SUMMARY

1. Suprarenal glands from forty-four women of child-bearing age who died by misadventure were divided into 'summer' and 'winter' groups, the summer group consisting of twenty-one from women who died in April\p=m-\September and the winter group of twenty-three from women who died in October\p=m-\March.

2. The widths and the nuclear densities of four cortical zones, the glomerulosa, the outer fasciculata, the inner fasciculata and the reticularis, were measured in each gland, and the data obtained for both summer and winter groups compared.

3. In the zona glomerulosa nuclear population was increased in summer, due to a significant increase in nuclear density. There was no significant change in the width of this zone.

4. In the outer zona fasciculata and in the zona reticularis there were no significant seasonal differences in width or in nuclear density.

5. In the inner zona fasciculata the nuclear population was considerably increased in summer, due to significant increases in both width and nuclear density.

6. The lipoid content appeared to be similar in summer and winter in each of the four zones.

7. It was suggested that the increase in nuclear population in the zona glomerulosa in summer might indicate an increase in output of sodium-retaining hormones which could account for the increase in water retention at that time of year, and that the increase in nuclear population in the inner zona fasciculata in summer was a manifestation of separate functioning of this region.

SUMMARY

A marked surge of luteinizing hormone (LH) occurs daily at 18.00 h in oestrogen-treated ovariectomized rats maintained under regular lighting from 06.00 to 20.00 h. The administration of p-chlorophenylalanine (PCPA), p-chloroamphetamine or 5,6-dihydroxytryptamine in treatments designed to cause a severe depletion of brain serotonin abolished this daily surge. Synthesis of serotonin may be temporarily restored in PCPA-treated animals by the administration of the serotonin precursor, 5-hydroxytryptophan. The effectiveness with which restoration of synthesis resulted in restoration of the LH surge varied according to the time at which the precursor was administered, the optimal time being 10.00 h. The results suggest that there is an essential, permissive function performed by serotonin in the production of the LH surge and that this function occurs during a critical period.

Ovariectomized rats in which <7% of the suprachiasmatic nuclei had been spared by bilateral radiofrequency lesions were distinguishable from those with >40% of the nuclei by their consistent failure to show the oestrogen-induced daily surge of LH, either with or without pharmacological manipulations of serotonin (5-HT), and also by their loss of the normal rhythmicity of drinking. Minor damage to structures adjacent to the suprachiasmatic nuclei was similar in both groups. The identical facility with which electrical stimulation of the preoptic area induced LH release in the two groups of animals suggested that they were not characterized by different degrees of damage to the preopticotuberal pathway. These results are considered in relation to evidence indicating that the suprachiasmatic nuclei represent the densest concentration of 5-HT terminals in the forebrain and also the site of a mechanism involved in the generation of circadian rhythms.

The concentrations of LH and prolactin in the serum in response to a single s.c. injection of oestradiol benzoate (OB, 1 μg) were studied in 21-day-old Wistar rats of different sexual status. In intact female rats, the total concentration of oestrogen in the serum reached a maximum level within 30 min of the injection, whereas the level of LH fell and remained low until a surge occurred 54 h later. However, the amount of prolactin in the serum increased within 1 h of injection of OB and thereafter showed diurnal fluctuations with evening peaks. Male rats castrated within 24 h of birth showed increases in the serum level of both LH and prolactin in response to OB, which were similar in timing to those seen in intact female rats. However, intact male rats failed to show an increase in the level of either LH or prolactin after treatment with OB, while neonatally androgenized female rats, although failing to show an increase in the level of LH, exhibited a prolactin response which was similar to that observed in intact female rats.

A further study was undertaken in rats which had received an initial injection of OB and a subsequent injection of progesterone 5 h before the expected female-type LH surge. In intact female rats, progesterone augmented the oestrogen-stimulated surge of LH, but this effect was not observed in either intact male or neonatally androgenized female rats. In all three groups, however, the level of prolactin in the serum was increased when compared with control animals which had received OB only.

The effect of progesterone, injected at various times after priming with OB, on the output of LH and prolactin was also studied in female rats killed 5 h after the second injection. Up to 21 h after injection of OB, progesterone had no effect on the level of LH or prolactin. If, however, progesterone was then injected during the middle of the light period, but not in the dark period, the concentrations of LH and prolactin increased. Thus there are critical times during a 24 h period when the gonadotrophin output of the oestrogen-primed female rat is responsive to the stimulatory effects of progesterone.

Female Wistar rats aged 21, 18 and 16 days were injected s.c. with oestradiol benzoate (OB) at 12.00 h on Day 1 and the concentrations of LH and prolactin in the serum were measured 54 h later at 18.00 h on Day 3; the effect of a second injection of OB, given at different intervals after the first, on the concentration of gonadotrophins in the serum was also investigated. In 21-day-old rats, surges of both LH and prolactin occurred at 18.00 h on Day 3; if a second dose of OB was given within 24 h of the first, then the expected surge of LH was diminished, but if it was given on the morning of Day 3 the surge was augmented. The concentration of prolactin was, however, unaffected by the second injection. In 18-day-old rats there were no surges of gonadotrophin in response to a single OB stimulus on Day 1; however, a second dose of OB given on the morning of either Day 2 or 3 caused surges of both LH and prolactin at 18.00 h on Day 3. No such response was obtainable in 16-day-old rats.

An injection of progesterone at 12.00 h on Day 3 after a priming dose of OB at 12.00 h on Day 1 enabled surges of LH to be elicited in 18-, 16- and 14-day-old female rats but not in younger animals; surges of prolactin could be elicited to a diminishing degree in 18-, 16-, 14- and 12-day-old rats but only in younger animals if larger doses of progesterone were administered.

In further groups of young female rats an injection of progesterone was given at 12.00 h on Day 3 after the animals had been primed at 12.00 h on Day 1 with diethylstilboestrol or another synthetic oestrogen (RU2858). The results obtained paralleled data from the previous experiment in which progesterone was given after priming with the natural steroid OB.

Although the capacity for cyclicity or acyclicity in rats is normally determined early in the neonatal period, these results suggest that mechanisms concerned with the surge response to a single OB stimulus are intact and functional by 21 days of age but not in younger rats; however, it is possible to facilitate both the LH and prolactin response as early as 14 days of age.

SUMMARY

The rate of [³⁵S]methionine incorporation into protein in discrete cerebral areas was measured before and after the administration of oestradiol benzoate (OB) to chronically ovariectomized rats. The circadian rhythm of incorporation which is normally seen in the intact cyclic female rat was deleted by ovariectomy. A daily rhythm of incorporation reappeared, however, in all the brain areas studied 30 h after a single injection of OB (20 μg), and was still present 12 days later.

The release of luteinizing hormone (LH) after administration of 20 μg OB was measured in chronically ovariectomized animals and was found to be biphasic. High levels of LH after ovariectomy were initially reduced by negative feedback, but this phase was followed 52 h later by a facilitation of LH release between 15.00 and 18.00 h. The facilitation of LH release at this time of day was still detectable 12 days after the initial injection.

The evidence for a functional link between the rhythm of neural activity which is reflected by [³⁵S]methionine incorporation, and the ability to 'time' the facilitation of LH release is discussed.

SUMMARY

Serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured by radioimmunoassay in groups of Wistar rats at 6-hourly intervals during the course of a 4-day oestrous cycle. In addition to a surge at about 18.00 h on the day of pro-oestrus, a circadian rhythm in LH levels was observed which was accentuated during metoestrus. FSH levels showed a protracted periovulatory rise which reached peak levels at about 03.00 h on the day of oestrus. A daily rhythm was not observed.

The incorporation of [³⁵S]methionine (injected subcutaneously 1 h before death) into the trichloroacetic acid-precipitable proteins of the anterior pituitary and of discrete brain areas implicated in the control of gonadotrophin release, was also measured. An increase in protein synthetic activity was observed in the anterior pituitary and the area of the median eminence on the evening of pro-oestrus with peak levels coinciding with the LH surge at 18.00 h on the day of pro-oestrus. An increase in protein synthetic activity relative to that in a 'control' area (the putamen) was observed in the preoptic area and in the amygdala 15–18 h before the LH surge. It is suggested that these changes in protein synthetic activity are associated with the train of neural and humoral events leading to ovulation.

After ovariectomy, protein synthetic activity in all areas investigated was at the low levels observed during the oestrous cycle. Daily injections of 20 μg oestradiol benzoate into intact females led to levels of protein synthesis as great as, or greater than, the maximal levels observed during the oestrous cycle.

Measurements of variations in total protein concentration/unit wet weight of pituitary or brain tissue showed that they were apparently unrelated to variations in protein synthetic activity. Protein concentration appeared to be high in all the brain samples taken at oestrus and low in those taken at metoestrus. Furthermore, superimposed upon a marked daily rhythm, there was a peak of protein concentration on the evening of dioestrus both in the preoptic area and in the area of the median eminence.

SUMMARY

Ovulation was delayed for 24 h after the administration of sodium pentobarbitone (Nembutal, 35 mg/kg body weight) at 14.00 h, before the critical period on the afternoon of prooestrus. The expected preovulatory surge of serum LH at 18.00 h of pro-oestrus was also delayed until 21.00 h on the following day; however, increased levels (> 12 ng/ml) were observed in 14 out of 23 animals (killed by decapitation) at 21.00 h on the day of Nembutal administration. The serum FSH rise observed on the morning of expected oestrus was extended after Nembutal treatment, and a further rise was noted 24 h later.

Peak levels of incorporation of ³⁵S from methionine into protein of the median eminence area (ME) and of the anterior pituitary (AP) which normally occur about the time of the preovulatory LH surge, were also delayed until 21.00 h on the day following Nembutal administration.

Neither ovulation nor the preovulatory gonadotrophin rises with their accompanying changes in incorporation in the ME and the AP, were altered by Nembutal administered after the pro-oestrous critical period.

Thus Nembutal, while blocking ovulation, inhibits the circadian rhythm of incorporation of ³⁵S from methionine in the brain as well as the peaks of incorporation in the median eminence and the anterior pituitary which accompany the normal preovulatory surges of gonadotrophin.

SUMMARY

The incorporation of [³⁵S]methionine into protein of the anterior pituitary and discrete brain areas was measured following the administration of antibodies to oestrogen, ovariectomy, or adrenalectomy on the afternoon of dioestrus. The antibody to oestrogen deleted the circadian rhythms of methionine incorporation normally observed in the various brain areas together with the peaks of incorporation normally observed in the median eminence area and anterior pituitary on the evening of pro-oestrus. The peaks of incorporative activity normally observed in the preoptic area and amygdala (relative to the putamen) at 03.00 h on the day of pro-oestrus were also deleted. Administration of the antiserum on the morning of pro-oestrus failed to alter the pattern of methionine incorporation normally observed on the evening of pro-oestrus.

Ovariectomy performed at 16.00 h of dioestrus blocked the preovulatory rise of luteinizing hormone (LH) (as did the antibody to oestrogen) and inhibited the peak of methionine incorporation normally observed in the anterior pituitary on the evening of pro-oestrus. However, for the peak in the median eminence to be inhibited, ovariectomy had to be performed on the morning of the preceding oestrus. Adrenalectomy alone, or adrenalectomy with ovariectomy, performed on the afternoon of dioestrus did not affect the levels of methionine incorporation in the brain or anterior pituitary at 18.00 h on the day of prooestrus.

Animals which had been ovariectomized and injected with 2·5 μg oestradiol benzoate on the morning of oestrus showed significantly increased levels of methionine incorporation in all the brain areas and the anterior pituitary at 18.00 h of expected pro-oestrus. The administration of antibody to oestrogen to a similar group of animals on the afternoon of expected dioestrus inhibited the rise at 18.00 h of expected pro-oestrus. The apparent discrepancy between the results obtained with ovariectomy and the antiserum appeared to be due to the ability of the antiserum to neutralize the activity of oestrogens retained by the tissues.

The present results suggest that the changes in incorporation of methionine into protein in the brain and anterior pituitary are brought about by the action of endogenous oestrogen: there appears to be a steady summative effect on the median eminence throughout the oestrous cycle together with a short-lived effect occurring during pro-oestrus and affecting the anterior pituitary.

ABSTRACT

In the mediobasal hypothalamus (MBH) of prooestrous rats or acutely ovariectomized oestrogentreated adults a marked but short-lived increase in adrenergic activity occurs at 16.00 h, 2 h before the oestrogen-dependent surge of gonadotrophins at 18.00 h. In this study oestrogen-stimulated (noon on day 1) 22-day-old female rats were used which are known to produce surge levels of prolactin at 18.00 h on day 2 and surges of both prolactin and LH at 18.00 h on day 3; although similar treatment of 18-day-old animals or oil-treated 22-day-old rats failed to produce these effects. Radioenzymatic assays of adrenaline concentrations and of the activity of its synthesizing enzyme (phenylethanolamine-N-methyl transferase; PNMT, EC 2.1.1.28) in the MBH of oestrogen-treated 22-day-old rats showed significant (P< 0·05–0·01) increases in both parameters at 16.00 h (i.e. 2 h before surge levels of gonadotrophins) on days 2 and 3 when compared with other times of day. Such effects were not seen in oil-treated 22-day-old animals or in oestrogen-treated 16-day-old rats. Noradrenaline and dopamine concentrations in the MBH of oestrogen-treated 22-day-old rats remained at baseline levels on days 2 and 3 with the exception of noradrenaline at 17.00 h on day 3 when levels appeared higher (P<0·05) than at either 15.00 or 16.00 h. Subsequent measurements of PNMT activity in oestrogen-treated 22-day-old rats at 4-hourly intervals throughout days 2 and 3 showed the presence of a clear circadian rhythm with peak levels occurring at 16.00 h. In conclusion, a temporal relationship (not necessarily specific) exists between increased adrenergic activity in the MBH of oestrogen-treated 22-day-old rats and a surge of gonadotrophins (LH and/or prolactin) 2 h later. This relationship apparently depends on an oestrogen-stimulated circadian rhythm of PNMT activity.

J. Endocr. (1986) 109, 45–51