Measurement of gonadotrophin and corticotrophin releasing activity of pituitary stalk blook and brain extracts under various physiological and experimental conditions

The aim of this thesis was to correlate changes in the content of (i) gonadotrophin-releasing hormone (GnRH) in brain extracts, pituitary stalk and peripheral plasma, and (ii) hypothalamic corticotrophin-releasing activity (CR-activity) with the patterns of secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH) and adrenocorticotrophic hormone (ACTH) from the anterior pituitary gland and corticosterone from the adrenal gland, under various physiological and experimental conditions. Most measurements were made on samples obtained from rats of the Wistar strain, but the results of some studies in the human are reported in Chapter 8.
Radioimmunoassay techniques were used to measure GnRH, LH, FSH and ACTH concentrations, while a competitive protein binding method was used to determine plasma corticosterone concentrations. CR-activity was assessed using an in-vitro assay involving short-term incubation of hemipituitaries: the amount of ACTH released into the medium was measured by radioimmunoassay.
The hypothalamic content of GnRH, pituitary ACTH, and pituitary and plasma LH and FSH were measured in male, female and androgenized female rats from foetal Day 15 to postnatal Day 65. CR-activity was also measured at a few times during the postnatal period. Hypothalamic GnRH was detectable as early as foetal Day 15, increased gradually until postnatal Day 2 and then steeply between Days 5 and 16, a period during which major maturational changes occur in the hypothalamo- hypophysial axis. T|he levels of GnRH were similar in both males and normal females until Day 41, after which the increase which had been occurring from Day 16 continued in the male but not the female. In female animals treated with testosterone propionate (TP) shortly after birth, the hypothalamic content of GnRH at Day 9 was significantly less than that in normal females and males of the same age. Between Days 16 - 22 levels reached those found in the normal females and males. The lower level of GnRH in the TP treated female was associated with pituitary gonadotrophin and plasma FSH levels which were lower than in the normal female until Day.3O, suggesting that TP inhibits the synthesis and release of GnRH and gonadotrophins. In the normal female, vaginal opening was associated with a marked drop in pituitary LH and FSH and hypothalamic GnRH, but in the TP-treated female vaginal opening occurred while hypothalamic GnRH and pituitary LH levels were still rising. The peak in pituitary LH and the rise in plasma LH seen on Days 3O and 41, respectively, in the normal female were each delayed by about 1O days in the TP treated female. Similarly, the peak in pituitary FSH on Day 22 in the normal female occurred on Day 3O in the TP treated female. The profile of plasma FSH concentrations in the latter resembled more closely that of the male. In all three types of animal plasma FSH dropped significantly between Days 5O and 65. This could be attributed to inhibition of FSH release but not synthesis in the male (since pituitary FSH rose markedly during this period). However, in the androgenized female pituitary FSH also fell significantly suggesting that both release and synthesis had been inhibited.
The Day 15 foetal pituitary also contained ACTH, and again levels of this hormone rose steeply between Days 5 and 9. In contrast to the gonadotrophins, there was a marked divergence between the pituitary content and concentration of ACTH, content rising while concentration remained relatively steady after Day 9. There was no major difference in pituitary ACTH levels between the three types of animal throughout the study; however, around Days 16 and 50 CR-activity was higher in the males and androgenized females compared with that in the normal females.
Hypothalamic CR-activity and GnRH content, and pituitary and plasma LH, FSH and ACTH were measured in adult male rats maintained under a lighting regime in which the lights were on for 14 h. Two experiments were carried out. In the first some of the animals were kept in a room, distant from the animal house, in which the lighting was reversed with respect to the external environment. In animals exposed to the reversed lighting regime, hypothalamic GnRH content and pituitary gonadotrophin concentrations were significantly lower than the values in males kept in the animal house where they were in close proximity to female rats. In the second experiment, which was carried out on animals which had all been kept in the animal house, there were no significant differences between the GnRH contents measured at 3-4 hourly intervals throughout the day. Pituitary LH and FSH contents, but not concentrations, were significantly elevated at 12.OO h. There was little difference between the experiments in CR-activity, plasma ACTH concentrations and profiles of pituitary ACTH content and concentration. As expected there was a diurnal rhythm in plasma corticosterone concentrations; the peak occurred between 15.OO - 18.OO h. The profiles of^plasma and pituitary ACTH were similar to that of plasma corticosterone. CR-activity dropped significantly between 12.OO and 16.OO h, but remained stable at the other times.
Hypothalamic GnRH and pituitary and plasma LH and FSH levels were also measured in female rats which had 4-day oestrous cycles. There were no significant differences between hypothalamic GnRH content. During pro-oestrus the mean GnRH content rose to reach a high level at 18.OO h at which time plasma LH concentration had risen sharply to a level consistent with the peak of the pre-ovulatory surge. Plasma FSH concentration also rose significantly between 15.OO and 18.00 h of pro-oestrus. At metoestrus and dioestrus plasma FSH levels were lower in the morning than in the evening.
Extracts of cerebral cortex from animals of either sex, both prepubertal and adult, contained no detectable GnRH. However, cortical extracts did exhibit CR-activity This may have been due to the release of substances which cross reacted with the anti-ACTH serum but which were not steroidogenic (assessed in-vivo).
These results suggest that (1) there is no diurnal rhythm in hypothalamic GnRH, (2) there may be a diurnal rhythm in pituitary gonadotrophin content in the male and in plasma FSH concentration on the days of metoestrus and dioestrus in the female, (3) if a surge of GnRH does occur on the afternoon of pro-oestrus, the rate of GnRH synthesis exceeds its release, and (4) the mechanism which regulates gonadotrophin secretion in the male may be affected by factors in the environment other than the length of daylight (e.g. presence of female animals). The data also support the view that the diurnal rhythm of corticosterone secretion is under hypothalamo-hypophys ia1 control.
The effects of sex steroid hormones on the responsiveness of the neural mechanism responsible for the secretion of GnRH were examined in the female rat. Responsiveness was determined on pro-oestrus by measuring the increments in content (concentration x volume/time) of immunoreactive GnRH of pituitary stalk blood produced by electrical stimulation of the medial preoptic area or median eminence. Ovariectomy on the morning of dioestrus reduced the GnRH response to preoptic stimulation while oestradiol benzoate (OB) or TP administered immediately after ovariectomy augmented the response. The facilitatory effect of TP was possibly due to its conversion'to an aromatized derivative since 5a-dihydrotestosterone monobenzoate was ineffective. Progesterone did not facilitate preoptic responsiveness and, when administered to animals ovariectomized at 12.OO h of pro-oestrus, reduced the GnRH response at 18.OO h of the same day. Stimulation of the median eminence produced a significantly greater increment in GnRH than stimulation of the preoptic area. The facilitatory action of OB on the GnRH response was less marked for median eminence compared with preoptic stimulation. The administration of ICI 46474 at 17.00 h of dioestrus did not reduce preoptic responsiveness on the morning of the next day, suggesting that this compound does not act as an 'antioestrogen 1 at the preoptic area.
In a second series of experiments, blood was collected from the cut pituitary stalk of pro-oestrous rats before and during electrical stimulation of either the anterior hypothalamic area, suprachiasmatic nuclei, hippocampus or amygdala. The mean increments in GnRH content of pituitary stalk plasma produced by median eminence (data from previous series, see above) stimulation was nearly twice that obtained by preoptic stimulation (data from Fink and Jamieson, 1976). Significant increments in GnRH content were also obtained by stimulating the suprachiasmatic nuclei or anterior hypothalamic area. However, the increments produced by stimulation of the anterior hypothalamic area were considerably less than that obtained by median eminence, preoptic or suprachiasmatic stimulation. Stimulation of the hippocampus or amygdala had no appreciable effect on GnRH content. The increments in GnRH content in animals subjected to simultaneous stimulation of the hippocampus and preoptic area were similar to that obtained by preoptic stimulation alone. However, the increment was reduced when simultaneous stimulation was preceded by ventral hippocampal stimulation.
Studies were also carried out on animals subjected to section of the preoptic roof immediately before implantation of electrodes in the preoptic area. Regular oestrous cycles resumed in all these animals after a mean (± S.E.M.) of 18.8 ± 2.O days (compared with 5.5 ± O.3 days in animals subjected to electrode implantation only). The increment in GnRH content produced by preoptic stimulation was similar in the roof-sectioned compared with intact animals. However, the facilitatory effect of oestrogen on the GnRH response to preoptic stimulation was significantly reduced in the roof-sectioned compared with intact animals. (The latter study was carried out on animals ovariectomized on the morning of dioestrus and immediately afterwards given a s.c. injection of either oil or OB). These results suggest that the hippocampus and amygdala do not exert an acute, direct effect on the GnRH release mechanism, but areas outside the diencephalon may play a role in mediating the facilitatory effect of oestrogen. In agreement with the findings of other workers, integrity of the dorsal afferents of the diencephalon did not appear to be essential for maintaining regular oestrous cycles.
Whether the priming effect of GnRH can be elicited by electrical stimulation of the medial preoptic area, or by i.v. infusion or multiple i.v. injections of the synthetic decapeptide was also investigated. All experiments were carried out on animals anaesthetized with sodium pentobarbitone at 13.3O h. In pro-oestrous rats, the LH response to the second of two electrical stimuli, 15 min in duration and separated by 6O min, was significantly greater than the response to the first stimulus. When synthetic GnRH was infused at a constant rate for 90 min, plasma LH increased gradually for the first 45 to 60 min after which it increased markedly. This enhanced secretion of LH did not occur in rats which were infused with the same total dose of GnRH, either 15 or 75 ng/lOO g body wt, over periods of 45 min or less. When a dose of 15 ng GnRH/100 g body wt was administered in 6 divided doses by i.v. injections, each separated by 15 min, there was a marked increase in plasma LH after 75 min. The profile of the mean plasma LH concentration in rats subjected to preoptic stimulation for 9O min was similar to that in rats infused for 90 min with GnRH, but the variation in response was much greater in the stimulated rats.
These results indicate that the priming effect can be elicited by endogenous as well as synthetic GnRH, and that whether GnRH reaches the pituitary at a constant rate or in a pulsatile manner, the factor is capable of increasing significantly the responsiveness of the gonadotrophs. The relevance of these findings with respect to the development of the spontaneous preovulatory LH surge is discussed. A priming effect could not be elicited by constant GnRH infusion in dioestrus rats; this supports the view that steroid hormones, especially oestradiol-17β, determine the magnitude of the effect. The LH response in male rats^ subjected to i.v. infusion of GnRH was much lower than in females. Pre-treatment with OB did not increase the response significantly, suggesting that this sex difference cannot be ascribed simply to low levels of plasma oestrogen in the male.
In a second series of experiments on the priming effect, designed to simulate the spontaneous pre-ovulatory surge of gonadotrophins, adult rats which had been anaesthetized with urethane, were subjected to either electrical stimulation of the medial preoptic area or intravenous infusion of synthetic GnRH for 6-7 h between 14.OO - 21.OO h of pro-oestrus. Plasma LH concentrations increased exponentially 3O-6O min after the start of stimulation or infusion, peaks being reached after 2-4 h (depending upon the strength of the stimulus or the dose of GnRH). The concentrations then fell, but in animals infused with GnRH, levels at the end were still significantly higher than those before the beginning of the infusion. Comparison of the profiles of plasma LH concentration with the levels of immunoreactive GnRH in pituitary stalk (preoptic stimulation study) or peripheral plasma (infusion study) suggests that the rising phase of the spontaneous LH surge could be due to increased responsiveness of the anterior pituitary to elevated, but relatively constant levels of GnRH in hypophysial portal blood. However, the declining phase of the spontaneous surge is possibly the result of a fall in both GnRH output and pituitary responsiveness.
For maintaining a constant level of immunoreactive GnRH in peripheral blood during long-term infusion, PBS-0.1% gel proved to be a better diluent than O.9% saline or male rat serum. At about 2OˆC the latter appears to degrade synthetic GnRH and/or affect its immunoreactivity.
The studies in the human may be divided into three experiments. In the first, the concentration of immunoreactive GnRH was determined in median eminence, hypothalamic, and cerebral cortical tissue obtained at post-mortem from patients with Huntington's chorea and from non-choreic (control) subjects. Preliminary studies showed that the slope of hypothalamic extract from control and choreic subjects, both male and female, was similar to that of the synthetic GnRH which was used as standard in the radioimmunoassay. This suggests that human hypothalamic tissue contains a substance immunologically similar to that in other species such as the rat, sheep, pig and rhesus monkey. Though the concentration of GnRH was highest in the median eminence, relatively large amounts of GnRH were also found in the preoptic nuclei. The mean concentration of GnRH in median eminence of female choreics was greater than in the female controls and the male choreics and controls. Median eminence concentration was lowest in control females (age range, 39 - 88). There was little difference between the mean concentrations of GnRH in the median eminence of the two male groups, but there was an age-related drop in the median eminence GnRH concentration of the male controls.
Measurement of peripheral plasma GnRH, LH and FSH during the menstrual cycle of normal women and at random times in males and females exhibiting one of several hypogonadal states, formed the basis of the second experiment. Because of the low, and in most cases undetectable quantities of GnRH measured in the peripheral circulation, it was not possible to correlate changes in release of this hormone with patterns of LH and FSH secretion, even though all of the normal women had mid-cycle peaks in gonadotrophin concentrations and the hypogonadal patients exhibited the expected plasma LH and FSH levels.
Measurement of ACTH and CR-activity in various extracts of two medullary thyroid carcinomas constituted the third experiment. While extraction with a butanol-acetic acid-water mixture or 2 N acetic acid appeared to reduce immunological ACTH concentration and CR-activity, saline extracts and lyophilized portions of the tumours contained high concentrations of immunoreactive ACTH and significant CR-activity was demonstrated in-vitro. However, it was not possible to measure significant ACTH and/or CR-activity in-vivo, though it is possible that insufficient tissue was injected to elicit the expected response.
Overall, these studies provide further evidence for the contention that pGlu - His - Trp - Ser - Tyr - Gly - Leu - Arg - Pro - Gly - NH2 (GnRH) may be the (or at least 'a') substance which, under physiological conditions, mediates neural control of gonadotrophin release. Studies on CR-activity revealed the problem of using an antiserum to ACTH which also cross reacts with ACTH fragments. As for the gonadotrophin aspect, investigation of the hypothalamic-pituitary-adrena1 axis would be greatly facilitated by the characterization and synthesis of a corticotrophin-releasing factor (if one, only, exists).