Recent Progress in Hormone Research: Proceedings of the 1973 Laurentian Hormone Conference

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PREFACE

The 1973 Laurentian Hormone Conference was held at Mont Tremblant, Province of Quebec, Canada, August 26-31. The proceedings of that meeting constitute this volume of Recent Progress in Hormone Research and illustrate anew the continuous gathering of investigative momentum and sophistication in the field of endocrinology. In accordance with longstanding tradition of the Laurentian Hormone Conference, the papers were carefully prepared masterpieces and the discussions spirited and penetrating. The program opened with the presentation of the 1973 Gregory Pincus Memorial Lecture by Professor Ernst Knobil.

Topics covered by the program included regulation of the gonadotropins in primates, their neutralization by specific antibodies, and their role in oocyte maturation. Attention then turned to the enzymatic interconversion of estrogens followed by a novel concept of the mechanism of steroid hormone action. On exploration of other new territory the thermogenic action of thyroid hormone was related to active sodium transport. Rounding out an exciting week was an updating on the structure, heterogeneity, and activity of many of the lesser known hormones or hormonelike substances such as the somatomedins A, B, and C, the epidermal growth factor, the insect hormones, parathyroid hormone 1,25-dihydroxycholecalciferol, and prostaglandins. All in all, it was a memorable week of excellent scientific fare.

Personally and on behalf of the Committee on Arrangements, I want to thank Drs. Griff Ross, Samuel Solomon, Kenneth Savard, Maurice Raben, Paul Munson, Louis Sherwood, Peter Ramwell, and John Potts, Jr. for skillfully chairing the sessions and guiding the discussions. It is a pleasure, also, to acknowledge the heroic labors of Miss Joanne Sanford and her associates, Mrs. Mina Rano and Miss Lucy Passalapi, in transcribing the lengthy discussions immediately after each session. The always helpful cooperation of Academic Press in producing Volume 30 is acknowledged with gratitude.

ROY O. GREEP,     Cambridge, Massachusetts

April 8, 1974

On the Control of Gonadotropin Secretion in the Rhesus Monkey¹²

E. KNOBIL,     Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Publisher Summary

This chapter discusses the regulation of gonadotropin secretion in the rhesus monkey. The time courses of the circulating gonadotropic hormones during the menstrual cycle of the rhesus monkey are ascertained after long and arduous methodological struggles and are essentially identical to those described in the human female. These are perceived as being the resultant of relatively continuous or tonic secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) interrupted once every 28 days, on the average, by an abrupt, massive discharge of these hormones, which is represented as cyclic secretion by analogy to the basic schema postulated for the rat. In the rhesus monkey, the peak of this midcycle gonadotropin surge precedes ovulation by 37 hours, on the average. Moreover, the tonic secretion of LH and of FSH, as reflected by their concentrations in peripheral plasma at times other than during the preovulatory surge, appears to be controlled by a classical negative feedback loop involving, among other components, the ovary and the gonadotrophs of the adenohypophysis.

I Introduction

We undertook our studies of the regulation of gonadotropin secretion in the rhesus monkey, a representative primate, unburdened by preconceived notions or firmly espoused hypotheses, if only because we were innocent of significant experience or insight regarding this aspect of adenohypophysial physiology in any species. I like to think that this deplorable ingenuousness may have facilitated our labors, and it is for this reason that our approaches to the problem, at least for a time, were deliberately unguided by comparative considerations. I apologize at the outset for this cowardly strategy and for the glaring omissions attributable to it.

The time courses of the circulating gonadotropic hormones during the menstrual cycle of the rhesus monkey (Fig. 1), which were ascertained after long and arduous methodological struggles (Neill et al., 1967; Monroe et al., 1970; Karsch et al., 1973c; Yamaji et al., 1973), are essentially identical to those described earlier in the human female. We perceived them as being the resultant of relatively continuous or tonic secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) interrupted once every 28 days, on the average, by an abrupt, massive discharge of these hormones which we viewed as representing cyclic secretion by analogy to the basic schema postulated for the rat (Schwartz, 1969). In the rhesus monkey, the peak of this midcycle gonadotropin surge precedes ovulation by 37 hours, on the average (Weick et al., 1973).

FIG. 1 Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, and progesterone throughout the normal rhesus monkey menstrual cycle normalized to the day of the mid-cycle LH peak (day 0). Each point represents the mean ±SE of 7 observations for FSH, 19 for LH, 11 for estradiol, and 7 for progesterone. Redrawn from Krey et al. (1973).

II Ovarian Control of Tonic Gonadotropin Sectretion

The tonic secretion of LH and of FSH, as reflected by their concentrations in peripheral plasma at times other than during the preovulatory surge, appears to be controlled by a classical negative feedback loop involving, among other components, the ovary and the gonadotrophs of the adenohypophysis. The interruption of this negative feedback loop by gonadectomy leads to prompt increases in plasma gonadotropin levels³ which, in 2 or 3 weeks, achieve a relative plateau representing mean concentrations approximately 10 times greater than those observed preoperatively (Atkinson et al., 1970). These elevated gonadotropin concentrations, as revealed by analysis of daily blood samples, are the resultant of striking, rhythmic, pulsatile discharges of the hormones at a frequency of approximately one every hour (Fig. 2), which for this reason and for want of a better term we have designated as being circhoral (Dierschke et al., 1970). These pulsatile injections of the gonadotropins into the circulation appear to be superimposed on a background of continuous secretion which contributes but little to the gonadotropin level found in ovariectomized animals. A similar discontinuity in gonadotropin secretion has also been described in several other species, including man (Yen et al., 1972), but the monkey differs from the latter in that the oscillations in circulating gonadotropins cannot be observed in intact animals, not even during the preovulatory surge (Weick et al., 1973).

FIG. 2 Circhoral, pulsatile patterns of plasma luteinizing hormone concentrations in ovariectomized rhesus monkeys. Blood samples were taken every 20 minutes. From Dierschke et al. (1970) with permission.

The mechanisms which eventuate in the circhoral, pulsatile discharges of the gonadotropic hormones from the pituitary continue to intrigue and to elude us. At the very least, they cannot be attributed to nonspecific periodic release of all the adenohypophysial hormones, such as might be occasioned by intermittent alterations in blood flow, since fluctuations in plasma growth hormone concentration, when these occur at all in ovariectomized monkeys, are not synchronous with the rhythmic increments in gonadotropin levels (Fig. 3).

FIG. 3 Luteinizing hormone and growth hormone concentrations measured in the same plasma samples in ovariectomized rhesus monkeys. From Dierschke et al. (1970) with permission.

The rather attractive, and initially compelling, possibility that the intermittent discharges of LH may be the consequence of an autoregulatory mechanism mediated by a short-loop negative feedback system (Motta et al., 1969) whereby circulating levels of the gonadotropin could control its own secretion was also explored (T. Yamaji and E. Knobil, unpublished observations). Ovine LH, human LH, and hCG were infused intravenously into ovariectomized monkeys in order to determine whether elevated plasma concentrations of exogenous gonadotropin could influence the pulsatile pattern of endogenous LH secretion (Fig. 4). Although a study of similar design permitted the unambiguous demonstration that growth hormone secretion is under autoregulatory negative feedback control in the rhesus monkey (Sakuma and Knobil, 1970), these experiments failed to provide any evidence in support of the view that the circhoral discharges of LH are initiated by the decline in circulating LH below some critical, threshold concentration. Since nonsimian LH was utilized in this study, however, the possibility remains that experimentally imposed increments in circulating RhLH could inhibit endogenous LH secretion in the monkey, but this argument is not particularly compelling because nonsimian LH preparations possess striking gonadal stimulatory activity in hypophysectomized rhesus monkeys (Knobil and Josimovich, 1961). We were therefore led, by processes of exclusion, to consider the possibility that the signals which eventuate in the circhoral pulsatile release of gonadotropins from the pituitary gland originate in the central nervous system, being relayed to the gonadotropins by packets of LH-releasing hormone (LRH) discharged into the pituitary portal circulation. This will be considered at greater length later in this discussion.

FIG. 4 Failure of exogenous human luteinizing hormone (a, b), human chorionic gonadotropin (c), and ovine luteinizing hormone (d) administration to inhibit endogenous luteinizing hormone (RhLH) secretion in ovariectomized rhesus monkeys. Horizontal bars (a, b, d) indicate the duration of intravenous infusion beginning at 0 time. The arrow shows the time of the single intravenous injection of hCG. Endogenous and exogenous LH concentrations were measured in the same plasma samples by the appropriate, specific radioimmunoassays.

Closure of the negative feedback loop by the intravenous injection or infusion of estradiol-17β into ovariectomized monkeys, with resultant plasma concentrations of the steroid well within the physiological range (Yamaji et al., 1972), leads to a prompt cessation of the pulsatile gonadotropin discharges and a resultant decline in the mean concentration of these hormones (Fig. 5). If plasma estradiol concentrations characteristic of those normally observed early in the follicular phase of the menstrual cycle (50–70 pg/ml) are maintained in ovariectomized animals for several days, mean gonadotropin levels which are consonant with this stage of the cycle are achieved (Karsch et al., 1973b). These observations, along with the finding that progesterone alone, regardless of the plasma concentrations achieved or the duration of its administration, is without significant influence on the pulsatile mode of gonadotropin secretion or on their mean circulating levels in ovariectomized monkeys (Yamaji et al., 1972), led us to the conclusion that estradiol is, in all likelihood, the primary ovarian component of the negative feedback loop which regulates tonic gonadotropin secretion (Karsch et al., 1973b). In some experimental circumstances, however, a synergism between estrogen and progesterone in suppressing gonadotropin secretion is clearly demonstrable (Karsch et al., 1973d). Although of considerable interest, the physiological significance of this interaction remains obscure since no inkling of its operation is evident in the course of the normal menstrual cycle (see Fig. 1).

FIG. 5 Inhibition of tonic luteinizing hormone (LH) secretion in ovariectomized rhesus monkeys by low physiological concentrations of estradiol maintained by constant, intravenous infusion beginning at 0 time. Plasma estrogen concentrations prior to infusion were undetectable. From Yamaji et al. (1972) with permission.

A totally different dimension in the ovarian control of gonadotropin production must be introduced at this juncture, albeit parenthetically, because its physiological significance remains to be delineated. In the course of validating the radioimmunoassay for RhFSH now used in our laboratory (Yamaji et al., 1973), it was found that preparations of this gonadotropin derived from pituitary glands of ovariectomized rhesus monkeys had more than twice the biological activity, relative to immunoreactive potency, as FSH preparations obtained from the pituitaries of intact animals (Peckham et al., 1973). This striking increment in biological activity was related to a slower disappearance of the pituitary FSH preparations derived from ovariectomized monkeys from the circulation of assay rats. Exclusion chromatography revealed a preponderance of FSH components of larger molecular size in these pituitary extracts when compared to those of intact animals, suggesting that differences in the structure of FSH may account for the differences in biological activity observed. These qualitative differences in pituitary FSH were also found in the sera of intact and ovariectomized rhesus monkeys, indicating that ovarian activity may control not only the quantity but also the molecular size and consequent biological activity of the FSH discharged into the circulation (Peckham et al., 1973). In view of the well-established relationship between the biological activity of glycoprotein hormones, their half-time in the circulation and the degree of sialylation (Morel et al., 1971; Van Hall et al., 1971), it is tempting to suppose that the influence of ovariectomy on the structure and biological activity of FSH is attributable to increases in sialic acid content. Preliminary observations suggest that the physicochemical characteristics of RhLH are similarly under ovarian control.

III Ovarian Control of the Preovulatory Gonadotropin Surge

The demonstration by Michel Ferin and his colleagues (Ferin et al., 1969) that the administration of an antiserum to estrogen blocked ovulation in the rat firmly established the long-held view that estrogen may play an essential, albeit permissive, role in the control of ovulation in this species (Everett, 1961, 1972). This important observation, coupled with the nearly contemporaneous findings that small quantities of estrogen administered to sheep (Goding et al., 1969; Scaramuzzi et al., 1970) and rats (Caligaris et al., 1971) led to an acute release of LH which resembled the spontaneous, preovulatory discharges of the hormone, lent credence to the notion that the unambiguous preovulatory rise in circulating estrogens during the primate menstrual cycle (see Fig. 1) may represent the critical stimulus for the initiation of the gonadotropin surge (Vande Wiele et al., 1970; Hotchkiss et al., 1971).

Our initial attempts to test this compelling hypothesis by the administration of estrogen to rhesus monkeys on the second or third day of the menstrual cycle in the hope of inducing premature LH surges were uniformly unsuccessful. In these early experiments estradiol-17β was given in single intramuscular or subcutaneous injections either in oil or as crystalline suspensions, resulting in rapid but short-lived elevations in plasma estrogen concentrations with peaks in excess of 2000 pg/ml. When, however, the increments in circulating estrogen were sustained for several days by substituting estradiol benzoate for the free alcohol and injecting it subcutaneously in oil, thus mimicking the time course of plasma estrogen concentrations normally observed during the late follicular phase of the cycle, LH surges (and FSH surges, as we were to find later; see Fig. 12a) indistinguishable from those occurring spontaneously were reproducibly elicited (Fig. 6). This stimulatory or positive feedback action of estrogen on LH and FSH secretion was invariably preceded by a decline in circulating gonadotropin levels attributable to the negative feedback action of the steroid. Clearly, the positive feedback effect of estrogen on gonadotropin release is not simply a function of some threshold plasma concentration of the steroid. It must also be critically dependent on a time component, but the precise duration of the effective stimulus, or its threshold for that matter, could not be ascertained from these experiments, although they did permit the conclusion that an increment in plasma estrogen concentration, to be effective in evoking an LH surge, had to be sustained for at least 12 hours and had to exceed approximately 60 pg/ml (Yamaji et al., 1971).

FIG. 6 The positive feedback action of (a) multiple injections (n = 4) and (b) a single subcutaneous injection (n = 7) of estradiol benzoate in oil in normal, intact female rhesus monkeys. Injections of 5 μg/kg (a) and 30 μg/kg (b) of estradiol equivalents were given at the arrows. The bottom panel shows the time course of exogenous estrogen superimposed upon endogenous estrogen levels. Note the initial negative feedback action of the steroid. LH, luteinizing hormone. From Yamaji et al. (1971) with permission.

FIG. 12 Blockade of estradiol-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) surges by the implantation of Silastic capsules containing progesterone. The Silastic capsules were inserted on day 2 of the cycle and estradiol benzoate (EB) was injected on day 4 as indicated by the arrows. All points represent means ± standard error of 5 or 6 animals. The small increment in circulating progesterone coincident with the gonadotropin surges in the controls (a) represents endogenous progesterone secretion in response to ovarian stimulation by the gonadotropins. The composite illustrated in (b) reflects the responses of 3 animals which had gonadotropin surges resembling those of the controls and of 2 monkeys which failed to respond to the estrogen stimulus. From Dierschke et al. (1973) with permission.

Further delineation of the strength-duration characteristics of the positive feedback action of estrogen on gonadotropin release necessitated a more precise experimental approach. This was provided by the use of segments of Silastic tubing, packed with crystalline estradiol-17β (Fig. 7) and sealed at both ends. The subcutaneous implantation of such capsules effect, within 1 hour, increments in plasma estrogen levels that can be maintained in remarkably stable fashion for a year or longer (Karsch et al., 1973b). The dynamics achieved by this method of estrogen administration are superior to those provided by constant intravenous infusion, at least in our hands, and can be viewed as step increases in plasma estrogen concentrations.

FIG. 7 A capsule consisting of a 4-cm length of Silastic tubing filled with crystalline estradiol-17β and sealed at both ends with Silastic elastomer. Such a capsule, when implanted subcutaneously in ovariectomized animals, achieves sustained plasma estradiol concentrations of approximately 70 pg/ml.

By varying the number of Silastic capsules implanted, one can vary, in highly predictable fashion, the magnitude of the estrogen increments achieved while their duration is simply delimited by removing the implants at a predetermined time, whereupon plasma estrogen concentrations decline exponentially to control levels within 1 hour. The entire procedure thus results in a square wave estrogen stimulus. An individual experiment representative of those performed in this study is illustrated in Fig. 8. In this animal, an estradiol-containing Silastic capsule was implanted subcutaneously, on day 3 of the menstrual cycle, at 0 time. During the 36 hours that the implant was left in place, a step increase in plasma estrogen concentration of approximately 225 pg/ml was maintained. The initial response to this increment, which is within the physiological range, was a decline in plasma LH concentrations, but, before the implant was removed and while circulating estrogen levels were still elevated, this negative feedback effect of the steroid was interrupted by the initiation of an LH surge. This discharge of LH cannot, therefore, as some authors have suggested (Monroe et al., 1972; Yen and Tsai, 1972), be attributed to a release of the pituitary from negative feedback inhibition consequent to a fall in plasma estrogen concentration. After removal of the implant, plasma LH levels continued to rise to a peak and then declined in a manner characteristic of spontaneous preovulatory LH surges. During this LH discharge, the expected fall in plasma estrogen concentration to preimplantation levels was interrupted by a sharp rise in the circulating level of the steroid. This is an increase in ovarian estrogen secretion in response to the stimulatory action of the induced LH surge and is not demonstrable in similarly treated ovariectomized animals (Yamaji et al., 1971; Karsch et al., 1973b). The precipitous increase in circulating estrogens which is coincident with the ascending phase of the gonadotropin surge during the normal menstrual cycle (see Fig. 1) is explicable on the same basis (Hotchkiss et al., 1971). Figure 9 summarizes experiments performed in 36 animals exposed to approximately the same increments in plasma estrogen concentrations (with resultant levels in the upper physiological range of 200–400 pg/ml) for varying lengths of time. When the duration of the stimulus was less than 36 hours, only the negative feedback action of the estrogen was evident. When the implants were removed, plasma LH concentrations promptly rose to, or somewhat above, control levels, but unambiguous LH surges were never observed. When the estrogen increment was maintained for 36 hours, however, unequivocal LH surges were induced in 6 of 11 animals, and all responded with typical LH surges when the implants were left in place for 42 hours. This sequence was delayed by approximately 12 hours when the plasma estrogen concentrations achieved by the implants were of a lower magnitude, but still within the physiological range (100–200 pg/ml), and was advanced by 12 hours when the system was exposed to grossly supraphysiological concentrations of the steroid (Fig. 10). These 12-hour time shifts support the view that the initiation of induced LH surges, like that of spontaneous ones (Weick et al., 1973), is not coupled to the diurnal, light-dark cycle as is the case in the rat and other rodents (Schwartz, 1970; Schwartz and McCormack, 1972).

FIG. 8 Representative experiment utilizing the Silastic implant technique in determining the strength-duration characteristics of the positive feedback action of estrogen. Note the square wave estrogen signal superimposed upon endogenous estrogen levels while the implant is in place. The estrogen peak coincident with the induced luteinizing hormone (LH) surge represents an ovarian response to the gonadotropin. The Silastic implant was inserted on day 3 of the menstrual cycle. From Karsch et al. (1973c) with permission.

FIG. 9 The duration of an increment in circulating estrogen and the induction of luteinizing hormone (LH) surges in rhesus monkeys. The stippled areas designate the mean serum estrogen concentrations before, during and after the imposition of an estrogen increment by the subcutaneous implantation, at 0 hours, of Silastic capsules containing estradiol-17β. Each point on the LH curves represents the mean ± standard error of the number of observations shown in parentheses. From Karsch et al. (1973c) with permission.

FIG. 10 The magnitude of the increment of circulating estrogen and the induction of luteinizing hormone (LH) surges. See legend to Fig. 9. The data in the middle of the figure are those from Fig. 9 and are included for comparison. From Karsch et al. (1973c) with permission.

Estradiol increments achieving less than 100 pg/ml were ineffective in eliciting LH surges, even when applied for as long as 120 hours (Karsch et al., 1973c). It follows that, in a physiological setting, the control system which responds to the positive feedback action of estrogen with a gonadotropin discharge is activated by increments in circulating estrogens which exceed a threshold of approximately 100 pg/ml and are sustained for approximately 36–42 hours. In sharp contrast, much smaller increments in plasma estrogen concentration entrain the negative feedback system which governs tonic LH secretion and do so within minutes (Yamaji et al., 1972; Karsch et al., 1973b). The large difference in the response time of these two feedback systems undoubtedly accounts for the initially perplexing observation that a single increment in circulating estrogen has both inhibitory and stimulatory actions on LH secretion. These experiments further reveal that the estrogen increment, to be effective, must be applied until the LH surge actually begins. If the implants were removed much before this occurred, the expected LH surge was either not initiated or was incomplete (Karsch et al., 1973c). This phenomenon prompted Dr. R. F. Weick, one of our colleagues, to describe this system as having a short memory.

It must also be noted that effective increments in circulating estrogen imposed by the implantation of estrogen-containing Silastic capsules elicit but one gonadotropin surge even when they are left in place for several weeks (D. J. Dierschke and E. Knobil, unpublished observations). In similar circumstances, the rat releases daily LH surges for a week or longer (S. J. Legan and F. J. Karsch, personal communication).

The positive feedback action of physiological increments in circulating estrogen, which results in the induction of gonadotropin surges essentially indistinguishable, both quantitatively and qualitatively, from those occurring spontaneously during the menstrual cycle, is fully demonstrable in ovariectomized rhesus monkeys (Yamaji et al., 1971; Karsch et al., 1973b) as well as in ovariectomized-adrenalectomized animals (Fig. 11). These findings provide compelling support for the view that, during the normal menstrual cycle, the rise in circulating estrogen during the follicular phase is the primary stimulus for the initiation of the gonadotropin surge, a response which does not require the presence of any other ovarian hormone or, in other than a permissive context, of any adrenal steroids. While progestins, in a physiological setting, may modify the dynamics of this response as has been argued by some (Swerdloff et al., 1972), such an action, if it exists at all, must be subtle indeed. On the contrary, progesterone is a remarkably effective inhibitor of estrogen-induced gonadotropin discharges whether these occur spontaneously during the menstrual cycle (Spies and Niswender, 1972) or are induced experimentally by the administration of estrogen (Dierschke et al., 1973). The low luteal phase levels of progesterone characteristic of the rhesus monkey menstrual cycle are sufficient to prevent the positive feedback action of estrogen although the negative feedback action of the latter steroid remains fully evident (Fig. 12).

FIG. 11 Negative and positive feedback actions of single 30 μg/kg injections of estradiol benzoate (arrows) on gonadotropin secretion in an adrenalectomized-ovariectomized (OVX-ADX) and an ovariectomized (OVX) rhesus monkey. Note the similarity between the two responses. LH, luteinizing hormone; FSH, follicle-stimulating hormone.

As may be predicted from all of the foregoing, the complete pattern of gonadotropin secretion, as observed during the normal menstrual cycle, can be replicated experimentally in ovariectomized animals by superimposing the positive feedback action of incremental estrogen concentrations, consequent to estradiol benzoate injections, upon the sustained negative feedback inhibition of gonadotropin secretion effected by the constant levels of the steroid released from Silastic implants (Fig. 13).⁴ Taken together, the foregoing observations have led us to the conclusion (Knobil et al., 1972; Karsch et al., 1973b) that the time course of gonadotropin secretion throughout the entire menstrual cycle is primarily directed by the cyclic changes in ovarian estrogen production known to occur during the cycle. This conclusion implies that, in primates, the clock or Zeitgeber which determines the timing of ovulation is not resident in the cranium, as it appears to be in the rat (Everett, 1961, 1964), but is embodied in the ovary which, for this reason, we have referred to on occasion as a pelvic clock. This view of the ovary is not a new one, having been popularized by Corner some 30 years ago (Corner, 1943), but was adumbrated by the brilliant studies of Everett, Sawyer, and their colleagues (Sawyer et al., 1949; Everett et al., 1949; Everett and Sawyer, 1950) which demonstrated that the timing of ovulation in the rat clearly depends on a stimulus from the central nervous system.⁵

FIG. 13 Replication, in an ovariectomized rhesus monkey, of the time course in circulating luteinizing hormone (LH) observed during the normal menstrual cycle by superimposing increments in plasma estrogen concentrations (estradiol benzoate injections at arrows) upon constant estrogen levels maintained by a Silastic implant containing estradiol-17β inserted on day 0. From Karsch et al. (1973b) with permission.

IV The Site of Feedback Actions of the Ovarian Hormones

As mentioned earlier, it was reasoned that the signals which eventuate in the pulsatile discharges of LH when the negative feedback loop is interrupted by ovariectomy must originate somewhere in the central nervous system (Dierschke et al., 1970). Although attempts to abolish these circhoral discharges of LH by deep pentobarbital anesthesia, a procedure which in the rhesus monkey inhibits the secretion of growth hormone in response to stress (Knobil and Meyer, 1968), were unsuccessful, we found that narcoleptic drugs, such as chlorpromazine or haloperidol, as well as the more specific α-adrenergic blocking agents phenoxybenzamine and phentolamine were strikingly effective in this regard (Bhattacharya et al., 1972). β-Adrenergic blockade, in contrast, was without effect on pulsatile LH release (Bhattacharya et al., 1972). Single intravenous injections of the α-adrenergic blocking agents were followed, within minutes, by a cessation of the pulsatile gonadotropin discharges and a resultant fall in the plasma concentration of these hormones (Fig. 14). This inhibition of LH secretion was usually maintained for several hours and was terminated by an abrupt resumption of the pulsatile secretory pattern. The time course of these responses to pharmacologic blockade was indistinguishable from that observed when small doses of estradiol were similarly administered as single intravenous injections (Yamaji et al., 1972) or introduced by continuous intravenous infusion (see Fig. 5).

FIG. 14 Effects of single, intravenous phenoxybenzamine injections on plasma luteinizing hormone (LH) concentrations in ovariectomized monkeys. From Bhattacharya et al. (1972) with permission.

These observations were in striking harmony with those of Schneider and McCann (1970) in the rat that α-adrenergic blocking agents, but not β-blocking agents, prevented the dopamine-induced release of LRH by hypothalamic fragments in vitro, an effect shared by estradiol. We concluded, therefore, that the negative feedback loop which controls LH secretion in the rhesus monkey similarly contains a hypothalamic dopaminergic and/or noradrenergic component which generates intermittent signals to the LRH-secreting neurons and that estrogen, but not progesterone, interferes with the action of the neurotransmitters on LRH release (Bhattacharya et al., 1972). Despite considerable effort, however, we were unable to stimulate FSH and LH secretion by the introduction of dopamine or norepinephrine into the third ventricle using a wide spectrum of doses and injection frequencies as well as recipient monkeys in a variety of endocrinological states (R. F. Weick, L. C. Krey, and E. Knobil, unpublished observations). But such negative results do not seriously challenge the view that the central nervous system is at least one of the sites at which estrogens exert a negative feedback effect.

A hypophysial site of action, on the other hand, was clearly suggested by the finding (Fig. 15) that the responses to a given dose of synthetic LRH are progressively diminished as circulating levels of estrogen are increased following the implantation of estradiol-containing Silastic capsules (Krey et al., 1973). A similar inhibitory effect of estrogen on LRH-induced gonadotropin secretion, implying a hypophysial site of action, has also been reported in the human female (Thompson et al., 1973).

FIG. 15 Means ± standard error of the maximal increments in plasma luteinizing hormone (LH) concentration above preinjection values observed within 30 minutes after the intravenous injection of 20 μg of LRH in 5 ovariectomized monkeys prior to and after the implantation of Silastic capsules containing estradiol-17β. The ranges of the resultant plasma estradiol concentrations are indicated under each mean. From Krey et al. (1973).

Pharmacologic approaches to the site of the positive feedback action of estrogen in the rhesus monkey have been equally ambiguous. Over the past several years we have attempted to block spontaneous and estrogen-induced gonadotropin surges by all the drugs which were shown to be effective in inhibiting tonic LH secretion in ovariectomized animals.

One of many such studies is illustrated in Fig. 16. The results were invariably negative. Propanolol, a β-adrenergic blocking agent, was also inert, as were other agents which had variously been reported to block ovulation in the rat (Everett, 1961; Sawyer, 1969), such as scopolamine and, most notably, sodium pentobarbital. Profound pentobarbital anesthesia, initiated immediately before estrogen administration and maintained by repeated intravenous injections for 36 hours, did not interfere with the initiation of typical LH surges at the appropriate time (Fig. 17). This treatment did, however, induce a discharge of prolactin which was followed by overt lactation within 24 to 36 hours (L. C. Krey, W. R. Butler, G. Weiss, K. H. Lu, and E. Knobil, unpublished observations). While these uniformly negative studies do not provide acceptable evidence against the view that estrogen acts at the neural level, there causing a release of LRH which eventuates in a discharge of gonadotropin, neither do they support such a hypothesis. Alternatively, estrogen may act at the level of the anterior pituitary gland and either directly stimulate gonadotropin release (Piacsek and Meites, 1966; Schneider and McCann, 1970) or increase the sensitivity of the gonadotrophs to ambient levels of LRH. If the latter mechanism were operative (Schally et al., 1971; Legan and Karsch, 1974), one would predict that the pituitary would become more responsive to exogenous LRH as the follicular phase of the menstrual cycle progresses and plasma estrogen concentrations rise. This was not the case (Fig. 18). In fact, the LH increment in response to a standard intravenous dose of synthetic LRH declined somewhat during this time (Krey et al., 1973). Increased responses to exogenous LRH were observed only when this substance was injected during the time of the spontaneous LH surge. Although plasma estrogen concentrations are also markedly elevated at that time, they surely cannot account for the increase in the sensitivity of the system to the LRH since, as was shown in the experiments summarized in Fig. 15, estrogen inhibits the efficacy of the exogenous decapeptide in causing LH release. Rather, these studies support the conclusion that the pituitary of the rhesus monkey is most sensitive to the action of exogenous LRH when the secretion of gonadotropins is most active, whether this occurs in the absence of estrogen, as in the untreated ovariectomized animal, or during the spontaneous LH surge when estrogens are markedly elevated (Krey et al., 1973). Furthermore, since the response to LRH was essentially the same during the luteal phase of the cycle, when plasma progesterone concentrations are maximal, and during the follicular phase of the cycle, when progesterone is immeasurable (Fig. 18), this simple study also suggests that the site of action of progesterone in blocking estrogen-induced gonadotropin surges is not at the pituitary level.

FIG. 16 ) to inhibit estrogen-induced LH surges in intact animals (•—•). Estradiol benzoate was injected on day 0 (arrow), the third day of the menstrual cycle. Each point is a mean ± standard error.

FIG. 17 Failure of deep sodium pentobarbital anesthesia, maintained for 36 hours (horizontal bar), to inhibit luteinizing hormone (LH) surges induced by the implantation of Silastic capsules containing estradiol-17β (arrows). Points are means ± standard error. The sharp increase in circulating prolactin levels (upper panel) induced by the anesthesia was followed, in most animals, by copious lactation within 24 to 36 hours.

FIG. 18 Means ± standard error of the maximal increments in plasma luteinizing hormone (LH) concentration above preinjection values observed in rhesus monkeys within 30 minutes after intravenous injections of 10 μg of LRH during the early follicular, late follicular, mid-cycle surge, and mid-lutel phases of the menstrual cycle. The number of observations is in parentheses. From Krey et al. (1973).

If these findings are taken at face value, and if one excludes the possibility of a direct stimulatory action of the steroid, it may further be argued, by exclusion, that since estradiol does not enhance the sensitivity of the pituitary to exogenous LRH, its positive feedback action must be at the level of the central nervous system. Such facile interpretations must be viewed with considerable caution, however, if only because the small and short-lived increments in circulating LH and FSH effected by even the largest doses of LRH cannot be equated with spontaneous or estrogen-induced gonadotropin surges (Krey et al., 1973). Our unsuccessful attempts to replicate such gonadotropin surges by repetitive intravenous injection, by continuous infusion or by the administration of depot preparations of the decapeptide only served to convince us that more direct approaches to the localization of the positive and negative feedback actions of estrogens on gonadotropin secretion were in order and that we could no longer evade the awesome prospect of assaulting the monkey brain.

V Surgical Localization of the Hypothalamicohypophysial Gonadotropin Control System

Our plan of attack was formulated in the light of the vast experience accumulated during the extensive explorations of the neural control of pituitary function in the rat (Szentágothai et al., 1972). Being fully aware of the pitfalls associated with every direct approach to the rat hypothalamicohypophysial system which had been employed in the past, we deliberately elected to begin with the grossest approach to the problem with the intent of mapping the general geography of the gonadotropin control system. Could we, for example, by placing large lesions in the hypothalamus delineate centers for the control of tonic and of surge gonadotropin secretion in the rhesus monkey, as had been so successfully achieved in the rat (Szentágothai et al., 1972) ? Could we then localize the sites of positive and negative feedback action of estrogens, of the agonists and antagonists of autonomic neural activity, and of the electrophysiological correlates of neuroendocrine function? We had decided to proceed by placing in the hypothalamus of adult female monkeys the largest electrolytic lesions compatible with life when Dr. Lewis Krey joined our laboratory as a postdoctoral research fellow. He proposed that we attempt, instead, the surgical method of Halasz and his co-workers (Halasz and Pupp, 1965; Halasz and Gorski, 1967), which was to be so extraordinarily fruitful in elucidating cognate problems in the rat. Dr. Krey was undeterred by our arguments that, while some rats may survive the insult of a large knife lowered into the hypothalamus and then rotated with the resultant partial or complete isolation of the medial basal hypothalamus, the rhesus monkey could never do so and would, in all probability, not even emerge from anesthesia. Our protestations not withstanding, Dr. Krey quickly demonstrated that this operation, as performed on a few cull monkeys, was not only compatible with life but seemed to be astonishingly benign, at least as compared to transpharyngeal hypophysectomy in this species (Knobil and Greep, 1959). With this unexpected intelligence at hand, the entire laboratory enthusiastically joined in initiating studies designed to determine whether in the rhesus monkey, as in the rat (Halasz, 1972), a hypophysiotropic area in the medial basal hypothalamus which controls tonic LH and FSH secretion is dissociable from a presumptive, more anterior region which governs the initiation of gonadotropin surges in response to the positive feedback action of estrogen.

The procedure currently used for the deafferentiation of the monkey hypothalamus is as follows. Animals are anesthetized with pentobarbital and placed in a plexiglass headholder. Lateral roentgenographs of the skull are then taken which provide an unobstructed view of the area between the anterior clinoid processes and the interaural line. The distance between these is measured and corrected for magnification. The animals are then transferred to a stereotaxic instrument for placement of a modified Halasz knife at coordinates calculated from this measurement. The knife consists of a stainless steel blade, 1.5 mm wide, which forms an arc 7 mm in height with a 3 mm radius of rotation. A bone flap is removed from the calvarium, exposing the sagittal sinus. After incision of the dura, the sinus is gently retracted and the knife is lowered through the longitudinal fissure to the appropriate coordinates. The proper placement of the knife is then verified by a second X-ray and corrected, if necessary (Fig. 19). The knife is then rotated using short, vertical strokes at 5-degree intervals, followed by continuous sweeps. The depth of the knife is adjusted, in the process, to conform to the contours of the sphenoid bone and, thus, to the ventral surface of the brain. At sacrifice, as long as 6 months postoperatively, the brain is perfused in situ with saline followed by 10% formalin in saline. After fixation for at least 24 hours, the brain is removed from the skull and fixed for an additional 24 hours in 20% ethanol. Serial, frozen sections (50 μm) are then made through the entire preoptic and hypothalamic areas, and alternate sections are stained with cresyl violet. The boundaries of the lesions are then reconstructed from projections of these sections. In general, the cuts begin in the optic chiasm or at its posterior aspect and extend posteriorly to the mamillary bodies forming a conical island with a circular base having the median eminence and the infundibulum at its center. The completeness of the operation was attested to on one occasion, by the startling fact that the disconnected medial basal hypothalamus actually fell out of a fixed brain as it was being prepared for sectioning (Fig. 20).

FIG. 19 Doubly exposed roentgenogram showing the anteroposterior limits of the excursion of the modified Halasz knife in the course of a complete medial basal hypothalamic disconnection. Note the clinoid processes and the sella turcica.

FIG. 20 The base of the brain of a monkey sacrificed 7 days after complete disconnection of the medial basal hypothalamus. The prominent hole just posterior to the optic chiasm resulted when the disconnected hypothalamic island, comprising the entire tuber cinereum, fell out in one piece when the formalin-fixed brain was being prepared for sectsioning.

When we had achieved near mastery of the foregoing technique after 35 unsuccessful attempts, we discovered, somewhat to our chagrin, that McHugh and Gibbs (1972) had recently reported the complete isolation of the medial basal hypothalamus in the rhesus monkey, using a very similar approach, but with the intent of studying appetitive behavior in this species.

Of the 11 animals in which complete disconnection of the medial basal hypothalamus has been verified histologically (L. C. Krey, W. R. Butler, and E. Knobil, unpublished observations), all but one have had frank diabetes insipidus, most have been overtly hyperphagic, and a few have lactated throughout the entire postoperative period. All of these animals studied in this regard (L. C. Krey, K. H. Lu, W. R. Butler, F. Piva, J. Hotchkiss, and E. Knobil, unpublished observations) appear to have normal or slightly elevated plasma growth hormone concentrations but fail to respond to stressful stimuli, such as vasopressin administration or insulin hypoglycemia, in the usual manner (Meyer and Knobil, 1966; Sakuma and Knobil, 1970), and their serum thyroxine levels fall promptly after the operation (W. R. Butler, J. Espinosa-Campos, L. C. Krey, and E. Knobil, unpublished observations).

For the purposes of this discussion, only two illustrative animals will be presented in some detail. The first (No. 694) is representative of a group of 4 ovariectomized rhesus monkeys which was studied before and after complete disconnection of the hypothalamus with especial reference to the control of tonic gonadotropin secretion. Figure 22 depicts the actual reconstruction of the anteroposterior dimensions of the lesion in this animal superimposed on a diagrammatic representation of a parasagittal section through the hypothalamic area of an adult rhesus monkey (Fig. 21). A frontal section through the infundibular region of the same animal is shown in Fig. 23. The completeness of the isolation of the medial basal hypothalamus was clearly evident from an examination of this section as well as every other in the series encompassing the entire region shown in Fig. 22. The circulating patterns of LH and FSH 42 days before and 49 days after the operation are shown in Fig. 24. Complete deafferentation of the medial basal hypothalamus had no significant effect on the mean plasma concentrations of the gonadotropins, or on the circhoral pulsatile rhythm of LH and FSH discharge. The administration of estradiol benzoate to this animal first resulted in negative feedback inhibition of gonadotropin secretion followed by a characteristic surge.

FIG. 21 Schematic parasagittal section through the hypothalamus of the rhesus monkey. The limits of the third ventricle at midline are shown by the dashed line. Hypothalamic nuclei are designated as follows: ARC, arcuate; DM, dorsomedial; HP, posterior hypothalamic; PV, paraventricular; SCH, suprachiasmatic; SO, supraoptic; VM, ventromedial. MM, medial mamillary body; PM, premamillary area; AC, anterior commissure and OCH, optic chiasm.

FIG. 22 Reconstruction (dashed line) from serial, coronal sections of the lesion in monkey No. 694 superimposed upon a diagrammatic parasagittal section of the hypothalamus. See Fig. 21 for identification of hypothalamic structures.

FIG. 23 Photomicrograph of a coronal section at the mid-tuberal level of the hypothalamus of monkey No. 694. The section was stained with cresyl violet. ×7.

FIG. 24 Time courses of plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations, monitored every 15 minutes for an 8-hour period, in an ovariectomized monkey 42 days before and 49 days after complete disconnection of the medial basal hypothalamus (MBH).

The second monkey (No. 632) is one of a series of 7 intact females with normal menstrual histories studied before and after complete isolation of the medial basal hypothalamus. The reconstruction of the lesion in the mid-sagittal plane is shown in Fig. 25. Once again, completeness of the disconnection was verified by examination of serial sections through this entire area. One of these, through the mid-tuberal region, is shown in Fig. 26. The consequences of the operation in this animal are depicted in Fig. 27. The complete disconnection was performed on day 23 of a normal ovulatory menstrual cycle, but, unfortunately, the menstrual period which undoubtedly followed went unrecorded. It may be seen, nonetheless, that the next expected gonadotropin surge occurred at the appropriate time and resulted in ovulation as evidenced by the sustained rise in plasma progesterone concentrations. Estradiol benzoate, administered on the third day of the succeeding menstrual cycle, elicited the expected surges in FSH and LH. Despite the fact that this and most other animals in this series lactated for a time after the operation, no notable, chronic changes in circulating prolactin levels were observed. The most salient conclusion to be drawn from these very recent studies is that the sites of both the positive and negative feedback actions of estrogen and, hence, of the central components of the control systems which govern tonic and surge secretion of the gonadotropic hormones must be resident within the medial basal hypothalamicohypophysial unit. While the relative roles of its neural and glandular elements remain to be determined, we are elated by the fact that the seemingly unambiguous results of these simple experiments have circumscribed our field of exploration considerably. We now know where to look!

FIG. 25 Reconstruction (dashed line) from serial, coronal sections of the lesion in monkey No. 632 superimposed upon a diagrammatic parasagittal section of the hypothalamus. See Fig. 21 for identification of hypothalamic structures.

FIG. 26 Photomicrograph of a coronal section at the mid-tuberal level of the hypothalamus of monkey No. 632. The section was stained with cresyl violet. ×7.

FIG. 27 Time courses of circulating gonadotropins, prolactin, and ovarian hormones before and after complete disconnection of the medial basal hypothalamus on day 0. Menstrual periods are indicated by M. The arrow on day 38 indicates the subcutaneous injection of estradiol benzoate in oil. The plus and minus signs in the top panel signify the presence or the absence of lactation as judged by the manual expression of milk from the nipples. LH, luteinizing hormone; PROG., progesterone; PRL, prolactin; FSH, follicle-stimulating hormone.

In any event, it would appear that the rhesus monkey, unlike the rat (Everett, 1961, 1964; Halasz, 1972), does not seem to require a signal, generated by the preoptic area of the brain, which is obligatory for the initiation of the preovulatory gonadotropin surge. Coincidentally, the preovulatory gonadotropin discharge in primates does not appear to be linked to the diurnal light-dark cycle but seems, rather, to be a direct response to the positive feedback action of estrogen on the hypothalamicohypophysial apparatus. These considerations are in harmony with our recent finding that pentobarbital, which is thought by some to prevent ovulation in rodents by blocking the signal from the preoptic area (Everett, 1964; Schwartz, 1969; Norman et al., 1973), has no effect whatever on the estrogen-induced gonadotropin surge in the rhesus monkey (L. C. Krey, W. R. Butler, G. Weiss, K. H. Lu, and E. Knobil, unpublished observations). They are also consonant with our observation (Karsch et al., 1973a) that male monkeys (Fig. 28), unlike male rats in like experimental circumstances (Neill, 1972), respond to the positive feedback action of estrogen with an LH surge as do females, since it appears to be the preoptic area which is rendered inoperative when rats are exposed to androgen during the critical stage of neural differentiation (Barraclough, 1966; Gorski, 1971; Libertun et al., 1973).

FIG. 28 Induction of luteinizing hormone (LH) surges in gonadectomized male and female rhesus monkeys by the administration of estradiol benzoate (EB) at a dose of 30 μg/kg. Prior to injecting EB, plasma LH concentrations were suppressed by the subcutaneous implantation of Silastic capsules containing estradiol-17β. Plasma levels of estrogen effected by these treatments are shown in the lower panels. Each point depicts mean ± standard error of 5 and 6 observations for the males and females, respectively. From Karsch et al. (1973a) with permission. Copyright 1973 by the American Association for the Advancement of Science.

It would seem, therefore, as we have already noted in considering the control of the corpus luteum (Knobil, 1973), that primates, in contrast to a number of other mammals, have evolved simpler solutions to the physiological problems posed by the control of gonadal function, as members of this genus have become decreasingly dependent on the dictates of their environment for the timing of reproductive processes.

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DISCUSSION

E. M. Bogdanove: I would like to retain some hope for the hypothesis that in all vertebrate species ovulation ordinarily requires two inputs to the hypothalamic pituitary (HP) system (steroid feedback and some sort of neural input to the basal hypothalamus) [E. M. Bogdanove, in Reg Mammal Reproduction (S. J. Segal et al., eds.), pp. 65–78. Thomas, Springfield, Illinois, 1973]. We do know that under some conditions each of these inputs may substitute for the other, at least partially [E. M. Bogdanove, in Reproductive Biology (H. Balin and S. Glasser, eds.), pp. 5–70. Excerpta Med. Found, Amsterdam, 1972]. Despite your convincing demonstration that estrogen treatment alone induced ovulatory surges of LH in rhesus monkeys in which all extrahypothalamic neural input had presumably been eliminated by surgical isolation of the HP unit from other brain regions, I think two questions must be answered before we can define the extent to which the monkey differs from the rat, or assert that only the steroid input to the HP unit is needed in the monkey. It is true that in the rat the neural signal originates, at least partly, outside of the hypothalamus; hypothalamic deafferentiation does block spontaneous ovulation. However, do we have convincing evidence either that estrogen can not induce an LH surge in the rat with an isolated HP system or that, in the monkey, no part of the presumed neural signal can originate within the hypothalamus? Also, do you know whether estrogen alone can induce an LH surge, either before or after hypothalamic deafferentiation, in any of the species in which the surge is normally triggered by coitus, such as the rabbit, cat, mink, ferret, or vole?

My second point may be merely semantic, but I would like to ask you to defend your use of the term positive feedback to describe this effect of estrogen. It is my impression that this term denotes a phenomenon in which quantal amplification results from the interaction between two or more units (A, B, etc.) such that a signal from unit A causes unit B to release a signal which then feeds back—causing A to increase its signal and thus, in turn, to cause B to increase its signal, etc. Such a situation must continue either ad infinitum (improbably) or until the feedback loop is somehow destroyed (opened).

As long as the increments in LH and estrogen appeared to develop in parallel, as they do in the intact female, the positive feedback model did seem to provide a quite reasonable explanation for the events of the LH surge. However, I think your own experiments with exogenous estrogen in the spayed monkey have denied the validity of this model. Here, although the second component (unit B, the ovary) was missing, and thus could not respond to the initiating LH signal, the pattern of pituitary activity was identical to that seen with the ovary present. This makes it very clear that once the LH surge begins, estrogen feedback has no further influence on it, since the same LH surge occurs regardless of whether it is accompanied by rising or falling levels of estrogen. During most of the time prior to the start of the surge, when estrogen does exert its effect(s), LH and estrogen levels clearly show an inverse relationship, suggestive of negative feedback. The only point at which this may not be true is very early in the surge (at the tail end of the estrogen prepotential), but I question whether we can yet assert that any particular estrogen–LH relationship is obligatory at this particular point. Your studies therefore seem to have shown that the relationship between the causative estrogen event and the ensuing LH surge is one in which these events need not coincide but can be temporally separated. If this is true, they