Neuroendocrinology of the menstrual cycle in humans

4 Neuroendocrinology of the Menstrual Cycle in Humans ROBERT W. SHAW

The mechanisms controlling the menstrual cycle and reproductive function have long eluded us and indeed still do. It has been during this century, and more so during this last decade, that the complex mechanisms within the hypothalamus and pituitary which control gonadotrophin release and hence ovarian function have gradually begun to be elucidated. Many of these recent advances stem from the progress made by biochemists, anatomists and physiologists essentially performing in vitro and in vivo animal experiments, but I would like to consider the information available from studies in the human female. HISTORICAL MILESTONES The observation that castration led to deleterious effects upon reproductive function dates back to antiquity, though its causation was unknown. Moreover it had been noted that seasonal and emotional factors could also influence reproduction and menstruation. In 1886 Marie observed that there was a co-existence between the disease acromegaly and a tumour of the pituitary gland; this gave rise to considerable interest in this gland with possible relevance of its association with other disease entities. Indeed Crowe, Cushing and Homans (1910) demonstrated that hypophysectomy caused atrophy of the reproductive organs in the dog, implicating an important role for the pituitary in the control of reproduction. During the 1920s and 1930s the relationship between the pituitary gland and the gonads came under greater scrutiny. This resulted in the postulation of the existence of a pituitary gonad-stimulating hormone, and work to determine its chemical characterization and physiological significance began in earnest. The complex nature of the hypothalamo-pituitary-gonadal axis hindered significant advances for many years until specific gonadstimulating hormones in the anterior lobe of the pituitary were isolated (Smith, 1926; Zondek and Aschheim, 1926). Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - -

Vol. 7, No. 3, November 1978.

531

532

ROBERT W. SHAW

Gonadal steroids (Moore and Price, 1932; Hohlweg and Junkmann, 1932) and environmental stimuli (Marshall, 1936) were shown to influence hypophyseal gonadotrophin secretion, but the experimental techniques of stimulation, ablation and transplantation within the central nervous system in the 1930s established the hypothesis for a hypothalamic control of gonadotrophin secretion. This control was thought to be mediated by neurohumoral substances originating in the region of the median eminence of the hypothalamus (Harris, 1937). It was not until the demonstration of luteinizing hormone (LH)-releasing activity by hypothalamic extracts of rats (McCann, Taleisnik and Friedman, 1960) that the concept of hypothalamic control of LH secretion became really established. A few years later the presence of follicle stimulating hormone (FSH)-releasing activity in hypothalamic extracts was reported (Igarashi and McCann, 1964; Mittler and Meites, 1964). It was a further decade before isolation (Schally et al, 1971) of the LHreleasing hormone (LHRH) was achieved and its subsequent determination of structure and synthesis (Matsuo et al, 1971a). The finding of FSHreleasing activity of this LHRH led to the formulation of the hypothesis for a single hypothalamic hormone controlling secretion of both LH and FSH from the pituitary gland with the suggestion that sex steroids might play a role in modulating the proportions of LH and FSH released. (For these reasons, LHRH is referred to hereafter as gonadotrophin-releasing hormone.) The isolated natural gonadotrophin-releasing hormone (GnRH) had the structural formula depicted in Figure 1 and is a small polypeptide containing 10 amino-acid residues with a molecular weight of 1181. In this chapter some of the mechanisms involved in the cyclic-gonadotrophin release in the adult human female will be discussed. Studies on neuroendocrine mechanisms involved in ovulation, especially those which involve surgery to the brain or require administration of drugs, cannot be performed in humans in the same manner that they have so expertly been performed in the rat and Rhesus monkey. However, despite these difficulties a great deal has been learned in the past decade of the control mechanisms of gonadotrophin release in the human female. In particular the role played by the ovarian steroid hormones oestradiol-17/3 and progesterone and their influence upon the release of GnRH from the hypothalamus and the pituitary release of LH and FSH with relevance to the control of the normal menstrual cycle have been studied in great depth. ANATOMY OF THE HYPOTHALAMIC-PITUITARY AXIS

The neurohypophysis is the portion of the hypothalamus of special interest to this chapter. It is divisible into three regions: (1) the infundibulum, which constitutes the floor of the third ventricle (often termed the median eminence) and part of the wails of the third ventricle, which is continuous with (2) the infundibular stem, also termed the pituitary stalk, which is continuous distally with (3) the infundibular process or posterior pituitary gland. The neurohypophysis is supplied by blood from three sets of arteries -- the superior, middle and inferior hypophysial arteries, each supplying the

533

NEUROENDOCRINOLOGY OF THE MENSTRUAL CYCLE

infundibulum, infundibular stem and upper infundibular process and infundibular process respectively. The capillaries in the infundibular stem and infundibular process are interconnected forming a dense network of vessels (Page, Munger and Bergland, 1976). The adenohypophysis consists of (1) the pars distalis or anterior lobe of the pituitary, (2) the pars intermedia, synonymous with the intermediate lobe, and (3) the pars tuberalis, which is a thin layer of adenohypophysial cells

H,i__,,,4 ~_t,n N'~i

Pyro- GLU

I'---" C--O NH

~ ~LcH2...CH

H IS

NH ,,.

CH-CH2~ C=O NH

SER

HO-CH-CH 2 C=O NH

CH-CH2 C--O

~

NH

CH2 NH ,,,,CH3 CH-CH~'CH~ C=O ~ ~CH3 NH,~ HH H NH .C-NH-~-(~- CH NH~2

OH

TYR

GLY LEU

~ ~ ~1 C"O

ARG

D C=O

PRO

CH

GLY- NH2

NH

Figure l. Amino acid sequence of porcine GnRH.

lying on the surface of the infundibular stem and infundibulum. The pars distalis does not normally receive an arterial vasculature but it receives blood through portal vessels. These vessels arise from those prominent upon the infundibular stem (long hypophyseal portal vessels) and from the short hypophyseal portal vessels which join capillaries in the infundibular stem and upper infundibular process. It is hence apparent that the sinusoids of the adenohypophysis receive blood that has first traversed capillaries residing in the neurohypophyseal complex. This unique relationship of vasculatures has provided a basis for the view that the hypothalamus regulates the secretion of adenohypophyseal hormones through neurohormonal mechanisms involving hypothalamic releasing factors (McCann and Porter, 1969).

534

ROBERT W. SHAW

HYPOTHALAMIC REGULATION OF PITUITARY SECRETION Considerable effort has been made in the past two decades to identify, characterize and synthesize the neurohumoral substances thought to be produced in the neural elements in the infundibulum (Schally et al, 1968; Burgus et al, 1969; Matsuo et al, 1971b; Brazeau et al, 1973; Vale et al, 1975). Several substances which can either stimulate or repress the rate of release of one or more hormones from the pars distalis have been found in the infundibular complex. Amongst these substances are oxytocin, vasopressin, dopamine, epinephrine, norepinephrine, gonadotrophin-releasing hormone (GnRH), thyrotrophin-releasing hormone (TRH), somatostatin, vasoactive intestinal peptide (VIP), neurophysins etc. However, the mere finding of these substances within the hypothalamus or the basis of a particular activity demonstrated in a biological test system, does not necessarily mean they have a role in the physiological regulation of the pars distalis. Regulation by GnRH GnRH has been demonstrated in the hypophyseal portal blood from rats, monkeys and rabbits using radioimmunoassays (Carmel, Araki and Ferrin, 1976; Eskay, Mical and Porter, 1977; Neill et al, 1977). Electrical stimulation of the pre-optic area of the brain of the female rat on the day of pre-oestrus increases GnRH concentration in portal blood (Chiappa, Sherwood and Fink, 1975; Eskay, Mical and Porter, 1977) and the stimulus will also cause a marked release of LH from the pars distalis. The administration intravenously of antibodies against GnRH prevents this LH release following pre-optic stimulation. These data provide evidence favouring a cause-andeffect relationship between GnRH release by the hypothalamus and LH release by the pars distalis. Modulatory role of catecholamines on GnRH secretion Studies by Markee, Sawyer and Hollinshead (1948) and Sawyer, Markee and Townsend (1949) indicated that LH release and ovulations were dependent upon drug affected neural stimuli of both cholinergic and adrenergic origins. Dopamine, norepinephrine and serotonin are all found within hypothalamic neurones, but alone none of these substances can induce LH or FSH release from pituitary cultures. However, if median eminence extracts are added to the pituitary cultures in the presence of dopamine, then a massive release of LH (Schneider and McCann, 1969) and of FSH (Kamberi, Schneider and McCann, 1970), greater than that induced by the median eminence extracts alone, can be induced. Injections of dopamine into the third ventricle, but not into the pituitary, will also bring about an elevation of serum LH levels (Kamberi, Mical and Porter, 1970). This release of LH by intraventricular dopamine can be prevented by oestrogens given two hours prior to the dopamine (Schneider and McCann, 1970). These experiments indicate that hypothalamic dopaminergic neurones synapse with neurosecretory neurones which secrete GnRH and stimulate its release. Adrenaline may also play a role in some steroid feedback mechanisms.

NEUROENDOCRINOLOGY OF THE MENSTRUAL CYCLE

535

The progesterone-induced release of LH in spayed rats, pre-treated from two days with oestradiol benzoate, was abolished by phenoxybenzamine (aadrenergic blocker) or haloperidol (a- and p-blocker) whereas propranalol (/3-blocker) had no effect. Thus an a-adrenergic mechanism and catacholamines were involved. Noradrenaline was found to be the substance involved in this positive feedback release (Kalra et al, 1972). Serotonin appears to have a predominantly inhibitory role in gonadotrophin release in experiments to date (for references see McCann and Ojeda, 1976). It should perhaps be considered that several neurotransmitters may be involved in the control of gonadotrophin release and the exact mechanisms involved have still to be elucidated. HORMONAL EVENTS DURING THE MENSTRUAL CYCLE

The availability of radioimmunoassays and competitive protein binding assays during the last few years has enabled us to assess accurately the profile of gonadotrophin and steroid hormone secretion during the menstrual cycle (Neill et al, 1967; Midgley and Jaffe, 1968; Mishell et al, 1971; Abraham et al, 1972). These changes are schematically shown in Figure 2. OQ Menses OvulatiOn

}

•

¢3 i

"

#i ® 6 o e° ° °o

• ."

t ." o~°

.. t

"°°

I

_-C~___ _~=-~¢J _ m "I~1-~

I •

".

| I

,

,"

...o-

I

ol

"-I

LH ~'

0 IProgesterone

Time in days

Figure 2. Diagrammatic changes in serum levels of LH, FSH, oestradiol-17/3 and progesterone during the menstrual cycle.

Ovulation is preceded by spontaneous surges of both LH and FSH and plasma concentrations of both gonadotrophins are greater during the follicular than the luteal phase. Most studies suggest that the increase in plasma FSH during the late luteal, which continues into the follicular, phase is relatively greater than that of plasma LH. The mid-cycle surge of" FSH does not always occur and sometimes does not coincide with the LH surge, though this is most often seen in abnormal cycles (Ross et al, 1970; Thorneycroft et al, 1974). The role played by FSH in the ovulatory process remains unknown

536

ROBERT W. SHAW

and its occurrence with the LH surge may simply be due to a common releasing hormone inducing release of both gonadotrophins. However, although LH alone, in the form of human chorionic gonadotrophin (hCG), can induce follicle rupture, there is some evidence to suggest that LH and FSH work in synergism such that when both are present the effective dose of each is markedly reduced (Harrington and Bex, 1970). Such an action may be one of the functions of the accompanying FSH surge at mid-cycle. Using single daily blood sampling techniques, it has been shown that serum oestradiol-17fl levels rise sharply over a two or three day period, reaching a peak which usually precedes by one day the occurrence of the gonadotrophin surge (Johansson and Wide, 1969; Abraham and Klaiber, 1971). When more frequent samples of blood are taken, marked fluctuations in oestradiol-17/3 are apparent, but again highest levels usually occur on the day preceding that of the LH surge (Korenman and Sherman, 1973). The available information strongly supports the theory that the rise in oestrogen during the late follicular phase is the trigger that sets off the pre-ovulatory LH surge. Plasma 17a-hydroxyprogesterone levels also undergo marked changes at mid-cycle, but detailed studies indicate that 17a-hydroxyprogesterone begins to rise with and not before the LH surge, and may be the first index of luteinization (Thorneycroft et al, 1974). Shortly after the peak of the gonadotrophin surge plasma progesterone begins to rise, and prior to the surge the ovary also secretes significant amounts of androgens, mainly androstenedione and small amounts of dehydroisoandosterone and testosterone (Vande-Wiele et al, 1970). The significance of these hormones on the induction or modification of the midcycle gonadotrophin surge is not known. The majority of workers have been able to detect a diurnal variation in LH and FSH secretion during the menstrual cycle. The withdrawal of blood samples at frequent intervals have revealed that the release of gonadotrophins occurs in an episodic manner with a frequency of about one to four hours (Midgley and Jaffe, 1971; Yen et al, 1972a). The frequency and amplitude of the LH fluctuations vary during the menstrual cycle (Yen et al, 1972a). Episodic fluctuation of gonadotrophin release also follows the administration of synthetic GnRH in man (Mortimer et al, 1974) suggesting that the mechanism which underlies this phenomenon may be located at the level of the pituitary. Estimations of prolactin concentrations in human plasma show that there is little change in the profile of this hormone during the menstrual cycle (Ehara et al, 1973; Friesen and Hwang, 1973; McNeilly and Chard, 1974). However, high concentrations of serum prolactin invariably produce anovulation, and in such patients abnormalities of oestrogen feedback have been demonstrated (Glass et al, 1975) suggesting an action of elevated prolactin levels at a hypothalamic and pituitary level. McNatty, Sawers and McNeilly (1974) found that the concentration of prolactin in ovarian follicular fluid varies considerably during the menstrual cycle, being significantly lower during the late follicular and early luteal phase. These authors also showed that prolactin inhibits the production of

NEUROENDOCRINOLOGY OF THE MENSTRUALCYCLE

537

progesterone by human granulosa cells in vitro. If further studies confirm that prolactin can modulate ovarian steroid secretion in concentrations similar to those found in the normal menstrual cycle, another important variable will have been introduced into our understanding of the regulations of the menstrual cycle.

FEEDBACK ACTION OF OVARIAN STEROIDS Evidence has accumulated which indicates that the hormones synthesized in the ovary may act on several brain centres, or the anterior pituitary gland directly, to modify their activity and participate through different types of feedback mechanisms (described as long and short loop, negative and positive) in the final regulation of gonadotrophin release.

Negative feedback of oestrogen Negative feedback control of pituitary LH and FSH release has been postulated for over 40 years. Moore and Price (1932) considered that the ovary and adenohypothesis were linked in a rigid system of hormonal interactions, sex steroids acting by directly suppressing gonadotrophin secretion. It has since been realized that oestrogen also exerts its effect on gonadotrophin release in the hypothalamus and on changes in the release of GnRH. In situations of low endogenous oestrogen production (menopause, ovarian failure) levels of gonadotrophin will rise if negative feedback is intact and conversely the stimulation of endogenous oestrogen production or the administration of exogenous oestrogen will lead to an initial suppression of circulating gonadotrophin levels. This negative feedback effect of oestrogen on gonadotrophin release can be clearly demonstrated in postmenopausal women given oestrogens (Nillius and Wide, 1970; Yen and Tsai, 1971; Wise, Gross and Schalch, 1973). The negative feedback effect is apparent within two hours of administration of an intramuscular injection of oestradiol-17p (Nillius and Wide, 1970) and is still present to a marked degree at 24 hours, with a gradual return to pretreatment levels by 36 to 48 hours despite the persistence of elevated levels of oestradiol-17/3 within the circulation, in many instances still exceeding the normal physiological mid-cycle peak values (Shaw, 1975a). A similar negative feedback response to acute administration of oestrogens is seen in normal women studied during the follicular phase of their cycles (Tsai and Yen, 1971; Yen and Tsai, 1972; Shaw, 1975b). This negative feedback effect of oestrogen is the main factor which maintains the relatively low basal concentrations of plasma LH and FSH (Vande-Wiele et al, 1970; Knobil, 1974). The threshold for the negative feedback action of oestrogen is set to bring about suppression of gonadotrophin release with only small increases in oestradiol-17p levels in the normal female. Certainly the levels of oestradiol17/J reached in the early follicular phase of the cycle, when this negative feedback stimulus appears to play an important role in reducing gonadotrophin levels, are not very high and of the range 100 to 200 pmol/1 (30 to 60 pg/ml). However, it would appear not only to be simply absolute levels of

538

ROBERT W. SHAW

oestradiol but more a relationship to time of maintenance of increased levels and rate of rise that determine the negative feedback stimulus as well as an individual's sensitivity. The site of action of oestradiol in this negative feedback control appears to be at both a hypothalamic and anterior pituitary level. At the hypothalamus steroids appear to decrease GnRH secretion (Davidson et al, 1976), whilst at the pituitary the inhibitory effect is exerted by decreasing the sensitivity of the gonadotropes to GnRH (McCann, 1974; Davidson et al, 1976) from experimental work in the rat.

Positive feedback of oestrogen The fact that under certain circumstances oestrogen may stimulate (positive feedback) rather than inhibit gonadotrophin release was first proposed by Hohlweg and Junkman in 1932. Since this original work, a number of workers have shown that oestrogen can stimulate LH release in experimental animals (see review by Everett, 1964). The administration of an appropriate dose of oestrogen also triggers LH release in women (Nillius and Wide, 1971; Leyendecker, Wardlaw and Nocke, 1972; Monroe, Jaffe and Midgley, 1972; Yen and Tsai, 1972; Shaw, 1975a, 1975b) and Rhesus monkeys (Knobil, 1974). Figure 3 shows the circulating levels of oestradiol-17p and the changes in serum LH and FSH in six normally menstruating females given an intraOestrooen Provocation Test in SIX normal females--2.~ mqms Oestradlol Benzoate im. u/l /~ Mean 20 "J:SEM

E

, . o . o FSH *---,I, E 2

3000

2000

12

o

......

r .........

r 0

Time hrs Figure 3. Serum levels of oestradiol-17/3, LH and FSH in 6 normal females given 2.5 mg oestradiol benzoate (EB) i.m. on Day 4 of their menstrual cycle.

muscular injection of 2.5 mg oestradiol benzoate on day 4 of their menstrual cycles. It can be seen that there is a rapid rise in circulating oestradiol-17fl levels to a peak between 8 and 16 hours and an accompanying negative feedback suppression of LH and FSH levels. Oestradiol-17fl values then begin to

NEUROENDOCRINOLOGY OF THE MENSTRUALCYCLE

539

fall gradually to reach pre-injection levels at about 72 hours. A surge of LH (and a smaller rise in FSH levels in four of the six subjects) began some 32 hours following the oestradiol injections and was similar in height and duration to that of the mid-cycle gonadotrophin surge. Other subjects were studied following administration of different doses of oestradiol benzoate (0.5 rag, 1 rag, 2.5 mg or 5 rag). LH surges were induced in 14 of the 17 subjects studied, whilst FSH surges accompanied the LH surges in only nine subjects (Shaw, 1975a). FSH surges accompanying oestrogen-induced LH surges in human studies were not found by Tsai and Yen (1971), Monroe, Jaffe and Midgley (1972) or Nillius and Wide (1972a), whereas Yen and Tsai (1972) and Cargille et al (1973) did report accompanying increases in FSH levels in some subjects. The timing of the gonadotrophin surges following oestradiol injections was extremely variable in our above studies, occurring between 36 and 72 hours after the intramuscular doses of oestradiol-17/3, 12 to 24 hours after termination of oral ethinyl oestradiol or intravenous oestradiol-17fl infusions (Tsai and Yen, 1971, 1972) and 48 to 72 hours following 1 mg oestradiol benzoate i.m. (Nillius and Wide, 1972a).

What of the dynamics of this positive feedback? The neuroendocrine changes responsible for the acute surge of gonadotrophins following oestrogen administration, to levels often equivalent to mid-cycle peak heights, are as yet undefined in the human female. There is little doubt that the pre-ovulatory rise in circulating oestradiol17/3 represents the triggering stimulus for the initiation of the mid-cycle gonadotrophin surge. In 1969, Ferin et al reported that the administration of an antiserum to oestradiol-17/3 blocked ovulation in the rat, but this effect could be overcome by diethylstilboestrol, a synthetic oestrogen which did not cross-react with the antibodies. In the ewe a single intramuscular injection of 10/~g oestradiol-17fl induces LH release within 8 to 12 hours (Goding et al, 1969). However, in primates a more prolonged exposure of the hypothalamic-pituitary unit to oestrogen is required. This oestrogen effect has been termed 'positive feedback', but whether this is a true positive feedback stimulus or merely the partial removal of an oestrogen negative feedback following a reduction in circulating oestrogen levels remains uncertain. The latter view gains support from the results of Tsai and Yen (1971, 1972), Monroe, Jaffe and Midgley (1972), Nillius and Wide (1972a) and Shaw (1975b), in which the acute surges of LH were not observed until a fall in circulating oestrogen levels had occurred. Other workers have reported the occurrence of gonadotrophin surges whilst oestrogen levels are maintained at constant elevated levels in human females on oral ethinyl oestradiol therapy (Yen and Tsai, 1971; Cargille et al, 1973), in Rhesus monkeys in which constantly elevated levels of oestradiol-17/3 were maintained by slow release from sialastic capsules (Karsch et al, 1973), and in ewes given a continuous intravenous infusion of oestradiol-17p (Plant and Ward, 1974). There is also evidence to show that the circulating level of oestrogen attained must exceed a minimum threshold stimulus to induce positive

540

ROBERT W. SHAW

feedback. In the Rhesus monkey a single injection of oestradiol-17//resulting in a large but short lived (less than 12 hours) rise in plasma oestradiol-17fl did not elicit an LH surge (Yamaji et al, 1971). Sub-threshold plasma oestrogela:~levels of less than 100 pg/ml were equally ineffective even when maintained for as long as 120 hours, whereas slightly higher levels of 150 pg/ml sustained for at least 36 hours would consistently induce LH release (Karsch et al, 1973). The dynamics of positive feedback in the human have not been so extensively investigated as have those in the Rhesus monkey, but from our own studies and those of other investigators now reported in the literature, it would seem that the dynamics observed in the Rhesus monkey apply to the human also. There would seem to be an activation delay of 32 to 48 hours from the commencement of oestrogen administration until the onset of the positive feedback induced gonadotrophin surge, a minimum threshold level to exceed and suggestive evidence in addition for a strength-duration aspect to this stimulus in the human female. Several factors can inhibit the stimulatory action of oestrogen on gonadotrophin release. Elevated plasma progesterone concentrations, achieved by parenteral administration of natural progesterone, block the positive feedback action of oestrogen in Rhesus monkeys (Spies and Niswender, 1972; Dierschke et al, 1973) and humans (Netter et al, 1973). During the luteal phase of the menstrual cycle oestrogen-induced LH release fails to occur in normal females (Shaw, 1975a; Van Look, 1976) and may be explained by the inhibitory effect of elevated circulating levels of endogenous progesterone at this stage of the cycle. Many subjects with amenorrhoea fail to elicit LH surges following exogenous oestrogen administration (Shaw, Butt and London, 1974, 1975a; Thompson, Karam and Taymor, 1974) and this defect of positive feedback may be an explanation for their anovulation. Following such oestrogen provocation tests, Shaw et al (1975) demonstrated that only those with intact positive feedback would ovulate following Clomiphene administration. Finally, studies have demonstrated that women with elevated serum levels of prolactin fail to release LH in response to an oestrogen provocation test (Glass et al, 1975; Aono et al, 1976). It is not clear if this failure of positive feedback is a result of the hyperprolactinaemia per se or an additional manifestation of the underlying hypothalamic-pituitary dysfunction causing the hyperprolactinaemia.

EFFECT OF PROGESTERONE ADMINISTRATION UPON GONADOTROPHIN RELEASE In experiments investigating the control of the menstrual cycle much of the work has concentrated on the role played by oestrogens. However progesterone, the other major ovarian steroid hormone, also undergoes cyclical changes throughout the menstrual cycle and may play a facilitatory role. A closer look at the changes in serum progesterone occurring around the time of the mid-cycle gonadotrophin surge by Leyendecker, Wardlaw and

NEUROENDOCRINOLOGY OF THE MENSTRUALCYCLE

$41

Nocke (1972) suggested that there was indeed a significant rise in progesterone preceding the onset of the gonadotrophin surge -- from 5 nmol/1 to 8 nmol/l.

Negative feedback action of progesterone Progesterone is less effective than oestrogen at inhibiting the release of gonadotrophins, although in large doses intramuscularly it can be used as an effective contraceptive agent by inducing anovulation. How then does this 'negative' feedback effect of progesterone work? In ovariectomized rats progesterone, even in large doses, has little effect on baseline LH release (Nallar, Antunes-Rodrigues and McCann, 1966) and, indeed, the elevated gonadotrophin levels in postmenopausal women are unaffected by progesterone administration alone (Nillius and Wide, 1971). Progesterone then has little or no negative feedback action on basal gonadotrophin release. Progesterone can, however, suppress the ovulatory surge of LH as has been demonstrated in human females administered synthetic progestagens (Larsson-Cohn et al, 1972). Further evidence for this action came from animal experiments. Progesterone given at the critical time in the oestrous cycle of the rat (afternoon or evening of dioestrus) blocks the spontaneous ovulation which would occur the next day (Zeilmaker, 1966) and prevents the ovulation induced by median eminence LHRH containing extracts (Stevens et al, 1970). The site of this negative feedback action seems to be principally within the hypothalamus, though more recent studies indicate some direct pituitary effect (Baker, Eskin and August, 1973). The principal suppressive, negative, feedback action of progesterone is thus upon the mid-cycle gonadotrophin surge and it may be responsible for its short 24 hour duration. Positive feedback of progesterone Progesterone or synthetic progestagens administered alone have also been shown to induce positive feedback releases of gonadotrophins. However, it appears that the stage in the cycle at which the progesterone is given is of paramount importance in its ability to advance ovulation in rats (Everett and Sawyer, 1949) or to induce LH surges (Nallar, Antunes-Rodrigues and McCann, 1966). Both these groups of workers found that only progesterone administered in late dioestrus could induce these effects; if given earlier or later in the cycle a blocking ovulation would occur. In postmenopausal women, progesterone alone fails to affect LH or FSH levels, but if these women have been previously treated with small doses of oestrogen and then given the progesterone, a release of LH is observed (Odell and Swerdloff, 1968). Reports of investigations into positive feedback action of progesterone in normal women have been few. We investigated the effect of administering 12.5 mg or 25 mg of progesterone intramuscularly upon subsequent gonadotrophin release at different phases of the menstrual cycle in normal women. In only one of three subjects given the progesterone during the early follicular phase of the cycle, and in one of four studied during the mid-follicular phase of the cycle, an LH

542

ROBERT W. SHAW

surge was induced commencing some 12 hours following progesterone administration. However, all three subjects studied on day 12 of the cycle demonstrated acute surges of LH, with a less apparent accompanying FSH surge. The surges commenced between 8 and 12 hours following the progesterone (Shaw, 1975a). Leyendecker et al (1976) published some results on the experimental study of positive feedback effect of progesterone and these workers were likewise unable to induce a surge of gonadotrophins in the early or mid-follicular phase of the cycle in normal females, but could do so in two women on days 12 and 13 of the cycle. These surges commenced some six hours following 30 mg injections of progesterone. However, they found that if they pre-treated their mid-follicular phase study subjects with an injection of 3 mg oestradiol benzoate 24 hours before the progesterone injection, they could then also induce a gonadotrophin surge during this phase of the cycle (Figure 4).

3 mg E 2- Benzoate

mlU/ml

I i.m.

I 3°rag P,oge, ,oo

60 50

-

4030-

20-

LH

100--

IIIII 6 12

I 1111 18 24 6

Ill Ifll 12 18 2/,

FSH I II1 6 12 1800

Figure 4. Serum concentrations of LH and FSH during the mid-follicular phase of the menstrual cycle in three women following injections of 3 mg of oestradiol benzoate and 30 mg of progesterone. From Leyendecker et al (1976) with kind permission of the authors and the editor

of Archiv fiir Gynakologie.

These data suggest that progesterone may play an additional role in the regulation of the LH mid-cycle surge. However, progesterone can only induce its positive feedback action on an oestrogen primed pituitary. Whether this progesterone positive feedback effect is due to an action directly at a pituitary level or via the hypothalamus and altered endogenous GnRH release has still not definitely been determined. From Docke and Dorner (1965, 1969), on the basis of hypothalamic lesioning and steroid implant experiments, an action in the anterior hypothalamus, at least in the rat, has been established. The relevance of these observations in terms of the mechanisms governing the initiation, regulation and termination of the spontaneous mid-cycle LH peak is questionable. Studies utilizing administration of an anti-progesterone antiserum failed to block ovulation in the rat (Ferin et al, 1969). It would appear, therefore, that elevated levels of progesterone in the peripheral

NEUROENDOCRINOLOGY OF THE MENSTRUALCYCLE

543

circulation of intact animals have an inhibitory rather than stimulatory effect on the initiation of the pre-ovulatory gonadotrophin discharge. However, this does not exclude an action of rising levels of progesterone having a facilitatory effect on the further development of the LH peak. THE GONADOTROPHIN-RELEASING HORMONE

The decapeptide LHRH was first isolated from porcine hypothalamii by Matsuo et al (1971b) and Schally et al (1971). The early work on FSH and LH releasing activity was carried out using crude extracts which almost certainly contained some LH, FSH and vasopressin. At first it was thought that the complex changes in FSH and LH seen throughout the menstrual cycle could only be explained by the presence of two separate hypothalamic releasing hormones (McCann and Ramirez, 1964; Schally et al, 1968; Harris and Naftolin, 1970). However, when porcine LHRH was obtained in a highly purified state it was found to release both LH and FSH in humans (Kastin et al, 1969). Similarly purified preparations of LHRH and FSHRH of human origin showed that LHRH and FSHRH activity could not be separated (Kastin et al, 1971). Thus the hypothesis that there was a separate LHRH and FSHRH was challenged. The question of whether the FSH-releasing activity was due to an intrinsic property of the whole molecule of LHRH or to contamination with FSHRH in its preparation was not fully solved until the structure and synthesis of porcine LHRH was determined. This synthetic preparation is able to release both LH and FSH in vitro and in vivo and these findings have led to the hypothesis of one releasing factor (termed either LHRH or GnRH) controlling the secretion of both pituitary gonadotrophins with a change in sex steroid environment modulating the proportion of LH and FSH released (Schally, Arimura and Kastin, 1971). Effect of GnRH administration in the human In early 1972 the first clinical trial utilizing synthetic GnRH in the human was published (Kastin et al, 1972) and the synthetic material was shown capable of releasing both LH and FSH, though FSH was released to a less marked degree. The variation in response between individuals to the same dose of GnRH could vary as much as eight- to ten-fold. The synthetic material was found to be equally as effective as the natural porcine GnRH (Arimura et al, 1973) and is effective when given subcutaneously, by a single intravenous bolus, via continuous intravenous infusion (GonzalezBarcena et al, 1973) or intranasally (London et al, 1973). Following intravenous administration, a significant rise in serum LH will be seen within five minutes, and sometimes of FSH, reaching a peak within about 30 minutes, but FSH peaks are often delayed further. LH release has a linear log-dose relationship (Haug and Torjesen, 1973) up to doses of 250/~g, but no such relationship can be found for FSH. In the female the magnitude of gonadotrophin release in individuals varies with the stage of the menstrual cycle, being greatest in the pre-ovulatory phase, less marked in the luteal phase and least in the follicular phase of any individual cycle (Yen et al, 1972b; Shaw et al, 1974). This variation is most

544

ROBERT W. SHAW

apparent when LH changes are studied following G n R H administration (Figure 5). This cyclical variation in response would appear to be related to changes in endogenous oestrogen and progesterone levels and this is supported by the data in Figure 6. In this figure we have plotted the LH responses to 100 Hg G n R H as the sum of increments at 20, 30 and 60 minutes following injection against serum oestradiol-17fl levels. A linear relationship was found for LH but no such relationship could be found for FSH. Thus in any individual subject, LH release appeared to be related to circulating oestradiol levels suggestive of a positive feedback action of oestradiol on releasable LH. o/i Doy 4

30-

Doy9

20

18

0 I

0

I

I

20 38

I

I

60

0

I

I

I

20 30

60 rains

Day H

50.

Doy22 --

~"

40

~

T

....

:LH

FSH

30

20

18

0 I

0

20 38 mins

Figure 5. LH and FSH release following 100/~g G n R H i.v. at different phases of the same menstrual cycle in 6 normal females (mean + 1 s.d.).

Effect of oestrogen pre-treatment on subsequent response to GnRH

Experimental manipulation of steroid hormone levels in blood or the administration of a potent anti-oestrogen (ICI 46474) had demonstrated that the rise in plasma oestradiol which preceeds the LH surge is essential for the increased responsiveness of the anterior pituitary gland in the pre-oestrous rat (Aiyer and Fink, 1974). Work with anoestrus ewes has also shown an augmenting effect of oestradiol at certain dose levels; and Reeves, Arimura

545

NEUROENDOCRINOLOGY OF THE MENSTRUAL CYCLE

and Schally (1971) have suggested that part of the stimulatory effects of oestrogens may be due to an increased pituitary responsiveness to GnRH. From these and other reports it became apparent that the dose of oestradiol given and the time of subsequent re-testing with GnRH are important determinants of the subsequent responses. In the amenorrhoeic human female conflicting evidence on the effects of oestrogens on GnRH response have been reported. Nillius and Wide (1972b) showed an enhanced LH response to 100 ~g GnRH i.v. in one woman with amenorrhoea who had received 100 gg ethinyl oestradiol daily for eight days at the time of the repeat test. Thompson, Arfania and Taymor (1973) treated

ly/ 150

= ,ooI "~

14

I 250

II 500

I 750

z 1000

i 1250

E2 pmol/I Figure 6. Association between LH response to GnRH and circulating levels of oestradiol-i 7/'3 in individuals tested during the same menstrual cycle.

three amenorrhoeic women with either 25 or 50/~g per kg body weight of oestradiol benzoate i.m. and then assessed the effect of 150/~g GnRH given 20 hours later. All three patients showed a marked suppression in LH response whereas FSH responses remained unchanged. Our own data have shown that in amenorrhoeic subjects the effect of oestrogen on subsequent GnRH tests is dependent upon positive feedback to oestrogen being intact. Only those subjects with intact positive feedback will demonstrate an augmented LH and FSH response to GnRH 48 hours following oestrogen administration (Shaw, 1976). In the normally menstruating female oestrogen pre-treatment produced a biphasic effect upon pituitary sensitivity to GnRH administration during the early follicular phase of the cycle (Figure 7). In these four women a GnRH test was performed immediately before and 4, 20 and 44 hours following 1 mg oestradiol benzoate i.m. The oestrogen pre-treatment produced suppression of LH release at 4 and 20 hours compared with the pre-treatment

546

ROBERT W. SHAW

response, but an augmented response is seen by 44 hours. FSH release showed a similar biphasic pattern of response. These results suggested an activation delay of this oestrogen priming effect. In a larger study of 18 normal females studied during the early or midfollicular phase of the cycle and given either 0.5 mg, 1 mg or 2.5 mg of oestradiol benzoate i.m., Shaw, Butt and London (1975b) demonstrated an augmentation of LH response in 15 of 18 and of FSH release in 14 of 18 of the

u/I 70

f r b

2'o

4'4

Time hrs alter [B inj

Figure 7. Biphasic effect of 1 mg oestradiol benzoate upon LH response following 100#g GnRH during the early follicular phase of the cycle.

subjects when re-tested 44 hours after the oestrogen administration. Apart from the amplifying responses to GnRH, oestrogen pre-treatment delayed the timing of the LH peak from 20 to 30 minutes in the control response to 60 or 90 minutes in the post-treatment response in those receiving the 2.5 mg oestradiol benzoate dose. This effect was only noticed in two of the 12 subjects who received either 0.5 mg or 1 mg oestradiol benzoate and may thus be dose related. If the ratio of LH:FSH release was studied it was found that there was a preferential release of LH following oestrogen priming (Shaw, Butt and London, 1975b). Overall there was found to be a direct correlation between sum of LH increments and circulating levels of oestradiol in the above studies (sum A LH = 7.30 + 0.08 E2, r ---- 0.61, P<0.001). This correlation also held for sum of FSH increments (sum A FSH ---- 2.20 d- 0.03 E2, r = 0.67, P<0.001). Other workers support the above data of an initial suppressive action of oestrogen on pituitary responsiveness to G n R H (Keye and Jaffe, 1974) followed by a later augmenting action (Jaffe and Keye, 1974) which is both concentration and duration dependent (Young and Jaffe, 1976). Oestrogen priming then has a biphasic effect with an initial and almost immediate suppressive effect (negative feedback) which may well occur

NEUROENDOCRINOLOGY OF THE MENSTRUAL CYCLE

547

directly at a pituitary level and prevents the self priming and activation effects of GnRH between the two pools of gonadotrophins. There is a gradual removal of this negative feedback effect with partial return to normal by 20 hours and full augmentation (or amplification) by at least 44 hours. P R I M I N G EFFECT OF GnRH

It has been shown that a single intravenous bolus of GnRH is highly effective in releasing both LH and FSH in normal female subjects, but the amounts released depend upon the dose of GnRH administered and upon the stage of the menstrual cycle. Results from in vitro experiments with pituitary cells in culture indicate that GnRH is involved not only in release of stored LH and FSH, but is of importance in maintaining synthesis of the gonadotrophins within the gonadotrophic cells (Steinberger, Chowdhury and Steinberger, 1973). Hence repeated exposure of the gonadotrophin producing cells to GnRH seems essential for the maintenance of adequate pituitary stores. When subjects are tested with repeat 100 ~g GnRH tests 4, 20 and 44 hours following an initial test during the early follicular phase of the cycle, a far greater response is observed in the first repeat test at 4 hours whilst the control, 20 and 44 hour tests show almost identical responses in terms of LH release (Figure 8) and FSH release.

.60 5o =: 4 0 ,,.,,.i

•

•

c

"~ 3 0 E

~, 20 10 o Time

hrs

Figure 8. Self-priming effect of repeat injections of 100 ~g GnRH during the early follicular phase of the cycle.

Rommler and Hammerstein (1974) in a more extensive study of this priming effect of GnRH in normal women showed that it was present if the time interval was between one and four hours, with the maximal effect being observed at two hours, and studies using continuous infusions of small doses

548

ROBERT W. SHAW

of GnRH (0.2/~g/min) have shown similar findings (Hoff et al, 1977). This action has been termed the self-priming effect of GnRH. Wang and co-workers (1976) published more intensive studies throughout the menstrual cycle and demonstrated a definite cycle-related effect of 'selfpriming' to GnRH which was most apparent in the late follicular phase and at the time of the spontaneous gonadotrophin surge. The effect was more marked in terms of LH release than of FSH. In vitro studies (Fink and Picketing, 1975) support the conclusion of in vivo studies that the priming effect of GnRH is due to a direct action of this hormone on the gonadotrophins. The priming effect of GnRH may also be of clinical importance. Thus the responsiveness of the pituitary gland to GnRH is enhanced in patients with hypergonadotrophic hypogonadism (Roth et al, 1972; Siler and Yen, 1973). Further, in patients with amenorrhoea (Nillius and Wide, 1972b) and anorexia nervosa (Palmer et al, 1975), a significant positive correlation was found between basal plasma gonadotrophin estimation and the increments which followed GnRH administration. In 1972, Roth et al stated that 'the degree of prior exposure of the gonadotropes to endogenous LRF appears to affect both the magnitude and the nature of the FSH and LH response to an acute dose of synthetic LRF'. This self-priming effect of GnRH is of extreme importance in understanding the physiological control mechanisms of gonadotrophin release. It also suggests that there are two pools of gonadotrophins, one readily releasable by initial exposure to GnRH, and a second reserve pool which is less readily released (Wang et al, 1976; Hoff et al, 1977). The exposure of this larger reserve pool to GnRH allows it to be more readily released by a subsequent exposure to GnRH and is suggestive of a transfer of gonadotrophins from one pool to the other. The stage of the cycle -- i.e. the endogenous sex steroid environment prevailing -- greatly influences this transfer and sensitivity of the pituitary to GnRH. Is there a surge of GnRH at mid.cycle?

The measurement of GnRH in peripheral plasma might greatly help the understanding of the feedback mechanisms concerned in controlling gonadotrophin release. Radioimmunoassays for GnRH have been reported (Jeffcoate et al, 1973; Nett et al, 1973). Experimental data have given conflicting results as to the occurrence of the hypothetical surge of GnRH during the pre-ovulatory gonadotrophin surge. Jutisz and Kerdelhue (1973) and Crighton et al (1973) reported elevated GnRH levels during the LH surge of the sheep. In the human female, Malacara, Seyler and Reichlin (1972) claimed that they were able to detect elevations in bioassayable GnRH in 6 of 36 women during the mid-cycle; Keye et al (1973) and Arimura et al (1974) have also reported peripheral estimations of GnRH in normal females with values between 2 and 60 pg/ml. Other workers have been unable to confirm these findings (Jonas et al, 1974; Kelch et al, 1975). The difficulties in obtaining specific antisera to GnRH which do not crossreact with peptides other than the decapeptide and in obtaining sensitive

NEUROENDOCRINOLOGY OF THE MENSTRUALCYCLE

549

enough assays to measure what would seem to be extremely low peripheral plasma levels of GnRH, make it difficult to exclude or confirm a surge of G n R H on the basis of the data to date. Nett, Akbar and Niswender (1974) report that substances in the hypophyseal portal vessel blood are diluted 500fold by the time they reach the external jugular vein. Experimental techniques of collecting hypophyseal portal blood - - such as that described by Zimmerman et al (1973) in the monkey - - may however be utilized to study G n R H levels in stalk blood during menstrual cycles, at least in experimental animals, and may provide the answer to the question of the physiological occurrence of G n R H surges.

Effect of progesterone upon pituitary response to GnRH Some increase in progesterone levels has been shown to occur on the day of ovulation in humans and it has been suggested that this hormone could play a role in inducing or modifying the mid-cycle LH surge (Leyendecker, Wardlaw and Nocke, 1972; Leyendecker and Nocke, 1973). Some workers experimenting in rats and anoestrus ewes have shown no change in response to G n R H after progesterone (Debeljuk, Arimura and Schally, 1972; Aiyer, Chiappa and Fink, 1974), whereas others using rats, rabbits and anoestrus ewes have shown suppressed responses (Hilliard, Schally and Sawyer, 1971; Arimura and Schally, i970; Plant and Ward, 1973). In the human, Thompson, Arfania and Taymor (1973) gave three amenorrhoeic females progesterone intramuscularly in doses of 3 to 5 m g / k g and performed a repeat G n R H test 20 hours later when circulating levels of progesterone were between 32 and 52 nmol/1 (9-15 ng/ml) and showed suppressed LH and FSH release. The doses used and steroid levels achieved in these studies were however more in keeping with those of the mid-luteal phase of the cycle. From our own studies on progesterone effect on G n R H response in normal females some interesting points arise. Groups of women were studied during the early follicular phase (days four to six) or mid-follicular phase (days eight to ten) of their cycles. Following a control response to 100 ~g G n R H they were given either a 12.5 mg or 25 mg injection of progesterone i.m. and re-tested 20 hours or 44 hours later. Circulating levels of progesterone at the time of the control test were 3 to 6 nmol/1 (0.9 to 1.8 ng/ml), 20 hours following progesterone 8 to 13 nmol/1 (2.5 to 3.5 ng/ml) and at 44 hours 4 to 7 nmol/1 (1.3 to 2 ng/ml). This progesterone pre-treatment induced quite marked alterations in LH and FSH release in response to G n R H stimulation. The increase in LH release after G n R H was dependent upon the stage in the menstrual cycle at which the tests were performed as well as the time of re-testing following progesterone administration. Patients in the mid-follicular phase showed greater increases than those studied in the early follicular phase (P<0.001) (Figure 9). The effect upon FSH release was less marked and only subjects in the mid-follicular group showed any significant increase in FSH output (P<0.01) (Shaw, Butt and London, 1975c). These differences in release of LH and FSH after progesterone pre-

550

ROBERT W. SHAW

treatment could not be explained by differences in basal LH, FSH or progesterone levels between the two groups, since these were similar in both pre- and post-progesterone responses. However, serum oestradiol-17/3 levels were significantly greater in the midfollicular group but did not significantly increase following progesterone administration. These higher levels of oestradiol presumably had had an oestrogen priming action on the pituitary gonadotropes making them more susceptible to a progesterone action than in the early follicular phase when little oestrogen priming had occurred.

LH Increments u/I 3O0

Sum

C

Control r e ~ - ~ l ~

T

After Progesterone

250 200 15C 100 50 ~ 0

C

Day4-6

, T

C

Day8-10

Figure 9. Effect of progesterone pre-treatment (12.5 nag) upon LH response to 100/ag GnRH.

In the groups of subjects tested 44 hours after progesterone administration when circulating progesterone levels had returned to pre-injection levels, no change in LH or FSH release after GnRH was induced, despite the pituitary having been previously exposed to elevated concentrations of progesterone. Thus the progesterone stimulus was only effective during the time of high circulating concentrations (Shaw, Butt and London, 1975c). These data have shown that small changes in serum progesterone can modify pituitary responses to GnRH in the human and that the degree of enhancement of response is dependent upon the duration of time of pretreatment, the stage of the cycle and the presence of an oestrogen primed pituitary gonadotrope. CONCLUSIONS Let us then look at these complex interlocked changes in ovarian steroids and pituitary gonadotrophins that occur throughout the menstrual cycle in relation to the data presented above.

NEUROENDOCRINOLOGYOF THE MENSTRUALCYCLE

551

The gonadotrophins, LH and FSH, are released from the pituitary in an episodic, pulsatile manner, with peaks every two to three hours. Several lines of evidence now lend support to the suggestion of a hypothalamic mechanism for this pulsatile gonadotrophin release: antisera to GnRH abolish the pulsatile release of LH and FSH and pulsatile LH release can only be observed with pulsed delivery of GnRH and not by constant infusion. These data suggest that the pulsatile release of the gonadotrophins is directed primarily by the episodic secretion of GnRH from the hypothalamus. The ovarian steroids oestradiol-17fl and progesterone can each induce a positive feedback release of gonadotrophins, in many ways similar to that seen at mid-cycle, but progesterone can only induce its effect on a previously oestrogen primed pituitary gland. There are also marked differences between oestrogen and progesterone in the activation delay of this positive feedback stimulus. There is presumptive evidence that these positive feedback stimuli of the ovarian steroids involve both a direct pituitary action with alteration in synthesis of gonadotrophins and sensitivity to GnRH preceeding an induced increase in hypothalamic release of GnRH. Studies on the effect of synthetic GnRH upon gonadotrophin release and the effect of altering the steroid environment and its resultant effect upon GnRH responses have also provided information into the control of the menstrual cycle. The pattern of gonadotrophin release from the pituitary in response to repeated pulses of submaximal doses of GnRH or constant infusions over several hours suggests the presence of two functionally related pools of gonadotrophins. The first 'primary pool' is immediately releasable whilst the secondary pool requires a continued stimulus input and also represents the effect of GnRH on synthesis and storage of gonadotrophins within the pituitary cell itself. The size or activity of these two pools of gonadotrophins represent pituitary sensitivity and reserve respectively which varies throughout the cycle and is found to be regulated by the feedback action of ovarian steroids and by the self, priming action of GnRH itselL Oestradiol preferentially induces the augmentation of reserve and impedes sensitivity to GnRH with a differential effect apparent for LH release. This effect of oestradiol is both dose and time related. With these effects in mind let us then try to explain the release of gonadotrophins observed throughout the normal menstrual cycle.

Follicular phase In the early follicular phase the immediately releasable pool and reserve pool of gonadotrophins are at a minimum. The increased release of FSH seen at this time, responsible for initiating follicle development, must indicate an increased output of endogenous GnRH with the removal of negative feedback action because of low levels of oestrogen and progesterone. As oestrogen levels rise, secreted from the developing follicle, negative feedback action of oestradiol reduces GnRH output. With these progressive increasing levels of oestradiol to the mid-follicular phase the quantitative estimates of the primary, immediately releasable, pool of gonadotrophins show a slight increase, but there is a far greater increase in the reserve pool. This

552

ROBERT W. SHAW

demonstrates the augmentation action of oestradiol primarily upon pituitary reserve. Since there is no massive out-pouring of gonadotrophins at this time, secretion of endogenous G n R H must be minimal, but this might also be evidence for the impedence of G n R H sensitivity by oestrogen.

Proposed mechanisms for the pre-ovulatory LH surge It is suggested from the data reviewed that during the late follicular phase of the cycle there is an increase in the amount of oestradiol secreted by the ovary. Under the influence of elevated plasma oestradiol concentrations the sensitivity of the gonadotropes to GnRH eventually reaches a point when G n R H can exert its full priming effect. The development of the self priming effect of G n R H at mid-cycle has the effect of transferring gonadotrophins from the secondary reserve pool to the primary releasable pool. The increased pituitary responsiveness to G n R H may be further enhanced by progesterone acting on a fully oestrogen primed gland. These changes result in the massive release of LH - - the LH ovulatory surge. It is possible that these events could occur even if the gonadotropes were exposed to a constant level of GnRH. However, a surge or increased secretion of GnRH, which would act synergistically with the changes in pituitary sensitivity, cannot be excluded and seems likely. The cellular mechanisms which underlie these changes in pituitary sensitivity to G n R H are unknown. However, it seems that the facilitatory effect of oestradiol requires that the pituitary be exposed to increased plasma oestradiol concentrations for many hours (Aiyer and Fink, 1974; Cooper, Fawcett and McCann, 1974; Knobil, 1974). The priming effect of G n R H (Fink and Pickering, 1975) and possibly oestradiol (Schneider and McCann, 1970) seem likely to depend for their action on de novo protein synthesis. Luteal phase A progressive decrease in sensitivity and reserve characterizes pituitary function from the mid-luteal to late luteal phase and into the early follicular phase of the next cycle. This is probably due to a progressive decline in oestrogen and progesterone on which sensitivity and reserve are dependent. It is therefore apparent that the functional state of the pituitary gonadotrope as a target cell is ultimately determined by the modulating effect of ovarian steroid hormones via their influence on sensitivity and reserve and the hypophysiotrophic effect of G n R H itself. Whilst our knowledge of the various feedback effects of ovarian steroids in the control of gonadotrophin release has greatly advanced in this last decade, particularly since the availability of synthetic GnRH, a true understanding of their exact mode of action at a pituitary and hypothalamic level must await an accurate measurement of G n R H throughout the complex changes of the menstrual cycle. This has yet to be achieved, but when it can be, not only will we be able to understand the mechanisms controlling the normal menstrual cycle, but many other perplexing anomalies in patients with disorders of menstruation may be explained.

NEUROENDOCRINOLOGY OF THE MENSTRUAL CYCLE

553

ACKNOWLEDGEMENTS My thanks to Mrs J. Pickering for the preparation of this manuscript. The original work of the author and his colleagues was supported by a grant from the Medical Research Council. In addition to the authors quoted in the text, I am indebted to the Editors of Archiv fiir Gyni~kologie for permission to reprint Figure 4.

REFERENCES Abraham, (3. E. & Klaiber, E. L. (1971) Plasma immunoreactive estrogens and LH during the menstrual cycle. American Journal of Obstetrics and Gynecology, 108, 528-531. Abraham, G. E., Odell, W. D., Swerdloff, R. S. & Hopper, K. (1972) Simultaneous radioimmunoassay of plasma FSH, LH, progesterone, 17-hydroxyprogesterone and estradiol17/3 during the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 34, 312-318. Aiyer, M. S. & Fink, G. (1974) The role of sex steroid hormones in modulating the responsiveness of the anterior pituitary gland to luteinizing hormone releasing factor in the female rat. Journal of Endocrinology, 62, 553-572. Aiyer, M. S., Chiappa, S. A. & Fink, G. (1974) Effect of ovarian steroids on pituitary sensitivity to luteinizing hormone releasing factor in the rat. Journal of Endocrinology, 61, xii. Aona, T., Miyake, A., Shiosi, T., Kinugasa, T., Onishi, T. & Kurachi, K. (1976) Impaired LH release following endogenous estrogen administration in patients with amenorrheagalactorrhea syndrome. Journal of Clinical Endocrinology and Metabolism, 42, 696-702. Arimura, A. & Schally, A. V. (1970) Progesterone suppression of LH-releasing hormoneinduced stimulation of LH release in rats. Endocrinology. 87,653-657. Arimura, A., Saito, M., Yaoi, Y., Kumasaka, T., Sato, H., Koyama, T., Nishi, N., Kastin, A. J. & Schally, A. V. (1973) Comparison of the effects of subcutaneous and intravenous injection of synthetic LH-releasing hormone (LH-RH) on serum LH and FSH levels in men. Journal of Clinical Endocrinology and Metabolism, 36, 385-388. Arimura, A., Kastin, A. J., Schally, A. V., Saito, M., Kumasaka, T., Yaoi, Y., Nishi, N. & Ohkura, K. (1974) Immunoreactive LH-releasing hormone in plasma: mid-cycle elevation in women. Journal of Clinical Endocrinology and Metabolism, 38~ 510-513. Baker, B. L., Eskin, T. A. & August, L. N. (1973) Direct action of synthetic progestins on the hypophysis. Endocrinology, 92,965-972. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. & Guillemin, R. (1973) Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science, 179, 77-79. Burgus, R., Dunn, T. F., Desiderio, D. & GuiUemin, R. (1969) Structure moleculaire du facteur hypothalamique hypophysiotrope TRF d'origine ovine: mise en evidence par spectrometric de masse de la sequence. Compte Rendu des Seance de l'Academie des Sciences (D), 269, 1870-1873. Cargille, C. M., Vaitukaitis, J. L., Bermudez, J. A. & Ross, G. T. (1973) A differential effect of ethinyl oestradiol upon plasma FSH and LH relating to time. Journal of Clinical Endocrinology and Metabolism, 36, 87-94. Carmel, P. W., Araki, S. & Ferrin, M. (1976) Pituitary stalk portal blood collection in Rhesus monkeys: evidence for pulsatile release of gonadotrophin hormone (GnRH). Endocrinology, 99,243-248. Chiappa, S. A., Sherwood, N. M. & Fink, G. (1975) Effect of sex steroid hormones on the release of LH-RF into rat pituitary stalk blood by electrical stimulation of the medial preoptic area. Journal of Endocrinology, 67, 37p-38p. Cooper, K. J., Fawcett, C. P. & McCann, S. M. (1974) Inhibitory and facilitatory effects of estradiol-17fl on pituitary responsiveness to a luteinizing-follicle stimulating hormone releasing factor (LH-RF/FSH-RF) preparation in the ovariectomised rat. Proceedings of the Societyfor Experimental Biology and Medicine, 145, 1422-1426.

554

ROBERT w . SHAW

Crighton, D. B., Foster, J. P., Holland, D. T. & Jeffcoate, S. L. (1973) Simultaneous determination of luteinizing hormone and luteinizing hormone releasing hormone in the jugular venous blood of the sheep at oestrus. Journal of Endocrinology, 59,373-374. Crowe, S. J., Cushing, H. & Homans, J. (1910) Experimental hypophysectomy. Johns Hopkins Hospital Bulletin. 21,127. Davidson, J. M., Damassa, D. A., Smith, E. R. & Cheung, C. (1976) Feedback control of gonadotrophin secretion in the rat. In Regulatory Mechanism of Male Reproductive Physiology (Ed.) Spilman, C. H. et al. pp. 151-168. Amsterdam: Excerpta Medica. Debeljuk, L., Arimura, A. & Schally, A. V. (1972) Effect of estradiol and progesterone on the LH release induced by LH-releasing hormone (LH-RH) in intact diestrous rats and anestrous ewes. Proceedings of the Society for Experimental Biology and Medicine, 139, 774-777. Dierschke, D. J., Yamaji, T., Karsch, F. J., Weick, R. F., Weiss, G. & Knobil, E. (1973) Blockade by progesterone of estrogen-induced LH and FSH release in the Rhesus monkey. Endocrinology, 92, 1496-1501. Docke, F. & Dorner, G. (1965) The mechanism of the induction of ovulation by oestrogens. Journal of Endocrinology, 33,491-499. Docke, F. & Dorner, G. (1969) A possible mechanism by which progesterone facilitates ovulation in the rat. Neuroendocrinology, 4, 139-149. Ehara, Y., Siler, T., Vanden Berg, G., Sinha, Y. N. & Yen, S. S. C. (1973) Circulating prolactin levels during the menstrual cycle: episodic release and diurnal variation. American Journal of Obstetrics and Gynecology. 117,962. Eskay, R. L., Mical, R. S. & Porter, J. C. (1977) Relationship between luteinizing hormone releasing hormone concentration in hypophyseal portal blood and luteinizing hormone release in intact, castrated and electrochemically-stimulated rats. Endocrinology. 100, 263-270. Everett, J. W. (1964) Central neural control of reproductive functions of the adenohypophysis. PhysiologicalReview, 44~ 373-431. Everett, J. W. & Sawyer, C. H. (1949) A neural timing factor in the mechanism by which progesterone advances ovulation in the cyclic rat. Endocrinology, 45,581-595. Ferin, M., Tempone, A., Zimmering, P. E. & Vande-Wiele, R. L. (1969) Effect of antibodies to 17/~-estradiol and progesterone on the estrous cycle of the rat. Endocrinology, 85, 1070-1078. Fink, G. & Pickering, A. (1975) Dependence of the priming effect of luteinizing hormone releasing factor (LRF) on RNA and protein synthesis. Journal of Physiology. London, 252, 73p-TSp. Friesen, H. & Hwang, P. (1973) Human prolactin. Annual Review of Medicine, 24, 251-270. Glass, M. R., Shaw, R. W., Butt, W. R., Logan-Edwards, R. & London, D. R. (1975) An abnormality of oestrogen feedback in amenorrhoea-galactorrhoea. British Medical Journal ill, 274-275. Goding, J. R., Catt, K. J., Brown, J. M., Kaltenbach, C. E., Cumming, I. A. & Mole, B. J. (1969) Radioimmunoassay for ovine luteinizing hormone. Secretion of luteinizing hormone during estrus and following estrogen administration in the sheep. Endocrinology, 85, 133-142. Gonzalez-Barcena, D., Kastin, A. J., Schalch, D. S., Bermidez, J. A., Lee, D., Arimura, A., Ruelas, J., Zepeda, I. & Schally, A. V. (1973) Synthetic LH-releasing hormone (LH-RH) administered to normal men by different routes. Journal of Clinical Endocrinology and Metabolism, 37,481-484. Harrington, F. E. & Bex, F. J. (1970) Ovulation in the rat as a result of synergism between follicle stimulating hormone and luteinizing hormone. Endocrinologica Japon&a, 17, 387. Harris, G. W. (1937) The induction of ovulation in the rabbit by electrical stimulation of the hypothalamo-hypophyseal mechanism. Proceedings of the Royal Society, 122, 374-394 (Series B). Harris, G. W. & Naftolin, F. (1970) The hypothalamus and control of ovulation. British Medical Bulletin, 26, 3-9. Haug, E. & Torjesen, P. (1973) Effect of synthetic luteinizing hormone and follicle stimulating hormone-releasing hormone (LH/FSH-RH) on serum levels of LH, FSH, thyrotrophin (TSH) and growth hormone (HGH) in normal male subjects. Acta Endocrinologica, 73, 465-474.

NEUROENDOCR1NOLOGY OF THE MENSTRUAL CYCLE

55S

Hilliard, J., Schally, A. V. & Sawyer, C. H. (1971) Progesterone blockade of the ovulatory response to intra pituitary infusion of LHRH in rabbits. Endocrinology. 88~ 730-736. Hoff, J. D., Lasley, B. L., Wang, C. F. & Yen, S. S. C. (1977) The two pools of pituitary gonadotrophin: regulation during the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 44,302-312. Hohlweg, W. & Junkmann, K. (1932) Die hormonal-nervose regulierung der funktion des hypophysen vorderlappens. Klinische Wochenschrift, 1~ 321-323. Igarashi, M. & McCann, S. M. (1964) A hypothalamic follicle stimulating hormone-releasing factor. Endocrinology, 74~ 446-452. Jaffe, R. B. & Keye, W. R. (1974) Estradiol augmentation of pituitary responsiveness to gonadotrophin-releasing hormone in women. Journal of Clinical Endocrinology and Metabolism, 39,850-855. Jeffcoate, S. L., Frazer, H. M., Gunn, A. & Holland, D. T. (1973) Radioimmunoassay of luteinizing hormone-releasing hormone (LH-RH) in blood and urine. Journal of Endocrinology, 59~ XII. Johansson, E. D. B. & Wide, L. (1969) Periovulatory levels of plasma progesterone and luteinizing hormone in women. Acta Endocrinologica, 62, 82-88. Jonas, A., Findlay, J. K., Goding, J. R., Burger, H. G. & de Kretser, D. M. (1974) Radioimmunoassay for gonadotrophin-releasing hormone (Gn-RH): its application to the measurement of Gn-RH in ovine plasma. Journal of Reproduction and Fertility, 36, 446. Jutisz, M. & Kerdelhue, B. (1973) In vitro Studies on Synthetic LHRH and its Assay using a Radioimmunological Method (Ed.) Gual, C. & Rosenberg, E. pp. 98-104. Amsterdam: Excerpta Medica. Kalra, P. S., Kalra, S. P., Krulich, L., Fawcett, C. P. & McCann, S. M. (1972) Involvement of norepinephrine in transmission of the stimulatory influence of progesterone on gonadotrophin release. Endocrinology, 90, 1168-1176. Kamberi, I. A., Mical, R. S. & Porter, J. C. (1970) Effect of anterior pituitary perfusion and intraventricular injection of catecholamine and indolamines on LH release. Endocrinology, 87, 1-12. Kamberi, I. A., Schneider, H. D. G. & McCann, S. M. (1970) Action of dopamine to induce release of FSH-releasing factor (FRF) from hypothalamic tissue in vitro. Endocrinology, 86, 278-284. Karsch, F. J., Weick, R. F., Butler, W. R., Dierschke, D. J., Krey, L. C., Weiss, G., Hotchkiss, J., Yamaji, T. & Knobil, R. (1973) Induced LH surges in the Rhesus monkey: strength-duration characteristics of the estrogen stimulus. Endocrinology, 92, 1740-1747. Kastin, A. J., Schally, A. V., Gual, C. Midgley, A. R., Bowers, C. Y. & Diaz-Infante, A. (1969) Stimulation of LH release in men and women by LH-releasing hormone purified from porcine hypothalami. Journal of Clinical Endocrinology and Metabolism, 29, 1046-1050. Kastin, A. J., Schally, A. V., Gual, C., Midgley, A. R., Arimura, A., Miller, M. C. & Cabeza, A. (1971) Administration of LH-releasing hormone of human origin to man. Journal of Clinical Endocrinology and Metabolism, 32, 287-289. Kastin, J., Schally, A., Guik, C. & Arimura, A. (1972) Release of LH and FSH after administration of synthetic LH-releasing hormone. Journal of Clinical Endocrinology and Metabolism, 34, 753-756. Kelch, R. P., Clemens, L. E., Markovs, M., Westhoff, M. H. & Hawkins, D. W. (1975) Metabolism and effect of synthetic gonadotrophin releasing hormone (GnRH) in children and adults. Journal of Clinical Endocrinology and Metabolism, 38~ 805-810. Keye, W. R. & Jaffe, R. B. (1974) Modulation of pituitary gonadotrophin response to gonadotrophin-releasing hormone by estradiol. Journal of Clinical Endocrinology and Metabolism, 38,805-810. Keye, W. R., Kelch, R. P., Niswender, G. D. & Jaffe, R. B. (1973) Quantitation of endogenous and exogenous gonadotrophin releasing hormone by radioimmunoassay. Journal of Clinical Endocrinology and Metabolism, 36, 1263-1267. Knobil, E. (1974) On the control of gonadotropin secretion in the Rhesus monkey. Recent Progress in Hormone Research, 30, 1-36. Korenman, S. G. & Sherman, B. M. (1973) Further studies of gonadotropin and estradiol secretion during the preovulatory phase of the human menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 36~ 1205-1209.

556

ROBERT W. SHAW

Larsson-Cohn, U., Johansson, E. D. B., Wide, L. & Gemzell, C. (1972) Effects of continuous daily administration of 0.1 mg of norethindrone on the plasma levels of progesterone and on the urinary excretion of luteinizing hormone and total oestrogens. Acta Endocrinologiea, 71,551-556. Leyendecker, G. & Nocke, W. (1973) Further studies on the regulation of ovulation by ovarian steroids. ActaEndocrinologica, 177 (Supplement), 17. Leyendecker, G., Wardlaw, S. & Nocke, W. (1972) Experimental studies on the endocrine regulation during the pre-ovulatory phase of the human menstrual cycle. Acta Endocrinologica, 71,160-178. Leyendecker, G., Wildt, L., Gips, H., Nocke, W. & Plotz, E. J. (1976) Experimental studies on the positive feedback effect of progesterone, 17a-hydroxyprogesterone and 20 a-dihydroprogesterone on the pituitary release of LH and FSH in the human female. Archiv fiir Gyniikologie. 221, 29-45. London, D. R., Butt, W. R., Lynch, S. S., Marshall, J. C., Owusu, S., Robinson, W. R. & Stephenson, J. M. (1973) Hormonal responses to intranasal luteinizing hormone releasing hormone. Journal of Clinical Endocrinology and Metabolism. 37,829-831. Malaeara, J. M., Seyler, L. E. & Reichlin, S. (1972) Luteinizing hormone-releasing factor activity in peripheral blood from women during the mid-cycle luteinizing hormone ovulatory surge. Journal of Clinical Endocrinology and Metabolism. 34~ 271-278. Marie, P. (1886) Review of Medicine (Paris), 6, 297. Markee, J. E., Sawyer, C. H. & Hollinshead, W. H. (1948) Adrenergic control of the release of luteinizing hormone from the hypophysis of the rabbit. Recent Progress in Hormone Research. 2, 117-132. Marshall, F. H. A. (1936) Sexual periodicity and the causes which determine it. Philosophical Transaction, Royal Society of London, Series B, 226, 423. Matsuo, H., Arimura, A., Nair, R. M. G. & Schally, A. V. (1971a) Synthesis of the porcine LH and FSH releasing hormone by the solid phase method. Biochemical and Biophysical Research Communications, 45,822-827. Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. & Schally, A. V. (1971b) Structure of porcine LH and FSH releasing hormone: I. The proposed amino acid sequence. Biochemical and Biophysical Research Communications, 43~ 1334-1339. McCann, S. M. (1974) Regulation of the seeretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH). In Handbook of Physiology, Vol. IV (Ed.) Knobil, E. T. & Sawyer, W. H. pp. 489-517. Baltimore: Williams and Wilkins. McCann, S. M. & Ojeda, S. R. (1976) Synaptic transmitters involved in the release of hypothalamic releasing and inhibiting hormones. In Reviews of Neuroscience, Vol. 2 (Ed.) Ehrenpreis, S. & Kopin, I. J. pp. 91-110. New York: Raven Press. McCann, S. M. & Porter, J. C. (1969) Hypothalamie pituitary stimulating and inhibiting hormones. Physiological Reviews, 49, 240-284. MeCann, S. M. & Ramirez, V. D. (1964) The neuroendocrine regulation of hypophyseal luteinizing hormone seeretion. Recent Progess in Hormone Research, 20, 131-181. McCann, S. M., Taleisnik, S. & Friedman, H. M. (1960) LH-releasing activity in hypothalamie extracts. Proceedings of the Society for Experimental Biology and Medicine, 104, 432434. McNatty, K. P., Sawers, R..S. & McNeilly, A. S. (1974) A possible role for prolactin in control of steroid secretion by the human graafian follicle. Nature, 250, 653-655. McNeilly, A. S. & Chard, T. (1974) Circulating levels of prolactin during the menstrual cycle. ClinicalEndocrinology, 3, 105-112. Midgley, A. R. & Jaffe, R. B. (1968) Regulation of human gonadotrophins: IV. Correlation of serum concentrations of follicle-stimulating and luteinizing hormones during the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 28~ 1699. Midgley, A. R. & Jaffe, R. B. (1971) Regulation of human gonadotrophins: X. Episodic fluetuation of LH during the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 33, 962-969. Mishell, D., Nakamura, R. M., Crosignani, P. G., Stone, S., Kharma, K. & Nagaya, Y. (1971) Serum gonadotropin and steroid patterns during the normal menstrual cycle. American Journal of Obstetrics and Gynecology, 111, 60-65.

NEUROENDOCRINOLOGY OF THE MENSTRUAL CYCLE

557

Mittler, J. C. & Meites, J. (1964) In vitro stimulation of pituitary follicle-stimulating hormone release by hypothalamic extract. Proceedings of the Society for Experimental Biology and Medicine, 117, 309-313. Monroe, S., Jaffe, R. & Midgley, A. R. (1972) Regulation of human gonadotropins. XII. Increase in serum gonadotropins in response to oestradiol. Journal of Clinical Endocrinology and Metabolism, 34, 342-347. Moore, C. R. & Price, D. (1932) Gonad hormone functions and the reciprocal influence between gonads and hypophysis with its bearing on the problem of sex hormone antagonism. American Journal of Anatomy. 50, 13-72. Mortimer, C. H., Besser, G. M., Hook, J. & McNeilty, A. S. (1974) Intravenous, intramuscular, subcutaneous and intranasal administration of LH/FSH-RH: the duration of effect and occurrence of asynchronous pulsatile release of LH and FSH. Clinical Endocrinology, 3, 19-26. Nallar, R., Antunes-Rodrigues, J. & McCann, S. M. (1966) Effects of progesterone on the level of plasma luteinizing hormone (LH) in normal female rats. Endocrinology. 79, 907-911. Neill, J. D., Johansson, E. D. B., Datta, J. K. & Knobil, E. (1967) Relationship between the plasma levels of luteinizing hormone and progesterone during the normal menstrual cycle. Journal of Cl#ffcal Endocrinology and Metabolism. 27, 1167-1173. Neill, J. D., Dailey, R. A., Tsou, R. C. & Tindall, G. T. (1977) Secretion of luteinizing hormone releasing hormone (LHRH) in monkeys. In Hypothalamic Peptide Hormones and Pituitary Regulation (Ed.) Porter, J. C. pp. 203-224. New York: Plenum Press. Nett, T. M., Akbar, A. M. & Niswender, G. D. (1974) Serum levels of luteinizing hormone and gonadotrophin releasing hormone in cycling, castrated and anoestrous ewes. Endocrinology, 94, 713-718. Nett, T. M., Akbar, A. M., Niswender, G. D., Hedlund, M. T. & White, W. F. (1973) A radioimmunoassay for gonadotrophin-releasing hormone (GnRH) in serum. Journal of Clinical Endocrinology and Metabolism, 36, 880-885. Netter, A., Gorius, A., Thomas, K., Cohen, M. & Joubinaux, J. (1973) Blocage du pic d'ovulation de LH et FSH par la progesterone a faibles doses chiz la femme. Annales d'Endocrinologie, 34, 430-435. Nillius, S. & Wide, L. (1970) Effects of oestrogen on serum levels of LH and FSH. Acta Endocrinologica, 65, 583-594. Nillius, S. & Wide, L. (1971) Induction of a mid-cycle peak of LH in young women by exogenous oestradiol-17/3. Journal of Obstetrics and Gynaecology of the British Commonwealth, 78, 822-827. Nillius, S. & Wide, L. (1972a) Estradiol-17p (E2) induced peaks of luteinizing hormone (LH) in women: temporal relationship between E2 and LH in serum. Abstract of the International Congress of Endocrinology, June 18-24, 1972. Washington, D.C. Nillius, S. J. & Wide, L. (1972b) The LH-releasing hormone test in 31 women with secondary amenorrhoea. Journal of Obstetrics and Gynaecology of the British Commonwealth. 79, 874-882. Odell, W. D. & Swerdloff, R. S. (1968) Progestogen-induced luteinizing and folliclestimulating hormone surge in postmenopausal women: a simulated ovulatory peak. Proceedings of the National Academy of Sciences. 61,529-536. Palmer, R. L., Crisp, A. H., MacKinnon, P. C. B., Franklin, M., Bonnar, J. & Wheeler, M. (1975) Pituitary sensitivity to 50 /~g LH/FSH-RH in subjects with anorexia nervosa in acute and recovery stages. British Medical Journal. i, 179. Page, R. B., Munger, B. L. & Bergland, R. M. (1976) Scanning microscopy of pituitary vascular casts. American Journal of Anatomy, 146~ 273-285. Plant, H. C. & Ward, W. R. (1973) Effect of progesterone on the pituitary responsiveness to luteinizing hormone releasing hormone (LHRH) in intact anoestrous ewes. Journal of Physiology, 232, 45p. Plant, H. C. & Ward, W. R. (1974) Effect of I.V. infusion oestradiol-17fl with and without prior progesterone treatment on the plasma luteinizing hormone and follicle stimulating hormone concentration in anoestrous ewes. Journal of Endocrinology, 61, V-VI (Proceedings).

558

ROBERT W. SHAW

Reeves, J. J., Arimura, A. & Schally, A. V. (1971) Changes in pituitary responsiveness to luteinizing hormone-releasing hormone (LH-RH) in anoestrous ewes pre-treated with estradiol benzoate. Biology of Reproduction, 4, 88-92. Rommler, A. & Hammerstein, J. (1974) Time-dependent alterations in pituitary responsiveness caused by LH-RH stimulations in man. Acta Endocrinologica, 184 (Supplement), 21. Ross, G. T., Cargille, C. M., Lipsett, M. B., Rayford, P. L., Marshall, J. R., Strott, C. A. & Rodbard, D. (1970) Pituitary and gonadal hormones in women during spontaneous and induced ovulatory cycles. Recent Progress in Hormone Research, 26, 1-48. Roth, J. C., Kelch, R. P., Kaplan, S. L. & Grumbach, M. M. (1972) FSH and LH response to luteinizing hormone-releasing factor in pre-pubertal and pubertal children, adult males and patients with hypogonadotropic and hypergonadotropic hypogonadism. Journal of Clinical Endocrinology and Metabolism. 35,926-930. Sawyer, C. H., Markee, J. E. & Townsend, B. F. (1949) Cholinergic and adrenergic components in the neurohumoral control of the release of LH in the rabbit. Endocrinology. 44, 18-37. Schally, A. V., Arimura, A. & Kastin, A. (1971) Gonadotrophin releasing hormone - - one polypeptide regulates secretion of LH and FSH. Science, 173, 1036-1038. Schally, A. V., Arimura, A., Bowers, C. Y., Kastin, A. J., Sawano, S. & Redding, T. W. (1968) Hypothalamic neurohormones regulating anterior pituitary function. Recent Progress in Hormone Research, 24, 497-588. Schally, A. V., Arimura, A., Baba, Y., Nair, R. M. G., Matsuo, H., Redding, T. W., Debeljuk, L. & White, W. F. (1971) Isolation and properties of the FSH and LH-releasing hormone. Biochemical and Biophysical Research Communications, 43,393-399. Schneider, H. P. G. & McCann, S. M. (1969) Possible role of dopamine as transmitter to promote discharge of LH-releasing factor. Endocrinology. 85, 121-132. Schneider, H. P. G. & McCann, S. M. (1970) Release of LH-releasing factor (LRF) into the peripheral circulation of hypophysectomized rats by dopamine and its blockage by oestradiol. Endocrinology, 87, 249-253. Shaw, R. W. (1975a) A study of hypothalamic-pituitary-gonadal relationships in the female. MD Thesis, Birmingham University. Shaw, R. W. (1975b) Oestrogen modulation of gonadotrophin release. Proceedings of the Royal Society of Medicine, 68, 73-75. Shaw R. W. (1976) Effect of oestradiol-17fl on LH-RH responses in amenorrhoeic subjects. British Journal of Obstetrics and Gynaecology, 83, 564. Shaw, R. W., Butt, W. R. & London, D. R. (1974) The oestrogen provocation test - - a means of assessing hypothalamic-pituitary function in amenorrhoea. Journal of Endocrinology, 63, 46. Shaw, R. W., Butt, W. R. & London, D. R. (1975a) Pathological mechanism to explain some cases of amenorrhoea without organic disease. British Journal of Obstetrics and Gynaecology, 82, 337-340. Shaw, R. W., Butt, W. R. & London, D. R. (1975b) Effect of oestrogen pretreatment on subsequent response to luteinizing hormone-releasing hormone (LH-RH) in normal women. ClinicalEndocrinology, 4, 297-304. Shaw, R. W., Butt, W. R. & London, D. R. (1975c) The effect of progesterone on FSH and LH response to LHRH in normal women. ClinicalEndocrinology, 4, 543-550. Shaw, R. W., Butt, W. R., London, D. R. & Marshall, J. C. (1974) Variation in response to synthetic luteinizing hormone-releasing hormone (LH-RH) at different phases of the same menstrual cycle in normal women. Journal of Obstetrics and Gynaecology of the British Commonwealth, 81,632-639. Shaw, R. W., Butt, W. R., London, D. R. & Marshall, J. C. (1975) The oestrogen provocation test: A method of assessing the hypothalamic-pituitary axis in amenorrhoea. Clinical Endocrinology, 4, 267-276. Siler, T. M. & Yen, S. S. C. (1973) Augmented gonadotropin response to synthetic LRF in hypogonadal state. Journal of ClinicalEndocrinology and Metabolism, 37, 491-494. Smith, P. E. (1926) Hastening development of the female genital system by daily homoplastic pituitary transplants. Proceedings of the Society Experimental Biology and Medicine, 24, 131. Spies, H. G. & Niswender, G. D. (1972) Effect of progesterone and estradiol on LH release and ovulation in Rhesus monkeys. Endocrinology, 90, 257-261.

NEUROENDOCRINOLOGY OF THE MENSTRUAL CYCLE

559

Steinberger, A., Chowdhury, M. & Steinberger, E. (1973) Effect of repeated replenishment of hypothalamic extract on LH and FSH secretion in monolayer culture of rat ant. pituitary cells. Endocrinology, 92, 12-17. Stevens, K. R., Spies, H. G., Hilliard, J. & Sawyer, C. H. (1970) Site(s)of action of progesterone in blocking ovulation in the rat. Endocrinology, 86, 970-975. Thompson, I. E., Arfania, J. & Taymor, M. L. (1973) Effects of estrogen and progesterone on pituitary response to stimulation by luteinizing hormone-releasing factor. Journal of Clinical Endocrinology and Metabolism, 37, 152-155. Thompson, I. E., Karam, K. S. & Taymor, M. L. (1974) Positive feedback effects of estrogen in amenorrboeic women. American Journal of Obstetrics and Gynecology. 118, 788-792. Thorneycroft, I. H., Sribyatta, B., Tom, W. K., Nakamura, R. M. & Mishell, D. R. (1974) Measurement of serum LH, FSH, progesterone, 17-hydroxyprogesterone and estradiol17/3 levels at four hour intervals during the periovulatory phase of the menstrual cycle. Journal of Ctinical Endoerinology and Metabolism. 39, 754-758. Tsai, C. C. & Yen, S. S. (1971) The effect of ethinyl estradiol administration during early follicular phase of the cycle on the other gonadotropin levels and ovarian function. Journal of Clinical Endocrinology and Metabolism. 33,917-923. Vale, W., Brazeau, R., Rivier, C., Brown, M., Boss, B., Rivier, J., Burgus, R., Ling, N. & Guillemin, R. (1975) Somatostatin. Recent Progress in Hormone Research, 31,365-397. Vande-Wiele, R. L., Bogumil, J., Dyrenfurth, I., Ferin, M., Jewelewicz, R., Warren, M., Rizkallah, T. & Mikhail, G. (1970) Mechanisms regulating the menstrual cycle in women. Recent Progress in Hormone Research, 26, 63-95. Van Look, D. F. A. (1976) Failure of positive feedback. In Clinics in Obstetrics and Gynaecology, Vol. 3, No. 3 (Ed.) Baird, D. T. pp. 555-578. London: W. B. Saunders. Wang, C. F., Lasley, B. L., Lein, A. & Yen, S. S. C. (1976) The functional changes of the pituitary gonadotrophs during the menstrual cycle. Journal of Clinical Endocrinology and Metabolism. 42, 718-728. Wise, A. J., Gross, M, A. & Schalch, D. S. (1973) Quantitative relationships of the pituitarygonadal axis in postmenopausal women. Journal of Laboratory and Clinical Medicine, 81, 28-36. Yamaji, T., Diersehke, D. J., Hotcbkiss, J., Bhattaehanja, A. N., Surve, A. H. & Knobil, E. (1971) Estrogen induction of LH release in the Rhesus monkey. Endocrinology, 89, 1034-1041. Yen, S. S. C. & Tsai, C. C. (1971) The biphasic pattern in the feedback action of ethinyl estradiol on the release of FSH and LH. Journal of Clinical Endocrinology and Metabolism. 33,882-887. Yen, S. S. C. & Tsai, C. C. (1972) Acute gonadotropin release induced by exogenous estradiol during the mid-follicular phase of the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 34, 298-305. Yen, S. S. C., Tsai, C. C., Naftolin, F., Vandenberg, G. & Ajabor, L. (1972a) Pulsatile patterns of gonadotrophin release in subjects with and without ovarian function. Journal of Clinical Endocrinology and Metabolism. 35,931-934. Yen, S. S. C., Vandenberg, G., Rebar, R. & Ehara, Y. (1972b) Variation of pituitary responsiveness to synthetic LRF during different phases of the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 35,931-934. Young, J. R. & Jaffe, R. B. (1976) Strength-duration characteristics of estrogen effects on gonadotrophin response to gonadotrophin-releasing hormone in women. II. Effects of varying concentrations of estradiol. Journal of Clinical Endocrinology and Metabolism, 42~ 432-442. Zeilmaker, G. H. (1966) The biphasic effect of progesterone on ovulation in the rat. Acta Endocrinologica, 51~ 461-468. Zimmerman, E. A., Carmel, P. W., Husain, M. K., Ferin, M., Tannenbaum, M., Frantz, A. G. & Robinson, A. G. (1973) Vasopressin and neurophysin" high concentrations in monkey hypophyseal portal blood. Science, 182, 925-927. Zondek, B. & Aschheim, S. (1926) Ueber die funktion des ovariums. Deutsche Medizinische Wochenschrift, 52, 343.