol increased progressively during the 48 h after pessary removal in control ewes and in ewes receiving cortisol and GnRH. At the end of infusion, serum concentrations of oestradiol did not differ (P > 0.05) between control (7.7 +/- 0.8 pg ml-1) ewes and ewes receiving cortisol and episodic GnRH (6.4 +/- 1.3 pg ml-1). Moreover, these values were significantly greater (P < 0.05) than the serum concentrations of oestradiol in animals receiving cortisol (1.0 +/- 0.4 pg ml-1) alone. Collectively, these data indicate stress-like concentrations of cortisol block or delay follicular development and the preovulatory surge of LH in sheep. In addition, episodic GnRH overrides cortisol-induced delay in follicular maturation.     

First postpartum ovulations and corpora lutea in ewes which lamb in the breeding season

Two experiments involving crossbred ewes which lambed during the breeding season were performed to determine whether: (a) the interval to first postpartum ovulation could be reduced by weaning or mastectomy; (b) there are differences in luteal structure and luteinizing hormone (LH) receptor concentration between first postpartum corpora lutea induced with GnRH and normal cycling corpora lutea and (c) pretreatment of postpartum ewes with progesterone would affect luteal LH receptor concentration and luteal phase serum progesterone concentration.In experiment I, the mean interval (±SEM) to the first postpartum ovulation was 22.3 ± 1.1 days and was not significantly altered by weaning or mastectomy. More than half of the ewes had small, short-lived peaks of serum progesterone associated with short-lived corpora lutea prior to the normal luteal phase rise of serum progesterone. In experiment II, 2 h after GnRH injection on day 18 postpartum, serum LH concentrations were higher in ewes which received progesterone treatment on days 13 and 14 than in control ewes. Progesterone treatment did not affect mean corpus luteum weight (157 mg) or concentration of LH receptors (0.95 fmol/mg) in first postpartum corpora lutea, but progesterone-treated ewes had significantly higher endogenous serum progesterone concentrations on days 21–24. GnRH-induced corpora lutea from postpartum ewes were lighter in weight, paler in color, had lower LH receptor concentrations and had a more regressed histological appearance than corpora lutea of a similar age from normal, cycling ewes.     

Progesterone pretreatment has a direct effect on GnRH-induced preovulatory follicles to determine their ability to develop into normal corpora lutea in anoestrous ewes

In two experiments carried out during seasonal anoestrus, Romney Marsh ewes were treated with small-dose (250 ng) multiple injections of GnRH at 2-h intervals with and without progesterone pretreatment. In Exp. 1, 8/8 progesterone-primed ewes ovulated and produced functionally normal corpora lutea compared with 2/9 non-primed ewes. Follicles were recovered from similarly treated animals 18 or 28 h after the start of GnRH treatment (at least 14 h before the estimated time of the LH peak) and assessed in terms of diameter, granulosa cell number, oestradiol, testosterone and progesterone concentrations in the follicular fluid, oestradiol production in vitro and binding of 125I-labelled hCG to granulosa and theca. There were no significant differences in any of these measures in 'ovulatory' follicles recovered from the progesterone-pretreated compared to non-pretreated animals. In Exp. 2, follicles were removed from similar treatment groups just before and 2 h after the start of the LH surge. Unlike 'ovulatory' follicles recovered from the non-pretreated ewes, those recovered from progesterone-pretreated ewes responded to the LH surge by significantly increasing oestradiol secretion (P less than 0.01) and binding of 125I-labelled hCG (P less than 0.05) to granulosa cells. Overall there was also more (P less than 0.05) hCG binding to granulosa and theca cells from progesterone-pretreated animals. Non-ovulatory follicles recovered from progesterone-primed ewes had more (P less than 0.05) binding of 125I-labelled hCG to theca and a higher testosterone concentration in follicular fluid (P less than 0.05) than did those from non-primed ewes. These results suggest that inadequate luteal function after repeated injections of GnRH may be due to a poor response to the LH surge indicative of a deficiency in the final maturational stages of the follicle.     

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.     

Steroid feedback inhibition of pulsatile secretion of gonadotropin-releasing hormone in the ewe

The long-term negative feedback effects of sustained elevations in circulating estradiol and progesterone on the pulsatile secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) were evaluated in the ewe following ovariectomy during the mid-late anestrous and early breeding seasons. GnRH secretion was monitored in serial samples of hypophyseal portal blood. Steroids were administered from the time of ovariectomy by s.c. Silastic implants, which maintained plasma concentrations of estradiol and progesterone at levels resembling those that circulate during the mid-luteal phase of the estrous cycle; control ewes did not receive steroidal replacement. Analysis of hormonal pulse patterns in serial samples during 6-h periods on Days 8-10 after ovariectomy disclosed discrete, concurrent pulses of GnRH in hypothalamo-hypophyseal portal blood and LH in peripheral blood of untreated ovariectomized ewes. These pulses occurred every 97 min on the average. Treatment with either estradiol or progesterone greatly diminished or abolished detectable pulsatile secretion of GnRH and LH, infrequent pulses being evident in only 3 of 19 steroid-treated ewes. No major seasonal difference was observed in GnRH or LH pulse patterns in any group of ewes. Our findings in the ovariectomized ewe provide direct support for the conclusion that the negative-feedback effects of estradiol and progesterone on gonadotropin secretion in the ewe include an action on the brain and a consequent inhibition of pulsatile GnRH secretion.