ABSTRACT

Treatment of Damline ewes with twice-daily i.v. injections of bovine follicular fluid during the luteal phase for 10 or 2 days before prostaglandin-induced luteolysis resulted in a delay in the onset of oestrous behaviour and a marginal increase in ovulation rate. During the treatment cycle, blood samples were withdrawn at 15-min intervals for 25 h from 08.00 h on days 1, 6 and 10 (day 0 = oestrus). At all three stages of the luteal phase, plasma FSH concentrations were suppressed relative to controls 3 h after the 09.00 h injection of follicular fluid and remained low until

06.00 h on the following day. In the 10-day treatment group LH pulse amplitude was significantly greater than that of controls on days 6 and 10. Pulse frequency remained high throughout treatment and was significantly higher relative to controls on day 10 despite normal progesterone levels. The results suggest that the higher pulsatile LH secretion during the luteal phase is due to reduced negative feedback effects of oestradiol occurring as a result of the follicular fluid-induced reduction in FSH.

J. Endocr. (1986) 111, 317–327

Several methods are now available for the immunoassay of insulin in plasma. Of these, the double antibody technique of Hales & Randle (1963) is especially convenient. However, in studies of plasma insulin concentrations in the sheep with method C of Hales & Randle (1963), plasma samples assayed undiluted consistently yielded higher values than the same plasma samples assayed after 1:4 dilution with buffer B of Hales & Randle (1963). Addition of ovine insulin (0–200μ-u./ml.) to ovine plasma showed that the sensitivity of the assay was greater in undiluted plasma than in buffer, the decrease in antibody-bound labelled insulin for a given increment in insulin concentration being greater in the plasma system. Addition of 0·01 m-EDTA (Morgan, Sorenson & Lazarow, 1964; Sheldon & Taylor, 1965) did not eliminate this discrepancy. Insulin standards in buffer did not appear to provide a suitable baseline for assay of insulin in undiluted ovine plasma.

SUMMARY

Plasma growth hormone (GH) concentrations in adult sheep and lambs were measured by radioimmunoassay. Mean values before feeding were 3·1 (s.e. − 0·4, + 0·5) ng./ml. in lactating ewes and 3·9 (− 0·5, + 0·7) ng./ml. in their lambs. In adult wethers the mean GH concentration before feeding was 2·3 (−0·2, + 0·3) ng./ml.

After one i.v. injection of 400 μg. ovine GH, the hormone disappeared from the plasma of adult sheep with a half-time of 7–8 min. Plasma GH concentrations decreased at a similar rate after the establishment of high concentrations of endogenously secreted GH. The results suggest a shorter half-life than has been reported for human GH in man. GH could not be recovered in an immunologically recognizable form in urine. Fasting for 4 days led to no systematic change in plasma GH concentrations. Feeding did not increase plasma GH. Intravenous injection of saline resulted in some increase in plasma GH in both ewes and lambs. In adult sheep intravenous glucose (0·25 g./kg.) did not cause any greater change than that seen after saline, but in lambs an increase occurred. Intravenous injection of insulin (0·25 unit/kg.) resulted in an increase in plasma GH at 15–30 min. and this effect was found in both lambs and ewes. Infusion of adrenaline at either 25 μg. or 50 μg./min. into adult wethers (average body wt 51 kg.) caused a decrease in plasma GH concentration. It was concluded that the changes in plasma GH concentration of sheep in a number of physiological situations differ from those reported to occur in man.

ABSTRACT

The effects of insulin-like growth factor-I (IGF-I), IGF-II and des(1–3)IGF-I, a potent IGF-I analogue, on the secretion of GH and IGF-binding protein (IGFBP) from cultured rat anterior pituitary cells were measured. IGF-I and des(1–3)IGF-I stimulated GH secretion at low concentrations (maximally effective at 1 and 0·1 μg/l respectively) and inhibited GH secretion at higher concentrations. The half-maximal inhibitory concentrations (IC₅₀) were approximately 20 μg/l and 1 μg/l for IGF-I and des(1–3)IGF-I respectively. Thus des(1–3)IGF-I was more potent than IGF-I in these effects on GH secretion. We postulate that the increased potency of des(1–3)IGF-I in affecting GH secretion is due to decreased binding of this peptide by pituitary IGFBP compared with IGF-I. In contrast with IGF-I and des(1–3)IGF-I, IGF-II did not stimulate GH secretion at low concentrations, but did inhibit GH secretion from pituitary cells with an IC₅₀ of approximately 20 μg/l.

Several IGFBPs ranging in molecular mass from 22 000 to 52 000 were detected in medium conditioned by cultured anterior pituitary cells. When measured by Western-ligand blotting and competitive ligand-binding techniques, these IGFBPs exhibited decreased binding of des(1–3)IGF-I compared with IGF-I and IGF-II. The production of IGFBP by anterior pituitary cells was stimulated by the addition of IGFs to the culture medium.

Journal of Endocrinology (1991) 130, 93–99

ABSTRACT

It has previously been shown that treatment of ewes with bovine follicular fluid (bFF) throughout the luteal phase of the oestrous cycle lowers plasma levels of FSH but increases the frequency and amplitude of the pulses of LH. Under these conditions, ovarian follicles grow to a maximum diameter of 2·7 mm and have a reduced capacity to release oestradiol. We have examined the nature of the gonadotrophin signals controlling follicular development in the normally cycling ewe and have investigated the effects of previous exposure to bFF on these signals and the follicular responses to them. Control ewes (n = l) were injected i.v. with 9 ml bovine serum and treated ewes were injected with 9 ml bFF, twice daily from days 1 to 10 of the luteal phase (day 0 = oestrus). The ewes were injected with prostaglandin analogue on day 11 of the cycle to induce luteolysis and the gonadotrophin patterns were studied in blood sampled from these animals every 10 min for up to 72 h during the subsequent follicular phase. Following luteolysis (and the end of bFF treatment), LH pulse frequency increased rapidly in both groups and reached 1 pulse/h within 6 h. Thereafter, pulse frequency increased marginally and reached 1 pulse/50 min by the onset of the LH surge. This pattern was not affected by previous treatment with bFF. In the control ewes, the amplitude of the LH pulses did not change significantly following luteolysis or at any time during the follicular phase, while the levels of FSH declined slowly until the onset of the surge. In the treated ewes, on the other hand, there was an immediate increase in both LH pulse amplitude and the concentration of FSH immediately after the end of bFF treatment at luteolysis, and they remained above control levels for 24 and 16 h respectively. Plasma prolactin levels did not appear to change around the time of luteolysis but showed a marked and significant diurnal rhythm (nadir around noon and peak around midnight) in both groups. The concentrations of prolactin were significantly (P<0·001) lower and the preovulatory peak was delayed and reduced in the bFF-treated ewes relative to controls. The onset of oestrus was also significantly (P<0·01) delayed by bFF treatment, but the ovulation rates did not differ between the groups. Furthermore, comparisons within or between groups revealed no significant relationships between any of the variables of plasma LH secretion during the follicular phase and the subsequent ovulation rate.

These observations provide a complete description of gonadotrophin patterns during the follicular phase of the ewe and confirm the suggestion that an increase in LH pulse frequency is the major driving force behind the follicular growth that ultimately leads to ovulation. On the other hand, it appears most unlikely that the pattern of LH secretion during the follicular phase has any influence on ovulation rate. The levels of FSH declined in the period leading up to the preovulatory surge, presumably as a consequence of rising peripheral levels of oestrogen (and/or inhibin). We also expected LH pulse amplitude to decline during the follicular phase because it has been proposed that pulse amplitude is also controlled by oestrogen. The absence of any significant fall in amplitude suggests that hypotheses about the control of LH secretion drawn from studies with ovariectomized ewes require further verification in the intact ewe. The effect of bFF on prolactin levels probably reflects the low rates of secretion of oestradiol by the small ovarian follicles in these ewes.

J. Endocr. (1988) 116, 123–135

SUMMARY

The effects of some oestrogens and progestogens on the glycerylphosphorylcholine (GPC) diesterase activity and the protein concentration of rinsings of the rat uterus have been studied in relation to changes in the weight of this organ. Both enzyme activity and protein concentration were increased by subcutaneous administration of oestradiol-3:17β, oestriol, oestrone and diethylstilboestrol to ovariectomized rats and these responses were inhibited by progesterone and 17α-ethyl-19-nortestosterone. The GPC diesterase activity and protein content of uterine rinsings were affected independently of each other and of changes in the uterine weight.

SUMMARY

The administration of 5 mg. ovine growth hormone (GH) per day for 4 weeks to adult sheep resulted in the retention of 0·5 g. nitrogen/24 hr. This is less than that reported to occur in man and dog with a similar dose of GH. Both plasma volume and red blood cell volume increased by 20 % during treatment. A small decrease occurred in the amount of sodium excreted, but no change in potassium excretion could be demonstrated. The water intake of the treated group declined during GH injections.

Plasma glucose concentrations increased slightly during treatment but there was no alteration in the concentration of plasma free fatty acids or ketone bodies.

Body weight did not change measurably with GH treatment. Wool growth decreased during treatment but then increased and remained above control values for at least 20 weeks.

SUMMARY

Growth hormone (GH) concentrations in the plasma of foetal lambs (range 20–150 ng/ml) were ten or more times those in the plasma of ewes during the last third of pregnancy. Blood samples obtained from foetuses with indwelling vascular catheters indicated that there was a steady rise in foetal plasma GH concentrations from 40–50 ng/ml at 100–110 days gestation to maximum values of 110–120 ng/ml between 130 and 140 days gestation and then a rapid decrease around the time of birth.

Growth hormone disappeared from the foetal plasma after hypophysectomy of the foetus and there was no evidence for a placental source of the hormone. Ovine pituitary growth hormone given by intravenous injection disappeared from the foetal plasma at a similar rate to the endogenous hormone following hypophysectomy. The rate of GH removal from foetal plasma was slower than that in lambs 4–12 days after birth.

Glucose infusion into foetal lambs did not alter their plasma GH concentration significantly, but infusion of the catecholamine, isoprenaline, decreased the foetal plasma GH concentration rapidly. Cessation of infusion was followed by very rapid restoration of the former high plasma GH concentration. The results indicate that the high foetal plasma GH concentration is maintained by active secretion of the hormone, but its function in the foetal lamb remains obscure.

ABSTRACT

The gonadotrophic requirements for the induction of ovulation and formation of a viable corpus luteum in two breeds of seasonally anoestrous sheep of differing fecundity was investigated.

Welsh Mountain (n = 20) and Damline ewes (n = 19) were given LH or FSH either alone or in combination. Luteinizing hormone was injected i.v. at an increasing frequency for 72 h (one injection every 3 h for 24 h, one every 2 h for 24 h, and one every hour for 24 h) and FSH was injected in an identical manner for the first 36 h of treatment.

Exogenous LH alone and in combination with FSH induced a preovulatory LH surge in all 19 ewes and ovulation in 18 out of 19 ewes of both breeds. However exogenous FSH alone was ineffective. The incidence of normal corpus luteum function in ewes induced to ovulate was low and not related to treatment, timing or magnitude of the LH/FSH surge. It is concluded that in both breeds studied (a) it is the infrequency of LH pulses which limits the development of preovulatory follicles during seasonal anoestrus, (b) that the requirement for FSH remains unknown, and (c) that the induction of inadequate corpora lutea during seasonal anoestrus reflects either defects in hormonal priming of the preovulatory follicle and/or inappropriate luteotrophic support after ovulation.

J. Endocr. (1986) 111, 181–190

ABSTRACT

Induction of ovulation early post partum in sheep is associated with a high incidence (30–40%) of premature luteolysis. The present study was designed to characterize oxytocin receptor levels, oxytocin-stimulated inositol phosphate (IP) turnover (second messenger) and oxytocin-stimulated prostaglandin F_2α (PGF_2α) release in the endometrium of post-partum ewes induced to ovulate 21 days after parturition and expected to exhibit a range of corpus luteal functions subsequently.

Ovulation was induced on day 21 post partum using a controlled internal drug release device and pregnant mare serum gonadotrophin, and uterine tissues were collected on days 5, 10 or 15 of the cycle (n = 4/day). A further 12 ewes whose interval from previous parturition exceeded 150 days were similarly treated and acted as controls. Measurement of daily peripheral progesterone concentrations revealed that while all control ewes exhibited normal luteal function, abnormal luteal function was evident in two, two and one post-partum ewes studied on days 5, 10 and 15 of the cycle respectively. Oxytocin receptor binding was detected (by receptor-binding assay and in-vitro autoradiography) in the endometrium and myometrium of post-partum ewes at all three stages of the oestrous cycle but only at day 15 in control ewes. To determine IP turnover, 100 mg caruncular endometrium was incubated in duplicate for 2·5 h with 10 μCi [³H]inositol and treated with 0 or 2 μmol oxytocin/l for 30 min, then [³H]inositol mono-, bis- and trisphosphates were quantified. Oxytocin stimulated total IPs in all day-5 and day-15 post-partum ewes, in three of four day-10 ewes and in all day-15 control ewes. Basal endometrial PGF_2α release measured in triplicate (100 mg/well) during a 2 h incubation was higher in post-partum versus control ewes on days 5 and 10 but not on day 15 of the cycle. Similarly, oxytocin stimulated PGF_2α release to varying levels at all stages of the cycle in post-partum ewes but only on day 15 in control ewes. Irrespective of the treatment group endometrial oxytocin receptor number was significantly (P < 0·001) correlated with oxytocin-stimulated IP turnover and PGF_2α release.

Thus the induction of ovulation and the subsequent luteal phase in post-partum ewes is against a back ground of high oxytocin receptor expression and enhanced PGF_2α release which in some ewes may contribute to abnormal luteal function.

Journal of Endocrinology (1993) 136, 17–25