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Reference: Murray Jamieson, (1974). Some aspects of the hypothalamic control of pituitary gonadotrophin secretion. DPhil. University of Oxford.Citable link to this page:


Some aspects of the hypothalamic control of pituitary gonadotrophin secretion

Abstract: The development of that branch of physiology known asneuroendocrinology has followed the increasing awareness thatenvirornmental influences, sensed through the exteroceptors of thecentral nervous system, play an important part in the control ofreproductive and other endocrinologically mediated activity in animals.The implication of pituitary gland activity in these processes in therat by Smith (1927, 1932), who developed a satisfactory technique forhypophysectomy, led to a search for the connections between the centralnervous system and the pituitary gland. It was soon established thatthe posterior lobe of the pituitary is controlled by neuronal pathwayspassing from the hypothalamus to that lobe, and investigation of controlof the anterior lobe of the gland centred at first around theestablishment of a similar neural pathway.The discovery that the anterior pituitary is almost devoid ofnerve fibres, and the failure of investigators to establish any formof autonomic nervous control drew attention to the blood vessels in thepituitary stalk first adequately described by Popa and Fielding (1930,1933). These vessels offered an alternative control pathway to the anteriorlobe and a number of investigators in the 1930's (Hinsey & Markee, 1933;Friedgood, 1936; Hinsey, 1937; Harris, 1937; Haterius, 1937) put forwarda tentative hypothesis of humoral control of the anterior pituitary. By[illegible], Harris was able to state the neurohumoral hypothesis confidentlyin the light of the accumulation of a good deal of supporting evidence.One of the most profitable techniques used by investigators inexperiments on the hypothalamo-hypophysial axis has been electricalstimulation in the central nervous system with concomitant observationof changes in activity of extra-neural structures. Early experimentswere crude, Marshall and Verney (1936) applying diffuse electricalstimuli to the heads or lumbar spinal cords of rabbits, and Harris(1936) carrying out similar experiments on rats. In both cases, theseexperiments indicated that some neural structures in the head had beenexcited, with consequent ovulation in the rabbits and pseudopregnancyin the rats.The crudity of the electrical stimulation techniques used atthat time meant that close and detailed study of small areas of thecentral nervous system was impracticable. With the development ofelectronic stimulators capable of producing accurate, stable trainsof stimuli, it became possible to examine hypothalamic areas whichmight be involved in control of anterior pituitary secretion. A greatvariety of electrode materials, stimulus waveforms and parameters ofstimulation have been used. Frequently, scant regard has been paidto the differentiation of electrical stimulation from electrical lesionproduction.Many studies have been performed on the activation of ovulationor of luteinising hormone (LH) secretion by the anterior pituitary infemale rats after electrical stimulation in the diencephalon, andit became evident that medial preoptic area was involved (Critchlow,1958) There are few reports however, of activation of LH release fromthe anterior pituitary consequent upon hypothalamic stimulation in maleanimals. No event comparable to ovulation was observable in male animals,so Moll and Zeilmaker (1966) and Quinn (1966) resorted to transplantingovaries into castrated males. Direct current (d.c.) stimulation in themedial preoptic area resulted, in both these studies, in changes in theovarian 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 medialpreoptic area stimulation in normal male rats, using a variety ofelectrode materials and stimulation parameters.The present studies provide direct evidence for the release ofLH from the anterior pituitary gland consequent upon electricalstimulation of the medial preoptic area of the normal male rat, andconfirms the preliminary studies of Burger, Fink & Lee (1972) whoachieved rises greater than 400% in peripheral plasma LH concentrationin three male rats after medial preoptic area stimulation. The optimalparameter for alternating current (a.c.) square wave stimulation arefrequency 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 electrodesare used, although the effects of a.c. stimulation with steel electrodes,which results in deposition of ferric ions in the tissues, are prolongedin comparison to the effects of a.c. stimulation with platinumelectrodes.Direct current (d.c.) stimulation of the medial preoptic areawith steel electrodes is highly effective in causing increasedsecretion of LH by the anterior pituitary. Again, there is considerabledeposition of ferric ions from the electrode tips. D.c. stimulationwith platinum electrodes is ineffective however, which provides furtherevidence that deposition of metallic ions in nervous tissue has potentstimulatory effects.The results of the variety of stimulus procedures used in thepresent study suggest that as in the female rat, the medial preopticarea is involved in the control of gonadotrophin release in the male.While there is no cyclical waxing and waning of gonadotrophin releasein the male rat, there is evidence that normal male sexual behaviour isdependent on an intact preoptic area (Lisk, 1968; Giantonio, Lund &Gerall, 1970).In considering the results of the present and previousstimulation experiments, caution is urged such that when reportingstimulation and lesion experiments not only should electrical parametersincluding coulombage of pulses and pulse trains be reported, but alsothe physical and chemical characteristics of the electrodes themselvesshould be described.Another form of evidence in support of the neurohumoralhypothesis of control of the anterior pituitary gland has been thediscovery in extracts of hypothalamic tissue of substances capableof affecting the secretion of anterior pituitary hormones. Evidencefor the occurrence of hypothalamic substance(s) which act so as torelease gonadotrophins was produced by Harris and co-workers (Harris,1960; Campbell, Feuer, Garcia & Harris, 1961) who showed that infusionof extracts of the hypothalamic median eminence directly into thepituitary gland was effective in inducing ovulation in rabbits.Characterisation of these so-called hypophysiotrophic factors dependedon 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 porcinehypothalami, they were able to extract and isolate a substance capableof releasing LH and FSH from the anterior pituitary. Structuraldetermination of this material as a decapeptide followed, and it wassynthesised (Matsuo, Arimura, Nair & Schally, 1971) Similar work withovine hypothalami was carried out by Guillemin and co-workers (Amoss,Burgus, Blackwell, Vale, Fellows & Guillemin, 1971). The availabilityof synthetic luteinising hormone-releasing factor (LH-RF) as it wascalled meant that assays using synthetic LH-RF as a standard becameavailable.In the present studies, in-vitro bioassays using short-termincubation 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 ofsynthetic LH-RF ranging from 1.9 ng/ml incubation medium to 33.0 ng/mlmedium caused release of significant amounts of both LH and FSH over asix hour incubation period. Satisfactory dose-response curves wereestablished in both assays.The principal desirable characteristics of an assay method arespecificity, precision, sensitivity, repeatability, convenience andeconomy. In both assay systems LH and FSH release is caused by highconcentrations of potassium, but the concentrations of potassium innatural materials tested in the assay are sufficiently low so as tonot interfere in this respect. In the female assay system monoaminesand octapeptides at dose levels that might be expected to occur inextracts of natural materials do not cause release of LH or FSH.A number of synthetic fragments of LH-RF have been tested forLH and FSH releasing activity in the female assay system. Only thenonapeptide (LH-RF 1-9) exhibited consistent activity. The importanceof the entire chain length of LH-RF for full gonadotrophin releasingactivity is underlined by the fact that removal of the C-terminalamino acid results in depression of releasing activity by up to 95%.Precision and sensitivity of both assay systems aresatisfactory, but there is considerable between assay variation. Interms of economy it is evident that the use of pituitaries fromoestrogen-progesterone treated ovariectomised animals enablesconsiderable saving in numbers of assay animals required.A radioimmunoassay (RIA) first described by Nett, Akbar,Niswender, Hedlund & White (1973) has been established in thislaboratory using 125I-labelled LH-RF as tracer. It appears to bespecific for the decapeptide LH-RF, and it is clear that it isappropriate to measure LH-RF concentrations in a variety of biologicalmaterials using this RIA. Regrettably, clear data on between-assayvariability is lacking in the assays described, though the qualitycontrol parameters observed in the assay suggest that it is reasonablyin control. The fact that intact plasma contains constituents whichaffect binding of hormone to antiserum means that care must be takenwhen interpreting results, especially when the LH-RF concentration islow.Measurements have been made of LH-RF concentration in plasmaof post-menopausal women and no elevation has been observed despitethe fact that LH secretion is generally high after the menopause(Yen, Tasi, Naftolin, Vandenberg & Ajabor, 1972). It is apparent alsothat coital activity in the male rat has no discernable effect onperipheral plasma LH-RF concentration in the first 20 min. after coitus.The availability of in-vitro bioassay and radioimmunoassayfor LH-RF has prompted an examination of hypophysial portal bloodcollected from the cut pituitary stalk of rats under variousexperimental conditions. The work of Fink and co-workers (Fink, Nallar& Worthington, 1966, 1967: Fink 1967a, 1967b) showed that LH-RF activitywas present in hypophysial portal plasma using in-vivo assays whilePorter and his co-workers (Kamberi, Mical & Porter, 1969, 1970;Ben-Jonathan, Mical & Porter, 1973) have used in-vitro assays to detectLH-RF activity in hypophysial portal blood.In the present studies LH-RF has been detected in ultra-filtratesof pituitary stalk plasma of male animals by in-vitro assay. While it wasonly possible to achieve parallelism of dose-response curves betweensynthetic LH-RF and stalk plasma ultrafiltrate in one of six assays, itis clear that stalk plasma ultrafiltrate contains material capable ofreleasing both LH and FSH from pituitary glands in-vitro.By RIA, LH-RF was detected in pituitary stalk plasma of bothmale and female rats. With respect to the male, the evidence thatstimulation of the medial preoptic area leads to significant increases inLH-RF secretion complements and accounts for the rises in peripheralplasma LH consequent upon medial preoptic area stimulation.In the female, while the contents of LH-RF in stalk plasmacollected at pro-oestrus and metoestrus are somewhat higher than thosein stalk plasma collected at oestrus and dioestrus, the differences arenot statistically significant. The surge of LH secretion on theafternoon of pro- oestrus and the smaller rise in peripheral LHconcentration at metoestrus in females, may therefore be due tochanges in sensitivity of the pituitary gland to constant or onlyslightly increased levels of LH-RF in hypophysial portal blood at thosetimes.As in the male, electrical stimulation markedly increased thecontents of LH-RF in pituitary stalk blood in the female. There is nosignificant evidence that the sensitivity of the hypothalamo-medianeminence-portal vessel axis varies during the oestrous cycle, thoughFink & Aiyer (1974) find that the sensitivity of the entire hypothalamo-hypophysialaxis to electrical stimulation is markedly enhanced on theafternoon of pro-oestrus.In conflict with the results of others (Burger, Fink & Lee,1972; Ben- Jonathan, Mical & Porter, 1973) no evidence of increasedsecretion of LH-RF was found in castrated female rats when compared withintact dioestrous controls.The electrodes and the optimal a.c. stimulation parametersused in the present studies do not appear to have deleterious affects,as female rats continue to cycle with electrodes implanted in thepreoptic area, and LH-RF secretion increases markedly in both of two30 min. stimulation periods separated by a rest period of 30 min.While preoptic area stimulation can more than double pituitary stalkblood flow rates, such blood flow increases cannot account for themagnitude of increases in LH-RF secretion during stimulation.The major problem encountered in these studies has been theenormous disparity between estimates of LH-RF concentration inpituitary stalk plasma made by bioassay and RIA methods.Explanations for this disparity include the possibilities thatthe results either of the bioassay or of the RIA are artefactual, orthat the synthetic preparation of LH-RF is not identical to thephysiological endogenous LH-RF, or that endogenous LH-RF exists inportal blood either in pro-hormone form or bound to a plasma protein.

Type of Award:DPhil Level of Award:Doctoral Awarding Institution: University of Oxford Notes:This thesis was digitised thanks to the generosity of Dr Leonard Polonsky

Bibliographic Details

Issue Date: 1974Identifiers

Urn: uuid:67e0cf26-fd28-45bc-b77a-1c03619ed7e7

Source identifier: 601870680 Item Description

Type: Thesis;

Language: eng Tiny URL: td:601870680


Autor: Murray Jamieson - institutionUniversity of Oxford oxfordCollegeMerton College - - - - Bibliographic Details Issue Date: 1974 - Id



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