Central adrenergic activity in the mediobasal hypothalamus is temporally related to surge outputs of gonadotrophins in oestrogen-stimulated infant female rats.

In the mediobasal hypothalamus (MBH) of pro-oestrous rats or acutely ovariectomized oestrogen-treated 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 less than 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 less than 0.05) than at either 15.00 or 16.00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that central epinephrine neurons participate in the control and regulation of neuroendocrine events during the estrous cycle.

Although evidence has shown that central epinephrine (E) neurons play an essential role in the control of preovulatory gonadotropoin surge in rats, their function and site(s) of action are unknown. These experiments were performed in an attempt to identify any changes in E concentration or activity that might take place in areas of the brain known to receive adrenergic axon terminals and to be associated with increased output of gonadotropins (LH, FSH, and PRL) during the estrous cycle. E concentrations were measured by radioenzymatic assay, and E activity was assessed by the linear rate of decline of E (RDE) which occurs 2 h after administration of the centrally active E synthesis inhibitor, SKF 64139. During the proestrous critical period (1500-1700 h), significant increases in both concentration and RDE occurred in the medial preoptic area (mPOA) accompanied by a smaller but significant increase in the RDE in the mediobasal hypothalamus (MBH); 4 h later (2100-2300 h), significant increases in both concentration and RDE were seen in the MBH. At estrus, although E concentrations were generally higher in both the mPOA and MBH than on other days of the cycle, the concentration and RDE in the MBH increased significantly between 1500-1700 h, while RDE in the mPOA increased again between 1700-1900 h. There were no significant changes in either E concentration or RDE in the mPOA or MBH at metestrus or diestrus or in the perifornical area at any of the times studied. Thus, these findings may be associated with the output of gonadotropins over the periovulatory period.

Hypothalamic adrenergic activity precedes the preovulatory luteinizing hormone surge in the rat.

A preovulatory surge of luteinizing hormone (LH) is stimulated by an increase in plasma oestrogen (E2) and a proestrous neural signal which occurs during the 'critical period' (CP). Adrenergic activity during the CP in brain areas concerned with LH release and receiving adrenergic nerve terminals, increased in the arcuate nucleus-median eminence area (AN-ME) during the CP (14.30-16.30 h), but not in the adjacent perifornical area. Pentobarbital injected prior to the CP inhibited the effect as did ovariectomy, while E2 replacement restored it. Thus increased adrenergic activity in the AN-ME may represent the neural signal that triggers the preovulatory LH surge [5].

Possible resolution of a paradox concerning the use of p-chlorophenylalanine and 5-hydroxytryptophan: evidence for a mode of action involving adrenaline in manipulating the surge of luteinizing hormone in rats.

Various functions involving the central nervous system can be manipulated by the sequential administration of p-chlorophenylalanine and 5-hydroxytryptophan, compounds which respectively inhibit and restore the synthesis of 5-hydroxytryptamine in the brain. An involvement of 5-hydroxytryptamine in the control of a particular function has been considered established when the effect of p-chlorophenylalanine on that function can be overcome by treatment with 5-hydroxytryptophan. This assumption is not, however, invariably substantiated when the functional consequences of other methods of depleting 5-hydroxytryptamine are considered; studies on the control of the daily surge of luteinizing hormone in oestrogen-treated ovariectomized rats present such a paradox. The surge can be prevented by p-chlorophenylalanine and restored by 5-hydroxytryptophan. Nevertheless, neurotoxin-induced lesions of the 5-hydroxytryptamine projections from the raphe nuclei are compatible with a normal occurrence of the surge. We have therefore examined the effects of p-chlorophenylalanine and 5-hydroxytryptophan on hypothalamic monoamines in oestrogen-treated ovariectomized rats and find that the drugs respectively suppress and elevate the concentration of adrenaline in addition to that of 5-hydroxytryptamine. Phenylethanolamine N-methyltransferase, the enzyme responsible for converting noradrenaline to adrenaline, is shown to be inhibited in vivo by p-chlorophenylalanine and in vitro by its metabolite, p-chlorophenylethylamine. The reciprocal effects of p-chlorophenylalanine and 5-hydroxytryptophan on the concentration of adrenaline are of particular interest since drugs which inhibit adrenaline synthesis can block the luteinizing hormone surge. It is proposed that when the 5-hydroxytryptophan-reversible effects of treatment with p-chlorophenylalanine are not reproduced by other procedures which deplete 5-hydroxytryptamine, the significant action of these compounds may involve adrenaline.

Effects of manipulating serotonin on the incidence of ovulation in the rat.

Previous studies have shown that while depletion of brain serotonin by the administration of p-chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, blocks the daily surge of LH in oestrogen-treated ovariectomized rats, restoration of serotonin synthesis by treatment with its immediate precursor, 5-hydroxytryptophan (5-HTP), at a critical time of day, reinstates the surge. The present study indicates that the experimental procedure involving serotonin depletion and its subsequent replenishment may also be used to control the preovulatory LH surge and ovulation in intact cyclic rats provided that (1) the PCPA is administered subcutaneously rather than intraperitoneally nad (2) the 5-HTP is given in conjunction with carbidopa, a peripheral decarboxylase inhibitor: the latter observation providing further evidence for a central role for serotonin in the control of ovulation. These precautions were unnecessary when oestrogen was administered at the same time as the PCPA. It appears that PCPA administered intraperitoneally results in a suppression of the preovulatory rise in oestrogen secretion (and may have additional deleterious effects at the level of the ovaries) and that 5-HTP, in the absence of supplementary oestrogen, may block ovulation by peripheral action after conversion to serotonin. This study indicates the need for caution when using pharmacological 'cocktails' to investigate neuroendocrine events underlying ovulation when the experiments are carried out in the presence of the ovaries.

Lesions of the suprachiasmatic nuclei and the serotonin-dependent phasic release of luteinizing hormone in the rat: effects on drinking rhythmicity and on the consequences of preoptic area stimulation.

Ovariectomized rats in which less than 7% of the suprachiasmatic nuclei had been spared by bilateral radiofrequency lesions were distinguishable from those with greater than 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.

Serotonin involvement in the control of phasic luteinizing hormone release in the rat: evidence for a critical period.

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-chloramphetamine 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.

The effects of serum oestrogen-binding components on the unbound oestradiol-17 beta fraction in the serum of developing female rats and on inhibition of [3H]oestradiol uptake by uterine tissue in vitro.

Total oestradiol concentrations in the serum of young female rats were high and decreased after about 21 days of age. High affinity serum oestradiol-binding components (EBP), however, fell steadily from 5 to 23 days of age while the unbound oestradiol-17 beta fraction, which was low early in development, increased between 21 and 28 days of age. Injection (i.v.) of immature (EBP-rich) oestradiol-free serum into 21-day-old female rats led to a decrease in the unbound oestradiol fraction and an increase in serum FSH concentrations. Incubation of uterine tissue with [3H]oestradiol, with or without the addition of diethylstilboestrol (DES), showed the rate and degree of total and DES-suppressible uptake of [3H]oestradiol to be greatest from buffer, less from adult (EBP-poor) serum and negligible from immature (EBP-rich) serum; moreover, there was a positive correlation between the degree of uptake of [3H]oestradiol and the unbound fraction of [3H]oestradiol in the incubate. It is concluded that, at least in young rats, oestradiol activity depends more on the availability of free oestradiol than on its total plasma concentrations.

Ovarian steroid effects on gonadotrophin output, and on the incorporation of 35S from methionine into protein of discrete brain areas and the anterior pituitary.

The effects of various ovarian hormones administered on the morning of pro-oestrus on gonadotrophin levels and the incorporation of 35S from methionine into protein of discrete areas of the brain and the anterior pituitary were investigated at 15.00 h of the same day in female rats. The hormones which were investigated in this study could be divided in general into two groups according to their actions. The first group, consisting of oestradiol-17 beta and progesterone, tended to advance the pre-ovulatory surge of luteinizing hormone (LH) by 3-6 h from 18.00-21.00 h, together with the peaks of [35S]incorporation in the median eminence area and the anterior pituitary which normally accompany the LH surge. The second group, consisting of the LH-stimulated reduced progesterone metabolites, 5 alpha-pregnane-3,20-dione (pregnanedione) and 20 alpha-hydroxy-pregn-4-en-3-one (dihydroprogesterone), tended to inhibit serum gonadotrophin levels as well as inhibiting the pro-oestrous increase of [35S]incorporation in the median eminence area and in the amygdala, but not in the preoptic area and the anterior pituitary. On the afternoon of pro-oestrus in intact animals, luteinizing hormone-releasing hormone or LH administration had the same effect on [35Si1incorporation in the brain as did the progesterone metabolites, though this effect was not observed if the animals had been ovariectomized a few hours beforehand. It is suggested that certain ovarian hormones are involved in the neural events which induce the pre-ovulatory LH surge, while others are associated with neural events which terminate the stimulus for the LH surge.

Serum gonadotrophin levels during development in male, female and androgenized female rats and the effect of general disturbance on high luteinizing hormone levels.

Serum LH, FSH and prolactin levels were measured in blood samples which were obtained by decapitation from groups of female, neonatally androgenized female and male Wistar rats at 2-day intervals from birth to maturity. An increase in serum FSH levels was observed between 4 and 24 days of age in both the female and androgenized female groups, while a much later increase, between 28 and 44 days of age, occurred in the males. Serum prolactin contrast, serum LH levels were in general low in all three groups of animals, although very high levels (greater than 7 ng/ml) were recorded in 22 out of 168 females and 8 out of 192 males between 4 and 28 days of age, as well as in adult males; occasional high LH levels were also seen in the androgenized females. The nature of the high serum LH levels was investigated in anaesthetized and unanaesthetized immature females by serial blood sampling using a number of techniques. Unexpectedly, only three out of 58 animals had high LH levels: two of these showed an episodic form of LH release during which levels increased to peak values and then declined within a period of about 30 min. On investigation it was found that general disturbance within the 45 min before decapitation could inhibit high LH levels in females aged between 23 and 30 days.

Luteinizing hormone-releasing hormone for induction of follicular maturation and ovulation in women with infertility and amenorrhea.

Five patients with primary infertility and secondary amenorrhea who did not respond to clomiphene with a gonadotropin or estrogen surge were treated with 500 mug of luteinizing hormone, follicle-stimulating hormone-releasing hormone (LH-RH), self-administered subcutaneously every 8 hours for 14 days. Of four patients who responded to this treatment, three showed follicular maturation, ovulation, and menses, although the luteal phase was deficient; in the fourth patient, follicular maturation and menses occurred without evidence of ovulation. For their second course of treatment these four patients were given LH-RH with the addition of human chorionic gonadotropin when the urinary estrogen levels indicated follicular maturation. All four patients responded with ovulation, an adequate luteal phase, and menses, without clinical indication of ovarian hyperstimulation. These results suggest that LH-RH may be a better alternative to human menopausal gonadotropin in the treatment of anovulatory infertility.

Effects of sodium pentobarbitone on serum gonadotrophin levels and on the incorporation of 35S from methionine into protein in the brain and anterior pituitary during the oestrous cycle of the rat.

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 pro-oestrus. 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 (less than 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 gonadotropin 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.