The role of changing pulse frequency in the regulation of ovulation

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1 Human Reproduction vol.8 Suppl.2 pp.57-61, 1993 The role of changing pulse frequency in the regulation of ovulation J.C.Marshall 1 and M.L.Griffin Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Sciences Center, Box 466, Charlottesville, VA 22908, USA 'To whom correspondence should be addressed Ovulatory cycles in women result from sequential stimulation of ovarian follicular development by pituitary follicle stimulating hormone (FSH) and luteinizing hormone (LH). In the follicular phase the initial FSH stimulus declines and LH secretion increases toward the mid-cycle ovulatory surge. During the luteal phase gonadotrophin secretion is reduced. This reflects the effects of ovarian steroids inhibiting the frequency of gonadotrophin releasing hormone (GnRH) secretion by the hypothalamus, and the direct effects of oestradiol and inhibin to reduce gonadotroph (FSH) secretion. The frequency of GnRH stimulation of the gonadotroph is a selective regulator of gonadotrophin synthesis, with slow frequency stimuli favouring FSH and faster frequency stimuli favouring LH secretion. Current research has only revealed a single gonadotrophin releasing hormone. Thus, the ability to change the pattern (particularly frequency) of GnRH stimulation of the gonadotroph is proposed as an important regulator of differential FSH and LH synthesis, and hence of ovulatory cycles. In some disorders of ovulation the ability to regulate GnRH pulse frequency appears to have been lost. Slow frequency GnRH pulses are consistently seen in women with hypothalamic amenorrhoea and hyperprolactinaemia. The reduced GnRH secretion appears to reflect increased hypothalamic opiate tone and can be rapidly reversed with an opiate receptor blocker. Other disorders associated with anovulation show rapid frequency GnRH secretion, and polycystic ovarian syndrome (PCO) is commonly associated with fast-frequency, high-amplitude LH (GnRH) pulses. Such a GnRH stimulus would favour LH and androgen production and the failure of ovarian follicular maturation in PCO may reflect inappropriate sequential FSH secretion. Thus, the ability to change the pattern of GnRH stimulation of the gonadotroph appears to be an important mediator of gonadotrophin synthesis and secretion, and hence of cyclic ovulation. Key words: frequency/gonadotrophins/gnrh pulses/ovulation Introduction Ovarian function in mammals is controlled by the combined action of the two pituitary gonadotrophins luteinizing hormone (LH) Oxford University Press and follicle stimulating hormone (FSH). Gonadotrophin releasing hormone (GnRH) regulates both the synthesis and secretion of LH and FSH. GnRH is secreted by the hypothalamus in an intermittent manner and in women the frequency of GnRH discharges varies from intervals of min to 3-5 h. This pulse secretion of GnRH is essential for stimulating transcription of the genes coding for the common a and specific LH /? and FSH 13 subunits. Similarly, a pulsatile GnRH stimulus is required to maintain gonadotrophin secretion, and fast frequencies or continuous GnRH stimulation desensitizes gonadotrophin secretion. The frequency of the GnRH stimulus can effect differential regulation of subunit gene expression (Dalkin et al., 1989; Haisenleder etai, 1991) and also plays a role in the secretion of LH and FSH; with fast frequencies favouring LH and slow frequencies FSH secretion. The pattern of pulsatile GnRH secretion varies during reproductive life with pulsatile release being evident in the ovine fetus and in the neonate. In children, low-amplitude, slow-frequency pulses are present during the pre-pubertal period and both frequency and amplitude increase to herald pubertal maturation. In women, the frequency of GnRH pulses also changes during ovulatory menstrual cycles, with pulse frequency gradually increasing during the follicular phase and amplitude also being increased at the mid-cycle surge. Following ovulation, pulse frequency slows and remains slow until the demise of the corpus luteum, when frequency again increases to begin the next wave of follicular maturation. In this chapter we examine the role of the ability to change the pattern of GnRH stimulation of die pituitary. The changing patterns of GnRH secretion during ovulatory cycles are reviewed and their potential role in the differential regulation of gonadotrophin synthesis and secretion examined. Several ovulatory conditions exist in which the pattern of GnRH pulse secretion appears to be invariable. GnRH pulse frequency may be persistently slow in hypothalamic amenorrhoea and hyperprolactinaemia, or persistently rapid as seen in a majority of patients with polycystic ovarian syndrome (PCO). The hypothesis is presented that me ability to secrete GnRH at a rapid frequency is regained at the time of puberty, and that thereafter the maintenance of repetitive ovulatory menstrual cycles requires the ability to slow this rapid GnRH frequency during the luteal phase of the cycle. Loss of these regulatory mechanisms may thus be a contributory cause to common forms of anovulation and amenorrhoea. Pulsatile GnRH secretion during pubertal maturation In pre-pubertal girls LH (by inference GnRH) pulse amplitude is low and pulses occur infrequently, every 3 4 h, widi minor 57

2 J.CMarshaU and M.L.Griffm augmentation occurring during sleep (Jakacki et al., 1982; Kelch et al., 1987; Hale et al., 1988; Wu et al., 1989, 1990). A marked amplification of this sleep-entrained secretion of GnRH heralds pubertal maturation. Initially, both the amplitude and frequency of GnRH pulses increase with the onset of sleep, continuing for several hours before declining prior to awakening. Over time increased GnRH pulsatile secretion persists through 24 h and the process of pubertal maturation is determined by the transition from low-amplitude, slow-frequency GnRH secretion to higher amplitude rapid frequency pulse secretion in late puberty. Gonadotrophin responses to exogenous GnRH change during this process, from a predominant FSH secretory response before puberty to the adult pattern of LH responses exceeding those of FSH. These changes in the pattern of GnRH stimuli may be one mechanism where a single hypothalamic releasing hormone (GnRH) can differentially modulate synthesis and secretion of LH and FSH. Studies in rodents have shown that slow frequencies (pulse intervals > 120 min) enhance FSH /3 transcription and subunit expression, whereas fast frequencies (15 60 min) predominantly increase a and LH /3 mrna expression. These changes are associated with appropriate altered LH or FSH secretory responses and suggest that the pattern of GnRH pulses is important in determining gonadotroph responsiveness to GnRH (Haisenleder etal, 1988; Marshall etal., 1991). GnRH pulse secretion during the menstrual cycle During the follicular phase of ovulatory cycles the initial elevation of serum FSH declines and LH increases to a peak at the midcycle LH surge. Follicular recruitment and maturation is initiated by FSH, secreted in response to the previous late luteal increase in GnRH pulse frequency at a time when oestradiol and inhibin in plasma are low (Santen and Bardin, 1973; Backstrom et al., 1982; Reame etal., 1984; Crowley etal., 1985; McLachlin et al., 1987). Subsequently, oestradiol secreted by the maturing follicle selectively inhibits FSH release and stimulates the increase in GnRH pulse frequency during the late follicular phase (Baird, 1978; Karsch et al., 1983, Djahanbakhch et al., 1984). This in turn increases plasma LH, which further stimulates oestradiol, and the mid-cycle LH surge results both from increased frequency and amplitude of GnRH secretion and from the positive effects of oestradiol and progesterone in augmenting LH responses to GnRH. GnRH pulse frequency increases from one pulse every min in the early follicular phase to a frequency of one pulse per hour or slightly faster during the late follicular phase and during the surge. Studies in primates have shown that pulsatile LH secretion is maintained during the surge, with each LH pulse increasing in amplitude on the ascending, and decreasing in amplitude on the descending, portion of the LH surge. In sheep, direct measurements of portal blood GnRH after administration of oestradiol have shown an increase in GnRH pulse frequency and amplitude (Moenter et al., 1991) and during the LH surge GnRH levels are continuously elevated and remain elevated as plasma LH declines (Moenter et al., 1990). This suggests that the frequency of GnRH pulses increases to become very rapid or even continuous and results in desensitization of LH secretion, 58 a potential mechanism for the termination of the LH surge. After ovulation, luteinization results in increased progesterone secretion which, together with oestradiol, acts as the hypothalamic level to reduce the frequency of GnRH pulses. In the first few days after ovulation, pulses occur every min, but progressively slowly, so that by the mid-luteal phase GnRH pulses occur at irregular amplitude every 3-5 h. This slowing of GnRH secretion results from the effects of oestrogen and progesterone acting at the hypothalamus to increase endogenous opioid tone, which is inhibitory to GnRH pulse frequency. Administration of progesterone during the follicular phase also slows GnRH pulse secretion (Soules et al., 1984), and administration of the opiate receptor blocker naloxone increases GnRH pulse frequency during the luteal phase. As noted above, the slow frequency of GnRH pulses would favour FSH synthesis, but FSH release is inhibited by the combined effects of oestradiol and inhibin from the corpus luteum. The net effect would be expected to be an increase in pituitary FSH content. In contrast, the slow irregular luteal GnRH stimulus would not be optimal for maintaining LH synthesis, but as LH release continues to occur, depletion of pituitary LH stores would be expected. With the demise of the corpus luteum, serum oestradiol, progesterone and inhibin concentrations fall, LH pulse frequency increases and plasma FSH rises due to removal of the inhibitory effects of oestradiol and inhibin and the longer plasma half-life of FSH. These data suggest that the ability to regulate pulsatile GnRH secretion is important in maintaining ovulatory cycles (Haisenleder et al., 1988; Marshall et al., 1991). Similar changes have been described in various species, but it should be noted that in GnRH-deficient humans or primates, administration of fixed doses of GnRH at fixed frequencies have been shown to induce ovulation in single cycles (Knobil et al., 1980; Leyendecker et al, 1980; Filicori et al., 1991). In most instances, the dose of exogenous GnRH used was supraphysiological and this may override the need to change the frequency of the GnRH stimulus. In addition, most studies involve one cycle and data are not available as to whether the ability to alter GnRH frequency is important in maintaining ovulatory cycles over prolonged periods of time. The slowing of pulse frequency in the luteal phase may be important, however, because administration of rapid GnRH frequencies causes deficient follicular development and impaired corpus luteum function in subsequent cycles (Lam and Ferin, 1987; Soules et al., 1987). Of note, the changes in GnRH stimulation in the pituitary during ovulatory cycles appears to involve similar mechanisms to those present during pubertal maturation. The follicular phase involves increasing GnRH pulse frequency and amplitude, and reversal in plasma gonadotrophin secretion, from a predominant FSH to a predominant LH response similar to the changes observed during pubertal maturation. The luteal phase involves irregular, slow GnRH pulse stimuli which may not be adequate to maintain LH synthesis, but FSH synthesis continues, thus effecting a reversal of the pattern of gonadotrophin in synthesis in the gonadotroph. This would increase FSH available for release, consequent upon removal of the inhibition of GnRH frequency and the selective inhibitory actions of oestradiol and inhibin with the decline of the corpus luteum (Marshall et al., 1991).

3 Gonadotrophin pulse frequency and ovulation Pulsatile GnRH secretion in anovulation Several common forms of anovulation and amenorrhoea are known to be associated with abnormalities of pulsatile GnRH secretion. Present data suggest that the abnormal GnRH stimulus, and specifically the inability to increase or reduce GnRH pulse frequency, may be causally related to anovulation in these women. Hypothalamic amenorrhoea Hypothalamic amenorrhoea (HA) is a common disorder which is diagnosed only after exclusion of pituitary and ovarian abnormalities. The amenorrhoea is often preceded by marked weight loss, strenuous exercise such as gymnastics or competitive running, psychological stress and occasionally the use of oral contraceptives (Schwartz et al., 1981; Santen et al., 1978). The disorder is reversible, and in a majority of women removal of the antecedent condition results in return of ovulatory menses within 12 months. Basal plasma concentrations of LH, FSH, prolactin and oestradiol are normal or low, and responsiveness to GnRH is maintained. Several recent studies have shown that the frequency of GnRH pulse secretion is markedly reduced in a majority of women with HA (Raem et al., 1985; Veldhuis et al., 1985; Crowley et al., 1985). GnRH pulse frequency (one pulse every 3-4 h) and irregular amplitude resemble the patterns present in the normal luteal phase, suggesting increased hypothalamic opioid activity. This is confirmed in some patients by the fact that i.v. administration of the opiate receptor blocker naloxone rapidly restores normal frequency GnRH secretion (Quigley et al., 1980; Sauder et al., 1984a; Khoury et al., 1987). In a majority of women with HA the anovulation appears to reflect the persistence of slow-frequency GnRH stimulation of the gonadotroph, which is inadequate to increase LH synthesis and secretion to the extent required for the production of an ovulatory LH surge. The demonstration that naloxone rapidly reverses this change suggests that the disorder reflects increased hypothalamic opioid activity, though the mechanisms of the latter are unclear. In some women the long-acting, orally active opiate receptor blocker naltrexone, when given for several weeks, has been shown to be effective in inducing ovulation (Wildt and Leyendecker, 1987). Hyperprolactinaemia When serum prolactin is elevated in women, anovulation and amenorrhoea commonly ensue. Detailed studies have revealed slow irregular patterns of GnRH secretion which increase to normal follicular phase patterns after suppression of serum prolactin by dopamine agonist (Klibanski et al., 1984; Sauder et al., 1984b). Interestingly, the mechanisms of reduced GnRH pulsatile secretion associated with hyperprolactinaemia also appear to involve excess hypothalamic opioid activity. Administration of naloxone results in a rapid increase in pulsatile GnRH secretion, even though serum prolactin remains elevated (Grossman et al., 1982; Cook et al., 1991). This then suggests that the elevated prolactin acts to increase hypothalamic opioid activity, which in turn reduces GnRH pulse frequency and removes the normal ability to increase pulse frequency that occurs during the follicular phase of an ovulatory cycle. Polycystic ovarian syndrome (PCO) PCO is a disorder of unknown aetiology associated with anovulation, hirsutism, obesity and multiple cysts in the ovaries. The clinical syndrome may reflect several underlying causes, but the excess androgen secretion is predominantly of ovarian origin and in a majority of women reflects increased LH secretion (Barens and Rosenfield, 1989; Ehrmann et al., 1992; Chang et al., 1983). Studies have demonstrated that the frequency and amplitude of LH pulses are increased in patients with PCO (Kazer et al., 1987; Waldstreicher et al, 1988), though not to supranormal levels. In most studies, GnRH pulse frequency is consistently present in PCO at a frequency of approximately one pulse per hour similar to that present during the late follicular phase of ovulatory cycles. In women with persistently enhanced pulsatile GnRH secretion, FSH synthesis and secretion would be expected to decrease, but LH synthesis and secretion would increase, with consequent enhanced androgen production by the ovaries the changes found in many women with PCO. We have recently developed the hypothesis that a relative inability of oestradiol and progesterone to inhibit GnRH pulse frequency may be causally related to the development of PCO. If a peri-pubertal girl were relatively insensitive to the action of progesterone (low non-ovulatory plasma levels seen in cycles soon after menarche) in reducing GnRH pulse frequency, GnRH pulse slowing would not occur and the cyclic changes described in normal cycles would be absent. In the absence of selective FSH secretion, subsequent follicular development would not occur, rapid GnRH pulse frequency would be maintained, leading to anovulation and a prolonged deficiency of oestradiol and progesterone, thus perpetuating the rapid post-pubertal GnRH secretion. We recently examined this hypothesis by administering oestradiol and progesterone to women with PCO who had elevated pulsatile LH secretion (Christman et al., 1991). In all women studied, concentrations of oestradiol and progesterone similar to those present in the normal luteal phase resulted in an initial decrease in GnRH pulse frequency (at 10 days), and a subsequent decline in LH pulse amplitude (by 20 days). Removal of oestradiol and progesterone was followed by an increase in GnRH pulse frequency with a selective increase in plasma FSH, with LH levels remaining low during the subsequent 7 days of the study. LH/FSH ratios were returned to unity and responsiveness to exogenous GnRH showed that LH responses were impaired more than those of FSH. This suggests that the 2-3 weeks of reduced frequency GnRH stimuli may have reduced LH synthesis. The selective increase in FSH after withdrawal of ovarian steroids suggests that FSH synthesis was maintained, with FSH release increasing in response to the subsequent increase in GnRH pulse frequency in the presence of low plasma oestradiol concentrations. Of interest, the selective increase in FSH secretion produced follicular maturation in all patients and ovulation in some. Further studies are required to determine whether prolonged reduction in GnRH pulse frequency (to remove the GnRH stimulus and produce the effect similar to the administration of a GnRH antagonist) may result in a greater percentage of women ovulating after withdrawal of oestradiol and progesterone. These observations in anovulatory patients suggest that 59

4 J.CMarshall and M.L.Griffin ovulation does not occur in the presence of persistently slow or a persistently rapid frequency of GnRH secretion. In both instances, the observed pulse frequencies are within the ranges present in ovulatory cycles, the difference being that in ovulatory cycles GnRH pulse frequency changes between the follicular and luteal phases of the cycle. This suggests that the ability to change the pattern of GnRH secretion, perhaps to allow differential synthesis and secretion of LH and FSH, is an important mechanism in maintaining repetitive ovulatory cycles. References Backstrom.C.T., McNeilly,A.S., Leask.R.M. and Baird,D.T. (1982) Pulsatile secretion of LH, FSH, prolactin, estradiol and progesterone during the human menstrual cycle. Clin. Endocrinol., 17, Baird.D.T. (1978) Pulsatile secretion of LH and ovarian estradiol in the follicular phase of the sheep estrous cycle. Biol. Reprod., 18, Barnes,R. and Rosenfield,R.L. 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