Some aspects of the hypothalamic control of pituitary gonadotrophin secretion

The development of that branch of physiology known as neuroendocrinology has followed the increasing awareness that envirornmental influences, sensed through the exteroceptors of the central nervous system, play an important part in the control of reproductive and other endocrinologically mediated activity in animals. The implication of pituitary gland activity in these processes in the rat by Smith (1927, 1932), who developed a satisfactory technique for hypophysectomy, led to a search for the connections between the central nervous system and the pituitary gland. It was soon established that the posterior lobe of the pituitary is controlled by neuronal pathways passing from the hypothalamus to that lobe, and investigation of control of the anterior lobe of the gland centred at first around the establishment of a similar neural pathway.
The discovery that the anterior pituitary is almost devoid of nerve fibres, and the failure of investigators to establish any form of autonomic nervous control drew attention to the blood vessels in the pituitary stalk first adequately described by Popa and Fielding (1930, 1933). These vessels offered an alternative control pathway to the anterior lobe and a number of investigators in the 1930's (Hinsey & Markee, 1933; Friedgood, 1936; Hinsey, 1937; Harris, 1937; Haterius, 1937) put forward a tentative hypothesis of humoral control of the anterior pituitary. By [illegible], Harris was able to state the neurohumoral hypothesis confidently in the light of the accumulation of a good deal of supporting evidence.
One of the most profitable techniques used by investigators in experiments on the hypothalamo-hypophysial axis has been electrical stimulation in the central nervous system with concomitant observation of changes in activity of extra-neural structures. Early experiments were crude, Marshall and Verney (1936) applying diffuse electrical stimuli to the heads or lumbar spinal cords of rabbits, and Harris (1936) carrying out similar experiments on rats. In both cases, these experiments indicated that some neural structures in the head had been excited, with consequent ovulation in the rabbits and pseudopregnancy in the rats.
The crudity of the electrical stimulation techniques used at that time meant that close and detailed study of small areas of the central nervous system was impracticable. With the development of electronic stimulators capable of producing accurate, stable trains of stimuli, it became possible to examine hypothalamic areas which might be involved in control of anterior pituitary secretion. A great variety of electrode materials, stimulus waveforms and parameters of stimulation have been used. Frequently, scant regard has been paid to the differentiation of electrical stimulation from electrical lesion production.
Many studies have been performed on the activation of ovulation or of luteinising hormone (LH) secretion by the anterior pituitary in female rats after electrical stimulation in the diencephalon, and it became evident that medial preoptic area was involved (Critchlow, 1958) There are few reports however, of activation of LH release from the anterior pituitary consequent upon hypothalamic stimulation in male animals. No event comparable to ovulation was observable in male animals, so Moll and Zeilmaker (1966) and Quinn (1966) resorted to transplanting ovaries into castrated males. Direct current (d.c.) stimulation in the medial preoptic area resulted, in both these studies, in changes in the ovarian transplants suggestive of ovulation. With the advent of assays, and in particular radioimmunoassay for LH (Niswender, Midgley, Monroe & Reichert, 1968) it has become possible to study the effects of medial preoptic area stimulation in normal male rats, using a variety of electrode materials and stimulation parameters.
The present studies provide direct evidence for the release of LH from the anterior pituitary gland consequent upon electrical stimulation of the medial preoptic area of the normal male rat, and confirms the preliminary studies of Burger, Fink & Lee (1972) who achieved rises greater than 400% in peripheral plasma LH concentration in three male rats after medial preoptic area stimulation. The optimal parameter for alternating current (a.c.) square wave stimulation are frequency 60 Hz, pulse amplitude 0.5 mA and pulse duration 0.5–1.0 msec. Release of LH occurs when both steel and platinum twin unipolar electrodes are used, although the effects of a.c. stimulation with steel electrodes, which results in deposition of ferric ions in the tissues, are prolonged in comparison to the effects of a.c. stimulation with platinum electrodes.
Direct current (d.c.) stimulation of the medial preoptic area with steel electrodes is highly effective in causing increased secretion of LH by the anterior pituitary. Again, there is considerable deposition of ferric ions from the electrode tips. D.c. stimulation with platinum electrodes is ineffective however, which provides further evidence that deposition of metallic ions in nervous tissue has potent stimulatory effects.
The results of the variety of stimulus procedures used in the present study suggest that as in the female rat, the medial preoptic area is involved in the control of gonadotrophin release in the male. While there is no cyclical waxing and waning of gonadotrophin release in the male rat, there is evidence that normal male sexual behaviour is dependent on an intact preoptic area (Lisk, 1968; Giantonio, Lund & Gerall, 1970).
In considering the results of the present and previous stimulation experiments, caution is urged such that when reporting stimulation and lesion experiments not only should electrical parameters including coulombage of pulses and pulse trains be reported, but also the physical and chemical characteristics of the electrodes themselves should be described.
Another form of evidence in support of the neurohumoral hypothesis of control of the anterior pituitary gland has been the discovery in extracts of hypothalamic tissue of substances capable of affecting the secretion of anterior pituitary hormones. Evidence for the occurrence of hypothalamic substance(s) which act so as to release gonadotrophins was produced by Harris and co-workers (Harris, 1960; Campbell, Feuer, Garcia & Harris, 1961) who showed that infusion of extracts of the hypothalamic median eminence directly into the pituitary gland was effective in inducing ovulation in rabbits. Characterisation of these so-called hypophysiotrophic factors depended on the development of reliable, specific, sensitive, direct assay methods.
After Schally and his co-workers (Schally, Arimura, Bowers, Sawano & Redding, 1968) had purified hundreds of thousands of porcine hypothalami, they were able to extract and isolate a substance capable of releasing LH and FSH from the anterior pituitary. Structural determination of this material as a decapeptide followed, and it was synthesised (Matsuo, Arimura, Nair & Schally, 1971) Similar work with ovine hypothalami was carried out by Guillemin and co-workers (Amoss, Burgus, Blackwell, Vale, Fellows & Guillemin, 1971). The availability of synthetic luteinising hormone-releasing factor (LH-RF) as it was called meant that assays using synthetic LH-RF as a standard became available.
In the present studies, in-vitro bioassays using short-term incubation of anterior pituitary glands from normal male animals (3-4 pituitaries/flask) and from ovariectomised animals (1 pituitary/ flask) pretreated with oestrogen and progesterone (Ramirez & McCann, 1963) have been established. In both cases, addition of doses of synthetic LH-RF ranging from 1.9 ng/ml incubation medium to 33.0 ng/ml medium caused release of significant amounts of both LH and FSH over a six hour incubation period. Satisfactory dose-response curves were established in both assays.
The principal desirable characteristics of an assay method are specificity, precision, sensitivity, repeatability, convenience and economy. In both assay systems LH and FSH release is caused by high concentrations of potassium, but the concentrations of potassium in natural materials tested in the assay are sufficiently low so as to not interfere in this respect. In the female assay system monoamines and octapeptides at dose levels that might be expected to occur in extracts of natural materials do not cause release of LH or FSH.
A number of synthetic fragments of LH-RF have been tested for LH and FSH releasing activity in the female assay system. Only the nonapeptide (LH-RF 1-9) exhibited consistent activity. The importance of the entire chain length of LH-RF for full gonadotrophin releasing activity is underlined by the fact that removal of the C-terminal amino acid results in depression of releasing activity by up to 95%.
Precision and sensitivity of both assay systems are satisfactory, but there is considerable between assay variation. In terms of economy it is evident that the use of pituitaries from oestrogen-progesterone treated ovariectomised animals enables considerable saving in numbers of assay animals required.
A radioimmunoassay (RIA) first described by Nett, Akbar, Niswender, Hedlund & White (1973) has been established in this laboratory using 125I-labelled LH-RF as tracer. It appears to be specific for the decapeptide LH-RF, and it is clear that it is appropriate to measure LH-RF concentrations in a variety of biological materials using this RIA. Regrettably, clear data on between-assay variability is lacking in the assays described, though the quality control parameters observed in the assay suggest that it is reasonably "in control". The fact that intact plasma contains constituents which affect binding of hormone to antiserum means that care must be taken when interpreting results, especially when the LH-RF concentration is low.
Measurements have been made of LH-RF concentration in plasma of post-menopausal women and no elevation has been observed despite the fact that LH secretion is generally high after the menopause (Yen, Tasi, Naftolin, Vandenberg & Ajabor, 1972). It is apparent also that coital activity in the male rat has no discernable effect on peripheral plasma LH-RF concentration in the first 20 min. after coitus. The availability of in-vitro bioassay and radioimmunoassay for LH-RF has prompted an examination of hypophysial portal blood collected from the cut pituitary stalk of rats under various experimental conditions. The work of Fink and co-workers (Fink, Nallar & Worthington, 1966, 1967: Fink 1967a, 1967b) showed that LH-RF activity was present in hypophysial portal plasma using in-vivo assays while Porter and his co-workers (Kamberi, Mical & Porter, 1969, 1970; Ben-Jonathan, Mical & Porter, 1973) have used in-vitro assays to detect LH-RF activity in hypophysial portal blood.
In the present studies LH-RF has been detected in ultra-filtrates of pituitary stalk plasma of male animals by in-vitro assay. While it was only possible to achieve parallelism of dose-response curves between synthetic LH-RF and stalk plasma ultrafiltrate in one of six assays, it is clear that stalk plasma ultrafiltrate contains material capable of releasing both LH and FSH from pituitary glands in-vitro.
By RIA, LH-RF was detected in pituitary stalk plasma of both male and female rats. With respect to the male, the evidence that stimulation of the medial preoptic area leads to significant increases in LH-RF secretion complements and accounts for the rises in peripheral plasma LH consequent upon medial preoptic area stimulation.
In the female, while the contents of LH-RF in stalk plasma collected at pro-oestrus and metoestrus are somewhat higher than those in stalk plasma collected at oestrus and dioestrus, the differences are not statistically significant. The surge of LH secretion on the afternoon of pro- oestrus and the smaller rise in peripheral LH concentration at metoestrus in females, may therefore be due to changes in sensitivity of the pituitary gland to constant or only slightly increased levels of LH-RF in hypophysial portal blood at those times.
As in the male, electrical stimulation markedly increased the contents of LH-RF in pituitary stalk blood in the female. There is no significant evidence that the sensitivity of the hypothalamo-median eminence-portal vessel axis varies during the oestrous cycle, though Fink & Aiyer (1974) find that the sensitivity of the entire hypothalamo-hypophysial axis to electrical stimulation is markedly enhanced on the afternoon of pro-oestrus.
In conflict with the results of others (Burger, Fink & Lee, 1972; Ben- Jonathan, Mical & Porter, 1973) no evidence of increased secretion of LH-RF was found in castrated female rats when compared with intact dioestrous controls.
The electrodes and the optimal a.c. stimulation parameters used in the present studies do not appear to have deleterious affects, as female rats continue to cycle with electrodes implanted in the preoptic area, and LH-RF secretion increases markedly in both of two 30 min. stimulation periods separated by a rest period of 30 min. While preoptic area stimulation can more than double pituitary stalk blood flow rates, such blood flow increases cannot account for the magnitude of increases in LH-RF secretion during stimulation.
The major problem encountered in these studies has been the enormous disparity between estimates of LH-RF concentration in pituitary stalk plasma made by bioassay and RIA methods.
Explanations for this disparity include the possibilities that the results either of the bioassay or of the RIA are artefactual, or that the synthetic preparation of LH-RF is not identical to the "physiological" endogenous LH-RF, or that endogenous LH-RF exists in portal blood either in "pro-hormone" form or bound to a plasma protein.