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A. N. Brooks and A. S. McNeilly

Summary. Sheep fetuses at day 70 of gestation (term = 145 days) were implanted subcutaneously with a biodegradable implant containing a luteinizing-hormone-releasing hormone (LHRH) agonist (buserelin) to investigate whether treatment with LHRH agonist would induce a state of desensitization of the fetal gonadotrophs and thus influence fetal gonadal development.

Treatment with the LHRH agonist for 35–40 days caused a significant reduction in mean fetal plasma concentrations of LH and follicle-stimulating hormone (FSH) compared with control fetuses. LH pulses were evident in control fetuses but were completely abolished by buserelin treatment. Furthermore, the pituitary content of LH and FSH was significantly depleted in fetuses implanted with LHRH agonist. A bolus intravenous injection of 500 ng LHRH given to control fetuses caused a rapid and significant increase in plasma LH and FSH concentrations which was sustained for at least 60 min after injection. Pretreatment with buserelin completely abolished the LH and FSH responses to a bolus injection of LHRH. There were no differences between the sexes in fetal gonadotrophin concentrations or pituitary sensitivity to LHRH in control or agonist-treated fetuses. Furthermore, buserelin treatment for 35–40 days had no effect on the morphological appearance of the fetal gonads when compared with control fetuses, at least to day 110 of pregnancy.

These results provide evidence for the induction of a state of densensitization of the LHRH receptors of the fetal pituitary gonadotrophs following long-term treatment with an LHRH agonist, but provide no evidence for a role for gonadotrophin secretion in gonadal development at this stage in fetal life.

Keywords: fetus; sheep; gonadotrophin; LHRH; agonist

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D. W. Miller, H. M. Fraser and A. N. Brooks

The hypothalamo–pituitary–gonadal axis matures during fetal life and may be susceptible to adverse effects. Model systems can be used to understand its physiological role. The aim of this study was to determine whether antibodies to GnRH, administered to the mother, can cross the ovine fetal–placental barrier and suppress fetal gonadotrophin secretion. Maternal passive immunization to GnRH in pregnant Greyface ewes (day 103 of gestation) resulted in GnRH antibody titres of from 1:6000 to 1:9000 after 1 day and a suppression of the pulsatile secretion of fetal LH after 2 days. Fetal FSH concentrations declined gradually over the 11 days of the experiment and were only significantly different from control animals immunized against BSA in male fetuses. The slower decrease in fetal FSH concentrations than in LH concentrations shows that the secretion of FSH, unlike that of LH, is not dependent on short term changes in GnRH release. The lack of a suppressive effect of the maternal GnRH immunoneutralization on female fetal FSH secretion may be due to removal of the negative feedback effect of oestradiol and, possibly, inhibin. There was no sexual dimorphism in the effect of maternal GnRH immunoneutralization on fetal GnRH antibody titres or fetal LH secretion. These findings show that maternal passive immunization against GnRH results in GnRH antibodies crossing the fetal–placental barrier and suppressing fetal LH and FSH secretion in males, but only suppressing LH secretion in the females. Although the lack of effect on FSH secretion in the females needs to be investigated further, the present study provides evidence of a non-invasive procedure for blocking fetal gonadotrophin secretion which may be used to investigate hypothalamo–pituitary–gonadal function during early gestation in sheep.

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A. N. Brooks, N. B. Haynes, K. Yang and G. E. Lamming

Summary. 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 < 0·01), LH episode frequency and episode height (P < 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.

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K-P. Yang, G. E. Lamming, N. B. Haynes and A. N. Brooks

Summary. 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 < 0·05 and <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.

Keywords: opiods; LH; melatonin; ewes

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A. N. Brooks, G. E. Lamming, P. D. Lees and N. B. Haynes

Summary. 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 methionineenkephalin analogue FK33-824 (250 μg/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 8 h) 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.

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K. Yang, N. B. Haynes, G. E. Lamming and A. N. Brooks

Summary. 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-17β. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17β or oestradiol-17β 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-17β, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17β. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17β 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-17β exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17β during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.

Keywords: opioids; LH; oestradiol-17β; progesterone; ewes

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R. E. Short, A. N. Brooks, A. R. Peters and G. E. Lamming

Summary. Injections of an opioid agonist (bremazocine) and/or an antagonist (quadazocine) were given to heifers during the luteal or follicular phase of the oestrous cycle. Quadazocine was injected (210 mg/injection) three times at 2-h intervals, and bremazocine was injected (0·45 mg/injection) every 15 min for 6 h. Blood samples were taken every 15 min beginning 6 h before treatments started and continued for 18 h.

LH secretion patterns were not affected by quadazocine in the luteal-phase heifers, but quadazocine and bremazocine had marked effects during the follicular phase. Quadazocine increased LH secretion by increasing peak height but not peak frequency. Bremazocine decreased LH secretion through both peak height and frequency. This decrease was of greater magnitude than the increase due to quadazocine. When quadazocine and bremazocine were given together, these effects were cancelled and none of the effects carried over into the bleeding period after treatments stopped. No apparent interruption of follicular maturation was detected since all follicular-phase heifers were detected in oestrus at normal intervals. We conclude that heifers in this experiment did not have an opioid-mediated mechanism for progesterone suppression of LH but that an opioid mechanism for modulating LH does exist during the follicular phase.

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A series of experiments was conducted to investigate the effect of basal feed level and increased feed intake during oestrus on ovulation rate, early embryo survival, and residual pituitary LH potency in the pig. All the gilts used were fed 1·8 kg food/day from puberty to second heat. Gilts whose food intake was reduced from 1·8 kg to 1·4 kg/day following second heat had more atretic follicles at third heat and had fewer CL and fewer embryos at Day 20 of gestation, than control gilts fed 1·8 kg throughout, but these differences were not significant. Increasing the feed intake of gilts fed only 1·4 kg between second and third heat to 3·6 kg on Day 1 of oestrus only resulted in CL counts intermediate between those of the other two groups. The number of mature and atretic follicles during oestrus was not affected. Anoestrus was more frequent in gilts whose feed intakes were reduced to 1·4 kg/day. Residual pituitary LH potency was higher in gilts receiving only 1·4 kg feed/day than in gilts fed 1·8 kg/day. Increasing feed intake to 3·6 kg on Day 1 of oestrus significantly (P<0·05) reduced residual pituitary LH potency in animals previously fed 1·4 kg/day, but had no significant effect on animals previously fed 1·8 kg/day.

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K A Brennan, G S Gopalakrishnan, L Kurlak, S M Rhind, C E Kyle, A N Brooks, M T Rae, D M Olson, T Stephenson and M E Symonds

Epidemiological and animal studies strongly indicate that the environment experienced in utero determines, in part, an individual’s likelihood of developing cardiovascular disease in later life. This risk has been further linked to impaired kidney function, as a result of compromised development during fetal life. The present study therefore examined the influence of maternal nutrient restriction (NR), targeted at specific periods of kidney development during early to mid gestation, on the mRNA abundance of receptors for glucocorticoid (GCR), growth hormone (GHR) and insulin-like growth factors-I (IGF-IR) and -II (IGF-IIR), and the IGF-I and -II ligands. This was undertaken in both singleton and twin fetuses. At conception ewes were randomly allocated to either an adequately fed control group or one of four nutrient-restricted groups that were fed half the control amount from 0 to 30, 31 to 65, 66 to110 or 0 to110 days gestation. At 110 days gestation all ewes were humanely euthanased and fetal kidneys and surrounding adipose tissue sampled. There was no effect of NR or fetal number on kidney weight, shape or nephron number, but the surrounding fat mass was increased in singleton fetuses exposed to NR for 110 days. An increase in kidney mRNA abundance with NR only occurred in singleton fetuses where IGF-IR mRNA was enhanced with NR from 66–110 days gestation. In twin fetuses, NR had no effect on mRNA abundance. However, for all genes examined mRNA expression was lower in the kidneys of twin compared with singleton fetuses following NR, and the magnitude of the effect was dependent on the timing of NR. In conclusion, the abundance of mRNA for receptors which regulate fetal kidney development are lower in twin animals compared with singletons following periods of nutrient deficiency. This may impact on later kidney development and function.