SUMMARY

Changes in plasma LH concentrations were followed in chickens of both sexes from hatch to sexual maturity using a radioimmunoassay. Mean levels of LH were lower in the females than in the males at all stages of development. These levels rose rapidly in both sexes during the first week after hatch to maxima of 6·5 ± 1·2 (s.e.m.) ng/ml (n = 6) in the males and 4·6 ± 0·6 ng/ml (n = 6) in the females. Thereafter levels of the hormone in the circulation stabilized in the males but fell over a period of 1 or 2 weeks in the females to 2·5–3 ng/ml.

Plasma LH levels started to rise steeply in both sexes when they were between 16 and 19 weeks old at the same time as there was an increase in the rate of comb growth. Afterwards in six of the males studied in detail the mean plasma LH level rose significantly (P < 0·01) over a period of 5–8 weeks from 8·1 ± 1·2 to 13·2 ± 1·9 ng/ml. In a parallel study on six females the rate of LH secretion increased for approximately 3 weeks and then decreased for about the same period forming a prepubertal LH peak. The first eggs were laid between 22 and 25 weeks of age when mean plasma LH levels had fallen to about 1·8 ng/ml. The mean plasma LH level in these hens when they were laying (1·8 ± 0·3 ng/ml) was significantly lower (P < 0·01) than when they were sexually immature (2·7 ± 0·3 ng/ml). The duration of the period of rapid comb growth in each bird was closely related in the males to the time during which prepubertal LH levels were rising rapidly, and in the females to the duration of the prepubertal LH peak.

Differences in mean plasma LH concentrations in individual birds of either sex before the onset of puberty appeared to be related to subsequent reproductive performance.

The basal hypothalamus of Coturnix quail contains gonadotrophin releasing factor (GRF) activity (Follett, 1970) but the precise location of the neurons producing the neurohormone(s) is unknown. Lesioning studies show that the destruction of either of two sites blocks photo-inducible testicular growth. One of these lies in the dorsal basal hypothalamus around the paraventricular organ and the other directly above the median eminence (Sharp & Follett, 1969). These two areas are morphologically distinct when considered either in terms of the distribution of monoamines (Sharp & Follett, 1968) or of tanycyte processes (Sharp, 1972).

To explore this problem further, pituitaries were taken from sexually mature quail and fragments of the cephalic lobes were implanted into the hypothalami of gonadectomized birds. By removing the negative feedback effect of gonadal steroids it was hoped to stimulate GRF activity. It was anticipated that, as in rats (Halász, Pupp & Uhlarik, 1962), implanted pituitary cells would

Peripheral blood samples were taken from laying hens at frequent intervals during various periods of the ovulatory cycle in order to detect small changes in the concentrations of progesterone and androgen which might be important in initiating the preovulatory release of LH.

Blood samples were taken from seven hens at 1 h intervals for 3 h when the ovary contained a mature (C₁) follicle and on another occasion, when the largest ovarian follicle was immature. The concentrations of progesterone and androgens in the plasma were 30% higher when there was a mature C₁ follicle present in the ovary than when there was not, but this increase was significant (P < 0·05) only for progesterone.

The concentrations of progesterone and androgens were also measured in blood samples taken at 30 min intervals during the 3 h before and after the initiation of the first preovulatory LH surge of a sequence. The hens were kept on a lighting schedule of 14 h light/day and the first LH surge of a sequence was initiated at the beginning of the dark period. Just after the onset of darkness there was a small increase in the concentration of LH in the plasma and a subsequent, larger preovulatory release of LH. The first increase in the level of LH was associated with a small rise in the concentrations of androgens and progesterone in the plasma while the preovulatory release of LH was accompanied by a much larger increase in the secretion of these steroids.

It is proposed that the increase in the level of LH in the plasma at the onset of darkness stimulates the maturing ovarian follicles to secrete progesterone and androgens and that the quantities of these steroids secreted (particularly of progesterone) depends on the maturity of the largest ovarian follicle. If the largest ovarian follicle is mature, then the increase in the level of LH in the plasma associated with the onset of darkness stimulates the secretion of a quantity of progesterone sufficient to cause the preovulatory surge of LH. A diurnal increase in the concentration of LH in the plasma could, therefore, be responsible for timing the preovulatory surges of LH so that they are only initiated at night.

Agricultural Research Council's Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh, EH9 3JS

(Received 20 June 1977)

In the maturing hen the concentration of luteinizing hormone (LH) in the plasma rises to a prepubertal maximum and then declines over a 2–3 week period until the first egg is laid (Sharp, 1975). Sharp (1975) postulated that increased secretion of progesterone by the developing ovary may be responsible for this fall in the level of LH since the large, yolky follicles, which are thought to be steroidogenic (Furr, 1969), develop over a 2–3 week period before the onset of lay (Wilson & Sharp, 1975). The present experiment was designed to test this hypothesis by direct measurement of the amount of progesterone and LH in the blood of growing hens.

Variations in the level of LH in the peripheral plasma were determined by an homologous radioimmunoassay for avian LH (Follett, Scanes &

ABSTRACT

An assessment was made of the possible role of hypothalamic dopamine in the regulation of changes in plasma prolactin and LH in laying and broody bantam hens. Specific dopamine-binding sites were identified, using [³H]domperidone, in the anterior pituitary gland and in the anterior and posterior hypothalamus. The mean concentrations of dopamine-binding sites in both parts of the hypothalamus were 59–66 fmol/mg protein and did not differ between laying and incubating hens. The concentration of dopamine binding sites in the anterior pituitary gland was significantly (P<0·001) greater in laying than in incubating hens (278 ± 46 compared with 420 ± 32 fmol/mg protein, n = 5).

The turnover rates of dopamine were compared in the anterior and posterior hypothalami of laying, incubating and nest-deprived hens. The turnover rates were estimated from the rate of accumulation of dopamine after inhibiting its catabolism using the monoamine oxidase inhibitor, pargyline, or by measuring the ratio of the concentrations of dopamine and its major metabolite, homovanillic acid. Both methods gave the same results. The turnover of dopamine was increased in the anterior but not posterior hypothalamus of incubating hens when compared with laying or nest-deprived hens.

These results show, for the first time in birds, that the anterior pituitary gland contains specific binding sites for dopamine and that the concentration of these binding sites is inversely related to the concentration of plasma prolactin. The marked increase in dopaminergic activity in the anterior hypothalamus of incubating hens may stimulate the release of unidentified prolactin-releasing factors and/or inhibit the release of LH by exerting an inhibitory influence in the area of the hypothalamus containing LHRH cell bodies.

Journal of Endocrinology (1989) 121, 67–74

SUMMARY

This study was undertaken in laying hens to investigate the possibility that a diurnal increase in the concentration of plasma corticosterone is directly responsible for timing the preovulatory surge of LH which results in the first egg of a sequence.

Provided that the ovary contained a mature follicle, i.m. injection of 0·5 or 2·0 but not 0·1 mg corticosterone/kg stimulated a preovulatory release of LH. The dose of 0·5 mg/kg was less effective than that of 2 mg/kg and induced release of LH in only four out of eight hens. However, it resulted in concentrations of plasma corticosterone which were outside the physiological range.

Variations in the concentrations of plasma corticosterone were measured in ten hens on two successive nights for 8·5 h starting at the onset of darkness. The birds were maintained on a lighting regimen of 14 h light/day. The hens were selected so that on the first night there was no preovulatory release of LH while on the second night there was the first preovulatory surge of LH of a sequence starting soon after the onset of darkness. No diurnal increase in the concentration of plasma corticosterone was observed during the first 6 h of darkness on either night nor was any increase seen before the preovulatory release of LH.

These observations suggest that corticosterone is not directly involved in the timing of the first preovulatory surge of LH of a sequence.

SUMMARY

Single intramuscular injections of 0·5 mg progesterone/kg resulted in increased LH secretion in laying hens but not in pullets with completely undeveloped sexual organs. Injections of the steroid were first able to stimulate LH release 8–10 weeks before the onset of lay when the comb, ovary and oviduct had started to grow and basal plasma LH concentrations were beginning to rise. At this time, injections of 10 μg synthetic LH-RH/kg resulted in an incremental change in plasma LH levels of around 26 ng/ml. A similar incremental change was observed after giving the same dose of LH-RH to pullets with no signs of sexual development.

Three to four weeks before the first eggs were laid, basal plasma LH levels started to fall, the pituitary became progressively more insensitive to synthetic LH-RH and injections of 0·5 mg progesterone/kg resulted in a reduced LH response. Ten μg LH-RH/kg caused incremental changes in blood levels of LH of less than 5 ng/ml.

The final stage of sexual maturation occurred during the week before the onset of lay and was characterized by a rapid growth of large yolky ovarian follicles and a further fall in the sensitivity of the pituitary to synthetic LH-RH. However, injections of 0·5 mg progesterone/kg resulted in a prolonged release of LH.

These observations are discussed in relation to the maturation of the positive feedback mechanism by which progesterone stimulates the secretion of LH.

MRC Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EH3 9ER and * Agricultural Research Council's Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh, EH9 3JS

(Received 22 August 1977)

The decapeptide luteinizing hormone releasing hormone (LH-RH) stimulates the release of luteinizing hormone (LH) in birds as well as in mammals (Van Tienhoven & Schally, 1972) and a substance immunochemically similar to LH-RH is present in the chicken hypothalamus (Jeffcoate, Sharp, Fraser, Holland & Gunn, 1974). Avian LH-RH has still to be isolated and sequenced, however, and there is some doubt about whether the decapeptide is the naturally occurring LH-RH in the bird (Jackson, 1971).

In the hen, release of LH is induced by the positive feedback action of progesterone (Wilson & Sharp, 1975) which is presumably associated with the release of chicken LH-RH. To gain further information about the identity of chicken LH-RH and to investigate the site

SUMMARY

The ability of intramuscular injections of gonadal steroids to exert a positive feedback action on LH secretion was investigated in the ovariectomized hen. Plasma LH was measured by radioimmunoassay.

Single injections of progesterone (dose range: 0·05–10 mg/kg) or oestradiol benzoate (dose range: 0·01–1 mg/kg) did not result in an increase in plasma LH concentration. After priming with 0·1 mg oestradiol benzoate/kg on alternate days for 7 days and with 0·5 mg progesterone/kg on days 5, 6 and 7, a single injection of progesterone on day 8 (dose range: 0·1–2 mg/kg) caused the plasma LH concentration to start increasing after 15 to 30 min. Peak LH concentration was reached around 1·5–2 h after injection. The magnitude of LH response to progesterone was dose related. In contrast, a single injection of oestradiol benzoate (dose range: 0·01–1 mg/kg) failed to stimulate LH release in the oestrogen– progesterone primed ovariectomized (O-P-OVX) hen. A single injection of testosterone (dose range: 0·1–2·0 mg/kg) failed to stimulate LH release in ten out of 12 O-P-OVX hens. A small increase in LH secretion was observed in the two remaining birds.

When oestrogen or progesterone was omitted from the priming schedule, a LH positive feedback response to a single injection of progesterone was not observed. Increasing or decreasing the amount of oestrogen or progesterone in the priming schedule modified the LH response to a single injection of progesterone on the day following the last priming injection. This suggested that a critical oestrogen to progesterone ratio was required to prime the LH positive feedback mechanism.

It is suggested that, in the hen, the release of LH is facilitated by the positive feedback effect of a combination of oestrogen and progesterone in a two-phase process. The first is the priming phase, which depends on the presence in the blood of oestrogen and progesterone; the second is the inductive phase, which depends only on an incremental change in plasma progesterone concentration. Oestrogen is not involved in the inductive phase.

SUMMARY

Testosterone, androstenedione, oestrone, oestradiol-17β or deoxycorticosterone acetate (DOCA) were injected intramuscularly at several dose-levels and at various stages of the ovulatory cycle, and subsequent changes in plasma LH concentration were measured by radioimmunoassay.

In 19 out of 24 hens, injection of 0·1, 0·5 or 1·0 mg DOCA/kg resulted in a mean maximal increase in plasma LH concentration of between 0·47 and 2·10 ng/ml. The magnitude of this response was not related to either the dose or the stage of the cycle at which the DOCA was injected. In the remaining five hens DOCA failed to stimulate LH secretion. Injection of either androstenedione, oestrone or oestradiol did not result in any increase in LH level in the circulation. In contrast, injection of 0·5, 1·0 or 2·0 mg testosterone/kg between 22 and 26 h after the terminal ovulation of a sequence resulted in mean maximal incremental changes in plasma LH level of 1·98 ± 0·17, 2·17 ± 0·21 and 2·41 ± 0·31 (s.e.m.) ng/ml from pre-injection values of 1·38 ± 0·16, 1·58 ± 0·30 and 1·43 ± 0·39 ng/ml (n = 7, 6 and 5, respectively). The interval between the injection and the resulting rise in LH level was inversely proportional to the dose. The same doses of testosterone injected between 0 and 8 h after ovulation failed to stimulate LH secretion. There was also no significant increase in LH levels after injection of 0·5 and 1·0 mg testosterone/kg between 8 and 9 h after ovulation. However, injection of 2 mg testosterone/kg at this time resulted in a small but significant (P < 0·05) increase in LH levels.

Since the largest ovarian follicle is more mature at 22–26 h after ovulation than at 0–9 h after ovulation, the ability of testosterone to cause the release of LH therefore appears to depend upon the degree of maturation of the ovarian follicle next due to ovulate.