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.

Failure of melatonin to influence endogenous opioid effects on LH secretion in the anoestrous ewe.

In May mature seasonally anoestrous ewes were implanted with melatonin which advanced the onset of cycles by about 1 month. The LH response to an opioid antagonist, WIN-3, was determined 5, 15, 25 and 60 days after melatonin implantation, by intravenous administration of WIN-3 (12.5 mg/dose) 4 times at 15-min intervals during both the 1st and the 5th hour of an 8-h treatment period. There was no effect of WIN-3 at 5, 15 and 25 days after melatonin implantation. At 60 days LH concentration and pulse frequency were significantly increased (P less than 0.05 and less than 0.01 respectively) in response to WIN-3 treatment, but only in those animals which had begun reproductive cycles, an effect known to be mediated by the presence of progesterone. We were therefore unable to find evidence to support the hypothesis that the influence of melatonin in advancing the breeding season may be via an opioidergic pathway.

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.

Gonadotropins--domestic animals.

The application of RIAs for the gonadotropins to the study of domestic animal reproduction has depended on the availability of sensitive and specific reagents, automated systems for increased throughput and an awareness of the assay errors. Illustrations are given on the measurement of seasonal, surge and episodic changes in LH secretion and the advantages gained by such investigation are outlined.

Endogenous opioid peptides and the control of gonadotrophin secretion.

The endogenous opioid peptides are a group of recently discovered compounds which occur in the brain of a wide variety of species. Originally named because of their opiate-like activity, they have since been demonstrated to have multifaceted actions, one of which appears to be the modulation of luteinising hormone (LH) secretion. Because of the prime position of LH in the ovulatory process, this role for the opioids has attracted considerable interest. Their mode of action is essentially one of suppression and they work by inhibiting the release of hypothalamic gonadotrophin releasing hormone. Through this mechanism they have been implicated in the suppression of LH secretion during the prepubertal period and the modulation of LH during the oestrous cycle. It is well established that gonadal steroids suppress LH secretion by negative feedback upon the hypothalamic-pituitary axis, and this action may be brought about, in part, through intermediary opioidergic neurones. Much of the research to date has been carried out upon laboratory rodents and primates, but there is evidence now accruing that the opioids have similar actions in domestic animals. Knowledge of the role of these compounds may therefore aid in the understanding of an area of commercial importance, namely the control of ovulation in farm livestock.

The effect of manipulating growth in sheep by diet or anabolic agents on plasma cortisol and muscle glucocorticoid receptors.

1. The cortisol status (total plasma cortisol concentration, free cortisol concentration, transcortin capacity) and the characteristics of skeletal muscle binding for cortisol and dexamethasone were examined in female lambs either implanted with Zeranol or trenbolone acetate or whose dietary intake was restricted. 2. The skeletal muscle glucocorticoid receptor had a high affinity for the glucocorticoid triamcinolone (relative binding affinity 0.85) and cortisol (relative binding affinity 0.51) with virtually no affinity for trenbolone. 3. Trenbolone acetate treatment reduced the binding capacity of sheep skeletal muscle for cortisol within 2 d of implantation. The other treatments had little effect except a small reduction in the animals where food intake was restricted. Similarly, binding capacity for dexamethasone was reduced by trenbolone acetate treatment but was not affected by the other treatments. This reduction in trenbolone acetate-treated animals is, at least in part, due to a reduction in glucocorticoid receptors. 4. Transcortin capacity was elevated by Zeranol treatment but reduced with diet restriction or trenbolone treatment. 5. No support for the suggestion of free cortisol concentration being important in the growth-promoting mechanism of trenbolone or Zeranol was obtained. 6. Although insulin concentrations were not significantly altered by treatment (P greater than 0.05), when combining all the animals there was evidence of a negative correlation between total cortisol/insulin value (P less than 0.05) or free cortisol:insulin value and growth rate (P less than 0.001). Free cortisol was negatively correlated to growth rate (P less than 0.05) and transcortin capacity positively correlated (P less than 0.01).

Opioid modulation of LH secretion in the ewe.

Administration of opioid agonists and antagonists and measurement of resulting hormone changes were used to study the possible effects of opioids on reproductive function in the ewe. Intravenous administration of the long-acting methionine-enkephalin analogue FK33-824 (250 micrograms/h for 12 h) to 3 ewes during the follicular phase of the oestrous cycle depressed episodic LH secretion. This effect was reversed by administration of the opiate antagonist naloxone (25 mg/h) in combination with the FK33-824 treatment; in fact LH secretion was enhanced by the combined regimen. Naloxone (25 mg/h for 12 h) administered alone to 3 ewes in the follicular phase also enhanced LH secretion. In 3 animals treated with FK33-824 during the follicular phase, progesterone remained basal for 14 days after treatment, suggesting that ovulation was blocked. Jugular venous infusion of naloxone (25, 50 or 100 mg/h for 8h) into 5 ewes during the early and mid-luteal phase of the cycle resulted overall in a significant increase in mean plasma LH concentrations and LH episode frequency. To investigate whether endogenous opioids suppress LH release in seasonally anoestrous sheep, naloxone was infused intravenously into mature (25, 50 or 100 mg/h for 8 h) and yearling ewes (12 . 5, 25 or 50 mg/h for 8 h) during early, mid- and late anoestrus and plasma LH concentrations were measured. In the mature ewes, there was a trend for naloxone to increase LH values during the early anoestrous period but naloxone was without effect during mid- and late anoestrus. In the yearlings, naloxone infusion consistently increased plasma LH concentrations as a result of a significant increase in LH episode frequency. These experiments indicate that endogenous opioid peptides probably modulate gonadotrophin secretion during both the follicular and luteal phases of the oestrous cycle. However, the follicular phase of the sheep cycle is of short duration, and there may be residual effects of luteal-phase progesterone during this period. Secondly, there may be an age-dependent effect of naloxone on LH secretion during seasonal anoestrus in the ewe, with opioids playing a part in the suppression of LH in young but not in mature animals.

Ovarian steroid involvement in endogenous opioid modulation of LH secretion in seasonally anoestrous mature ewes.

In June, 16 mature ewes were ovariectomized and allocated to four groups: 1, saline; 2, naloxone; 3, progesterone implant plus naloxone; 4, oestrogen implant plus naloxone. Steroids were implanted at the time of ovariectomy. At 5 days after ovariectomy, the animals were intravenously infused with saline for 8 h and naloxone (50 mg/h) in saline for 8 h the following day. Three intact ewes were given naloxone in a similar way. During infusions and for 8 h on the day after naloxone, jugular venous blood samples were taken every 15 min and assayed for LH. Naloxone resulted in significant increases in mean LH concentration (P less than 0.01), LH episode frequency and episode height (P less than 0.05) in Group 3 ewes, but was without effect in any other group. These results provide evidence that the progesterone status of the ewe affects its response to naloxone, that progesterone negative feedback on LH release may be mediated by an opioid system, and that increased oestradiol negative feedback during seasonal anoestrus is unlikely to work via increased opioid inhibition of LH.

Evidence that the onset of the breeding season in the ewe may be independent of decreasing plasma prolactin concentrations.

Ten ewes of each of two breeds, Dorset Horn (long breeding season) and Welsh Mountain (short breeding season), were given subcutaneous oestradiol-17 beta implants and then ovariectomized. Another 10 ewes of each breed were left intact. On 3 May 1982, all the ewes were housed in an artificial photoperiod of 16L:8D. After 4 weeks, half of the ewes of each breed and physiological state were abruptly exposed to a short-day (8L:16D) photoperiod while the others remained in long days (16L:8D). The time of onset of the breeding season was significantly (P less than 0.05) advanced in ewes switched to short days (12 August +/- 10 days) compared to those maintained in long days (4 September +/- 14 days). Dorset Horn ewes began to cycle (20 July +/- 7 days) significantly (P less than 0.001) earlier than Welsh Mountain ewes (19 September +/- 6 days). Disparities in the time of onset of cyclic activity in ewes of different breeds and daylength groups were echoed in disparities in the time at which plasma LH and FSH concentrations rose in oestrogen-implanted, ovariectomized ewes of the same light treatment group. Prolactin concentrations showed an immediate decrease in ewes switched to short days, but remained elevated in long-day ewes. Since the breeding season started in the presence of high prolactin concentrations in long-day ewes, it seems unlikely that prolactin is an important factor determining the timing of the onset of cyclic activity.

Ovarian oestrogen-dependent maturation of the LH/FSH surge mechanism during prepubertal development in the gilt.

An involvement of ovarian secretions and in particular oestradiol-17 beta in the maturation of the positive feedback mechanism controlling gonadotrophin surge secretion was studied in prepubertal gilts. The LH/FSH responses to an intramuscular injection of age- and body weight-related doses of oestradiol benzoate (OB) were compared in intact gilts at 60 days of age with or without oestradiol-17 beta pretreatment from 30 to 52 days of age. Four further groups of gilts were challenged with OB at 160 days and were intact, ovariectomized at 60 days, ovariectomized at 60 days and given oestrogen therapy from days 60 to 130 or ovariectomized at 130 days. A significant increase in the magnitude of LH surge responses to OB and a decrease in the time to the first consistent period of surge secretion in intact gilts at 160 compared to 60 days of age confirmed earlier studies and is considered to represent a real maturational change in positive feedback activity. A longer response interval was also present in the majority of ovariectomized gilts. Furthermore a significant reduction in the magnitude of OB-induced LH responses at day 160 occurred in gilts ovariectomized at day 60 compared to those ovariectomized at day 130 and with intact control animals. Oestrogen therapy after ovariectomy at day 60 effectively restored the magnitude of the LH response however. It is concluded that maturation of the positive feedback mechanism is ovarian, and probably oestrogen, dependent.

Evaluation of LH-RH stimulation of testosterone as an index of reproductive status in rams and its application in wild antelope.

In rams a positive correlation (P less than 0.001) existed between average testosterone levels from 30-min blood sampling for 18 h and average testosterone levels of samples taken 0, 1 and 2 h after injection of LH-RH administered 90 min after anaesthesia. Attempts were therefore made to assess testosterone status by LH-RH challenge and limited blood sampling in animals immobilized in their natural habitat. In impala (Aepyceros melampus) territorial males had higher plasma testosterone values than did bachelors after LH-RH challenge (8.1 compared with 2.6 ng/ml, P less than 0.05). In blesbok (Damaliscus dorcas), the relationship was less clear, but testicular volume was correlated with plasma testosterone concentration and with testicular responsiveness measured by testosterone produced per unit of LH (P less than 0.001 and P less than 0.05, respectively). The LH-RH challenge technique therefore has value as a measure of testicular function and permits study of ungulates in their natural environment.

Long-term rhythms of testicular volume and plasma prolactin concentrations in rams reared for 3 years in constant photoperiod.

Two groups of 6 rams were kept under constant photoperiod consisting of short days (8 h light (L): 16 h dark (D); Group S) and long days (16 h L:8 h D; Group L) from 4 to 38 months of age. Five other rams were reared under a photoperiod representative of that occurring naturally (Group N). Testis size and plasma prolactin concentrations were obtained weekly. These data were subjected to time series analysis. The results indicated that there were persistent periodic excursions in both parameters measured. In Group N, the average cycle length for both testis volume and plasma prolactin was about 1 year and the peaks in plasma prolactin preceded those in testis volume by about 18 weeks. Rams from Group L also showed rhythmical changes in these parameters with periodicities of around 35 weeks and it is suggested that these cyclic changes may constitute true endogenous circannual rhythms; again the prolactin peaks preceded those of testis volume by about 18 weeks. Overall, rams from Group S had excursions of testis growth of a similar magnitude to those of Group L but the changes were less regular than those of Group L. Plasma prolactin was significantly lower in Group S than in Group L and there was little evidence for rhythmicity. It is proposed on the basis of the temporal relationship between peaks of prolactin and testis volume in Groups N and L, that prolactin may play a role in the timing of the reproductive cycle in the ram.

The effect of rearing under a long or short photoperiod on testis growth, plasma testosterone and prolactin concentrations, and the development of sexual behaviour in rams.

Two groups of 6 rams were maintained under constant photoperiodic conditions consisting of short days (8 h light: 16 h dark; Group S) and long days (16 h light; 8 h dark; Group L) from 4 to 20 months of age. Five other rams were reared under a photoperiod representative of that occurring naturally (Group N). Testis size, plasma testosterone and prolactin concentrations were monitored weekly and sexual behaviour tests were carried out at regular intervals. Over the treatment period Groups S and L did not differ in terms of testis growth or plasma testosterone. Both groups had a phase of testis growth and increased testosterone followed by a decline and the temporal patterns for the two groups were equivalent. Sexual behaviour was slower to develop in Group L than in Group S, indicating that photoperiod can affect the development of sexual behaviour irrespective of peripheral plasma testosterone concentrations. Plasma prolactin levels showed a cyclic variation in Group L and were significantly higher overall than in Group S rams. This, together with a trend towards negative correlations between prolactin concentrations and sexual behaviour in Group L, indicates that prolactin may be involved in the effect of photoperiod on sexual behaviour. The presence of a cycle of testicular growth and of hormone concentrations in young animals under constant photoperiod tentatively suggests that these cycles are endogenous. The constant photoperiod did, however, affect the animals because the cycles which occurred in Groups S and L were out of phase with those of Group N by about 4 months.

Plasma progesterone and gonadotrophin concentrations and ovarian activity in post-partum dairy cows.

Plasma progesterone concentrations in jugular vein blood samples collected every other day after calving from 13 Friesian dairy cows indicated that ovarian cyclic activity was initiated by 16.6 +/- 1.1 (s.e.m.) days post partum, except for 1 cow which did not resume cyclic activity until Day 98 post partum. Rectal palpation of the ovaries indicated that a developing follicle was recognizable at a mean time of 15.7 +/- 2.0 days after calving. During the first oestrous cycle after parturition there was a significantly shorter period when plasma progesterone levels were elevated than during the next 2 cycles. Concentrations of progesterone, LH, FSH and prolactin were determined for 4 cows, in blood samples taken every 6 h from 2 to 36 days post partum. Tonic LH release was lower during the first 10 days than subsequently, but the lack of change in pattern for FSH suggests dissimilar control mechanisms for these hormones during this time. Three cows showed evidence of a resumption of ovarian cyclicity during the sampling period: in 2 there was an initial LH surge of a magnitude which would normally give rise to ovulation, followed 4 days later by an increase in plasma progesterone lasting only 5 and 9 days. This progesterone was considered to be of follicular origin. A second LH surge was followed by the presence of a corpus luteum.

Serum glucocorticoids, growth hormone and insulin and plasma glucose in bulls given prostaglandin E2 or F2 alpha 1.

Plasma glucose and serum insulin, growth hormone and glucocorticoid concentrations were determined in five yearling bulls given (im) 5, 15 or 30 mg prostaglandin E2 (PGE2), 30 mg prostaglandin F2 alpha(PGF2 alpha) or saline. Jugular blood was collected at frequent intervals around the time of injection and at .5--hr intervals from 1 to 9 hr after injections. Thirty milligrams PGE2 and 30 mg PGF2 alpha each caused 15- to 20-fold increases in serum glucocorticoids. Glucocorticoids increased with increasing doses of PGE2. Although PGE2 and PGF2 alpha each increased blood growth hormone, this effect was about twofold larger after PGE2. By contrast, PGE2 depressed serum insulin about 50% for 1 hr, then insulin increased about sixfold until 3 to 4 hours. Blood serum insulin increased after PGF2 alpha, but this effect only approached significance (P less than .10). Plasma glucose increased about 10 mg/100 ml after PGE2, but was not affected significantly by PGF2 alpha. Thus, the effects of PGE2 and PGF2 alpha on hormones which control glucose metabolism differ markedly. We speculate that PGE2 caused a twofold increase in growth hormone secretion within 10 to 20 min, that increased growth hormone induced increased blood glucose within 1 to 2 hr and that increased glucose caused increased insulin secretion at 2 to 4 hr, but we cannot rule out a transitory (1 hr) suppressive effect of PGE2 directly on the pancreas.

Effect of the odour of female rat urine on plasma testosterone concentrations in male rats.

Testosterone levels were significantly higher in males, regardless of sexual experience, exposed to the odour of urine from females than in those exposed to saline or kept in ordinary groups.