ABSTRACT

We have monitored the 24-h profiles of plasma melatonin concentrations between birth and 10 weeks of age, in lambs which were delivered to, and suckled, either pineal-intact (control group) or pinealectomized (pinealectomized group) ewes. Between 0 and 2 weeks of age, plasma concentrations of melatonin in lambs suckling either intact or pinealectomized ewes were highest at 01.00 h. At this age, however, there was no significant difference in the mean plasma concentrations of melatonin between the entire dark and light phases in lambs in either the control group (dark, 39·7 ± 6·0 (s.e.m.) pmol/l; light, 39·5 ± 8·1 pmol/l) or the pinealectomized group (dark, 79·8 ± 43·3 pmol/l; light, 60·9 ±8·7 pmol/l). Between 3 and 4 weeks of age, however, a diurnal rhythm in plasma melatonin concentrations was clearly present in the lambs in both the control and pinealectomized groups (control group: dark, 164·1 ± 5·6 pmol/l; light 26·2 ± 2·5 pmol/l; pinealectomized group: dark, 52·7± 8·0 pmol/l; light, 19·1 ± 5·3 pmol/l; P<0·001). Between 3 and 10 weeks of age, plasma concentrations of melatonin in the dark phase were significantly (P<0·05) lower in the lambs suckling pinealectomized ewes than in the control group. In both the control lambs and lambs suckling pinealectomized ewes, the mean plasma concentrations of melatonin in the dark and light phases increased significantly (P<0·05) between 3 and 6 weeks after birth.

In conclusion, we have demonstrated that a clear diurnal plasma rhythm in melatonin concentrations does not emerge until 3–4 weeks of age in lambs suckling either pinealectomized or intact ewes. We have also measured lower melatonin concentrations during the dark phase in lambs suckling pinealectomized ewes when compared with lambs suckling pineal-intact ewes between 3 and 10 weeks of age.

Journal of Endocrinology (1990) 125, 97–102

The corpus luteum was removed from tammar wallabies during either lactational or seasonal quiescence, and the pouch young were replaced on the teat after lutectomy. Follicular development resumed and the animals came into oestrus 11–14 days later. Injections of oil, progesterone or androstenedione did not prevent follicular growth, although ovulation only occurred in one animal, but treatment with oestradiol-17β, either alone or mixed with progesterone, inhibited the development of follicles. These results suggest that oestrogen, of luteal origin, is responsible for the suppression of follicular development during lactational and seasonal quiescence.

ABSTRACT

Exogenous cyclic adenosine nucleotides increase gonadotrophin-releasing hormone (GnRH) receptors in intact cultured rat pituitary cells in a similar manner to that observed with GnRH itself. In this study the calcium and microtubule dependency of GnRH receptor up-regulation was examined in vitro. Treatment of pituitary cells in Ca²⁺ and serum-containing media with either GnRH (1 nmol/l), K⁺ (58 mmol/l) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) for 7–10 h routinely resulted in a 50–100% increase in GnRH receptors. Incubation of pituitary cells with the calcium channel blocker verapamil, for 7 h, or the calcium chelator EGTA, for 10 h, had no effect on basal receptor levels but prevented the increase in GnRH receptors stimulated by either GnRH, K⁺ or dbcAMP. Luteinizing hormone release measured with the same stimulators over a 3-h period was prevented by both verapamil and EGTA. Calcium ionophore (A23187) increased GnRH receptors by 40–60% at low concentrations (10 and 100 nmol/l) while higher concentrations (10 and 100 μmol/l) reduced receptor levels. Luteinizing hormone release was not increased by receptor-stimulating concentrations of A23187, but was by higher concentrations (10 μmol/l). None of these pretreatments, for up to 10 h, impaired the subsequent LH response of the cells to increasing doses of GnRH.

Vinblastine (1 μmol/l did not affect basal receptor levels but markedly reduced the increase in GnRH receptors stimulated by GnRH, K⁺ and dbcAMP. This concentration of vinblastine had no effect on LH release. These results indicate that receptor stimulation by GnRH, K⁺ and dbcAMP is a calcium-dependent process requiring the integrity of the microtubule system and there is a different calcium requirement for the processes of GnRH receptor up-regulation and LH secretion.

J. Endocr. (1985) 107, 49–56

During the past few years it has become clear that anterior pituitary glands from different species of animal contain different proportions of physiologically active substances. Loeb and Friedman [1933], in comparing the anterior pituitary glands of various species, distinguished two groups according to their contents of gonadotrophic and thyrotrophic hormones. In one class, in which they placed ox, sheep, and pig, the gland possessed high thyrotrophic activity and high gonadotrophic activity of the type considered to be due to the 'luteinizing hormone' of the gonadotrophic complex. In the second class, in which they placed the cat, rat, guineapig, and rabbit, the pituitary gland contained little thyrotrophic hormone but a larger proportion of the follicle-stimulating constituent of the gonadotrophic complex. In this second class it is possible also to place the horse and adult man [Rowlands, 1936; Henderson and Rowlands, 1938; Cope, 1938]. An association between the thyrotrophic and the luteinizing

ABSTRACT

We have investigated the effect of fetal hypophysectomy on the localization of dopamine B-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and enkephalin-containing peptides in the fetal sheep adrenal, using immunocytochemical techniques.

Staining with anti-DBH was observed throughout the adrenal medulla in the intact (140–146 days of gestation) and hypophysectomized fetal sheep (147–164 days of gestation) and the newborn lamb (10–12 days after birth). In the adrenal medulla of the lategestation intact fetal sheep and newborn lamb, positive staining with anti-PNMT was observed in the peripheral rim of medullary cells adjacent to the adrenal cortex. After hypophysectomy, there was intense positive staining with anti-PNMT in the peripheral adrenal medullary cells and a small and variable proportion of central adrenal medullary cells were stained with anti-PNMT. In the adrenal gland of the intact fetal sheep and the newborn lamb, there was intense staining with anti-enkephalin in the peripheral rim of adrenal medullary cells. Staining with antienkephalin was less intense in the central medullary cells of the adrenal gland of the intact fetal sheep and the 10- to 12-day-old newborn lamb, and many unstained central medullary cells were present. After hypophysectomy, intense positive staining with antienkephalin was observed throughout the entire fetal adrenal medulla. Therefore, the fetal pituitary, either directly or indirectly through the adrenal cortex, plays a role in regulating the pattern of localization of both PNMT and enkephalin in the fetal sheep adrenal.

Journal of Endocrinology (1989) 121, 425–430

ABSTRACT

We have investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation.

Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138–139 days, either ACTH(1–24) (10·5 μg/0·24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0·9% (w/v) NaCl alone (0·24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0·2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2·0 × 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H₂O₂, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk).

The total adrenal content of immunoreactive Met-O-Enk was significantly greater in the Hx + ACTH group (326·2 ±66·7 (s.e.m.)ng/adrenal) when compared with either the Intact + sal group (152·7 ±44·0 ng/adrenal) or the Hx + sal group (112·1 ±20·8 ng/adrenal). In the adrenal glands from all fetuses immunoreactive Met-O-Enk was found in four molecular weight ranges: < 12 kDa, 12–7 kDa, 7–3 kDa and < 3 kDa. There was no significant difference between the Hx + sal and Hx + ACTH groups in the proportion of immunoreactive Met-O-Enk present in each of the molecular weight ranges in the adrenals and therefore the data from these groups were combined for further statistical analysis. The proportion of immunoreactive Met-O-Enk in the > 12 kDa range was significantly less in the Intact + sal group (5·5 ±2·3%) when compared with the hypophysectomized sheep with or without ACTH replacement (18·7 ± 4·5%).

These data demonstrate that fetal hypophysectomy alters the molecular weight profile of Enk-containing peptides in the adrenal of the fetal sheep and whilst ACTH replacement in the hypophysectomized fetus does not alter the post-translational processing of the Enk-containing peptides, it stimulates an increase in the total amount of immunoreactive Met-O-Enk in the fetal adrenal in late gestation.

Journal of Endocrinology (1992) 134, 369–375

Abstract

Parturition in the sheep is preceded by an increase in the synthesis of prostaglandins by intrauterine tissues. Prostaglandin G/H synthase (PGHS) is the central enzyme involved in prostanoid production. Its expression is enhanced during late gestation in the ewe. Recent studies have identified two PGHS isozymes, termed PGHS-1 and PGHS-2. The labour-associated expression of the two isozymes of PGHS in the sheep has not been characterized.

This study investigated the changes in expression of immunoreactive PGHS-1 and PGHS-2 in ovine amnion and placenta following glucocorticoid-induced labour. Ewes underwent surgery to implant fetal and maternal vascular cannulae and uterine electromyogram electrodes between 118 and 125 days of gestation. Fetal sheep were administered either the glucocorticoid betamethasone (n=5) or saline (control n=6) by direct transabdominal intrafetal injection. Ewes from the betamethasone-injected group were killed in the first stage of labour as indicated by uterine electromyographic activity. Ewes from the saline-injected group were killed at the same time to obtain age-matched control tissue. The time taken to euthanasia following induced-labour onset in the glucocorticoid-injected animals was 56·6 ± 0·8 h post-injection.

Plasma endocrine profiles in the maternal and fetal circulation following glucocorticoid injection were comparable to those observed following normal spontaneous delivery. At post-mortem, amnion and cotyledons were collected in liquid N₂ and stored at −70 °C. Solubilized tissue extracts were prepared and analysed by Western blots using polyclonal antibodies to PGHS-1 and PGHS-2 isozymes. Fetal amnion contained PGHS-1 isozyme at day 133 of gestation, as demonstrated in the saline-injected animals. Slightly higher PGHS-1 immunoreactivity was observed following induced-labour onset, although this did not reach statistical significance (P>0·05). PGHS-2 enzyme was not detectable in amnion. PGHS-2 expression was also not induced following labour onset.

In contrast, PGHS-2 demonstrated enhanced expression following glucocorticoid-induced labour in ovine cotyledon. This tissue contained PGHS-1 enzyme, but immunoreactive levels were minimal and demonstrated limited regulation at labour.

These data suggest that the previously reported rise in placental PG production at term in the sheep is predominantly due to increased expression of the PGHS-2 isozyme. This suggests that PGHS-2 contributes to PG production at term labour in sheep or is induced by the mechanisms controlling ovine parturition. PGHS-1 isozyme is produced constitutively in ovine amnion and may contribute to the gestational increase in PG formation by intrauterine tissues.

Journal of Endocrinology (1996) 151, 125–135

ABSTRACT

Acid–ethanol precipitation and gel filtration at acidic pH have been widely used to extract circulating binding proteins for insulin-like growth factor (IGF-I and IGF-II) from plasma or serum samples before radioligand assay for the respective IGFs. Gel filtration on Sephadex G-50 at neutral pH of neutralized acid– ethanol extracts of fetal and adult ovine plasma which had been incubated with ¹²⁵I-labelled IGF-I or ¹²⁵I-labelled IGF-II revealed that significant amounts of the IGF-binding protein activity survived the acid– ethanol extraction procedure.

Radioimmunoassay for IGF-I in acid–ethanol extracts of plasma samples from fetal, neonatal and adult sheep yielded results which depended upon the method used for separation of the antibody-bound IGF-I tracer from the free IGF-I tracer. Acid gel filtration of ovine fetal and adult plasma was found to remove completely the IGF-binding protein activity. Radioimmunoassay for IGF-I in samples of fetal, neonatal and adult sheep plasma that had undergone acid gel chromatography yielded consistent results for both methods that were used to separate antibody-bound IGF-I tracer from the free tracer.

Radioreceptor assays for IGF-II were similarly highly perturbed by the presence of binding protein in acid–ethanol extracts of ovine fetal and adult plasma. We conclude that acid–ethanol extraction can not be used reliably for the removal of IGF-binding proteins, and that only acid gel filtration is a completely safe and valid method.

J. Endocr. (1988) 119, 453–460

ABSTRACT

Castrated prepubertal lambs were hypophysectomized and then treated with GH and testosterone either alone or in combination over a series of 3-week treatment periods. Hypophysectomy resulted in a rapid reduction in skeletal growth rate which could be reversed by the administration of either GH (4IU three times a week for 3 weeks) or testosterone propionate (10 mg daily for 3 weeks). When GH or testosterone treatment was withdrawn, skeletal growth fell to the post-operative rate. Combined treatment with both GH and testosterone was no more or less effective than either hormone given singly. The order of administration did not have any effect on the growth rate. Circulating concentrations of insulin-like growth factor-I (IGF-I) were reduced by hypophysectomy, but neither GH nor testosterone treatment, alone or in combination, had any effect on IGF-I concentrations. Concentrations of IGF-II rose following hypophysectomy, and again were not affected by any of the hormonal replacement treatments.

In conclusion, both GH and testosterone could stimulate skeletal growth in the hypophysectomized lamb without any alteration of circulating IGF concentrations, and testosterone can clearly stimulate skeletal growth in the complete absence of GH.

Journal of Endocrinology (1989) 121, 563–570