I to

Journul of Sreroid Biochemisrry. Vol. 11. pp. 98 987 Pergamon Press Ltd 1979.Printed in Great Britain

30. Central Control of Steroid Production REGULATION OF GONADAL STEROID RHYTHMS IN RATS PUSHPA S. KALRA

and SATYA P. KALRA Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A. SUMMARY This

review clearly demonstrates the coupling of the adrenal and gonadal hormone rhythmicities. The physiological significance of this coupling is subject to considerable speculation. It is possible that adrenal secretions provide a transient period of quiescence or heightened gonadal secretion which may be important in reproductive performance and synchronization with other biological clocks. A breakdown in this interrelationship between the endocrine glands due to factors such as environment, stress (internal or external) or others may herald reproductive senescence. These studies also firmly establish the concept that gonadal steroids are able to modify hypothalamic LHRH activity. In this respect, while androgens and oestrogens were equipotent there was an indication that the central field of androgen action as compared to that of oestrogen may be restricted to the basal hypothalamus. The preoptic area (POA) LHRH neurons and their innervation to the organum vasculosum of lamina terminalis (OVLT) were resistant to steroid action. This raised the possibility of the existence of more than one population of LHRH neurons. Our studies suggest that only those LHRH neurons which project into the median eminence possess the ability to respond to changes in steroid titre. The complexity of hormonal interrelationships has become increasingly evident with the utilization of specific radioimmunoassay procedures for the quantitation of blood hormonal levels. According to the established concepts gonadal function in adult mammals is controlled by a mutual reciprocal relationship between secretions of the gonads, hypothalamus and pituitary gland. Recent investigations of the temporal organization of hormonal secretions have shown that gonadal hormone secretion is also dependent upon the complex interplay between the intrinsic rhythmicities in various components of endocrine glands of the reproductive system and their integration with the oscillations in other endocrine secretions. Utilizing radioimmunoassay procedures to measure hormone levels during the past 5 years, we have carefully analyzed the temporal sequence of gonadal hormone secretions in the rat [l-3]. Remarkable rhythmicities in the secretion of hormones by the gonads were evident. At present our efforts are directed towards an understanding of the underlying anatomical and physiological bases of these daily periodicities. The results of our endeavours along these lines have been reviewed previously [4]. In this communication we present evidence to show that adrenal secretions may exercise a modulating influence on the gonadal hormone secretions. Several hypotheses have been advanced to explain how gonadal steroids control the pituitary gonadotropin secretions. Extensive investigations have documented the congruency of luteinizing hormone releasing hormone (LHRH) containing pathways and the steroid concentrating neurons in the diencepha-

Ion [S]. However, little is known about the mechanism by which gonadal steroid titres modulate the secretion of these peptidergic neurons in the rat. The second part of this communication summarizes our results on the interaction between gonadal steroids and hypothalamic LHRH activities. CIRCADIAN PERlODlClTlES

IN SERUM

LEVELS OF CONADAL STEROIDS (i) Female rats During the estrous cycle, serum progesterone (P) levels displayed a circadian rhythm characterized by lowest levels around noon which were followed by higher rates of secretion so that levels were maximal at the start of the dark period [l, 23. The amplitude of this circadian rhythm was variable; smallest at estrus and progressively greater on diestrus II, diestrus I and proestrus. There was an apparent reciprocal relationship between the secretory patterns of P and oestradiol (E2) since nadirs in serum E2 concentrations were detected during periods of increased P levels. This suggested that elevated P levels may suppress E2 secretions. We .tested this possibility by examining the effect of exogenous progesterone on ovarian E2 production in uiuo [l]. Administration of ovulation-inhibiting doses of P at different times during the estrous, cycle resulted in the prompt and prolonged suppression of E2 without alterations in serum LH levels (Fig. 1). It is well known that in female rats the ovaries secrete substantial amounts of progesterone, although a small contribution from the adrenals has also been

981

982

KALRA

P. S.

PROGESTERONE

6

I

and S. P.

KALXA

TREATMENT

OIESTRUS

I

0800

DIESTRUS

II

OS00

DIESTRUS

JI

I800

DILSTRUS

It

2300

LJ CONTROL

DIESTRUS I

DIESTRUS I[

PROESTFRUS

ESTRUS

Fig. 1. The effect of progesterone administration (4mg/rat) on oestradiol secretion in intact cyclic rats. Progesterone was injected at times represented by shading of the bars; rats were sacrificed at the times denoted on the abscissa. Numbers in parentheses represent the number of rats/group. Progesterone admi~tration at the times tested rapidty suppressed oestradiol secretion for up to 48 h. From Kalra and Kalra[l].

suggested [3,6,7j. The observation of copsistent daily P rhythms in the cycling rats [l,i] prompted us to distinguish the relative contributions of the adrenals and ovaries in the composition of these daily periodicities. Serum P levels were measured at close intervals during a 24-h period in Iong-term ovariectomized rats (Fig. 2). A distinct diurnal serum P rhythm was evi-

dent; atthough the amplitude was smaller in these rats the time sequence of secietion was similar to that observed in intact rats Cl]. This pattern of P secretion closely raernbled the well documented daily hypersecretion of adrenal corticoids [S]. On the basis of the observation that P ~min~tration suppressed ovarian E2 secretion (Fig. 1) it was postulated that the daily hypersecretion of adrenal P may suppress ovarian E2 secretion and this action may eventuate in the apparent daily rhythmicity in serum E2 concentrations during the estrous cycle [l]. (ii) Male rats

1 lsoo

moo

Fig. 2. Circadian rhythm of serum P con~ntratio~

H

s f

in ovariectomized rats, Although smailer in amplitude the temporal pattern of changes was similar to that in intact female and male rats. Serum LH and FSH values displayed

no significant fluctuations. (5-6 rats/group).

Inter~tingly, a similar reciprocal reIatio~hip between serum P and androgens was observed in intact male rats 133. P concentrations were lowest in the morining and started to increase from 13.30h to peak at 19.30 h and declined through the dark period. On the other hand, androgens, testosterone (T) and Sa-dihydrotestosterone (DHT), were elevated in the morning, peaked at 16.30 h and declined thereafter through 21.30 h during the period when P levels were elevated (Fig. 3). This reciprocal relationship between serum P and androgens Was also evident during the dark period when P declined continuously while T was increasing. Serum LH levels fluctuated randomly during the 24-h period, and presented no discernible relationship with the distinct daily fluctuations in androgen secretion. This observation, again, indicated that factors other than LH, possibly the adrenal se-

Gonadal steroid rhythms

983

Fig. 3. Serum tevels of LH, FSH, progesterone(P), testosterone (T) and dihydrot~t~terone (DH’D in male rats over a 24-h period. Distinct circadian rhythms were evident in P, T aad FSH ievels. S-6 rats/group. Modified from Kalra and Kalra[3].

may be responsible for producing the daily serum androgen periodicity. In order to further assess the role of the adrenals in modulation of gonadal hormone secretion in males, a series of experiments were designed to disrupt the hypoth~~~pituit~adrenal axis. (a) EJiects of anterior-hypothalamic deufferentatian on sewn testosterone and progesterone rhythms. The temporal organ~ation of the various components of the adrenal ghrcocorticoid system has been extensively studied. The hormonal variables in the hypothalamo-pituitary-adrenal axis have been shown to possess a 24-h periodic&y [S]. Based on studies involving lesions and deafferentations [9, lo], it is evident that the neural links between the preoptic-anterior hypothalamic area (POA-AHA) and the medial basal hypothalamus (MBH) comprise the common final pathway in the central regulation of adrenal glucocorticoid periodicities. In view of these observations, we examined the effects of severing of the neural links between the POA-AHA and the MBH (by Hal&z-type knife) on stero~-ho~one rhythms in male rats (Table 1). The reproductive system and gonadotropin release were normal in the deafferented rats [3], but the characteristic reciprocity in serum cretions,

T and P levels normally

observed in intact rats at 22.00 h was blunted by deafferentation thereby resulting in abolition of the rhythms in these rats. These results clearly imply that either the timings of daily troughs and peaks in serum T secretion were shifted or more likely the rhythmicity of testosterone secretion was abolished following disruption of the hypothalamo-pituitary-adrenal axis. (b) E@cts o~~~tr~~n or ~re~ieetomy on serum testosterone and prigesterone rhythms. As in the case of females, the serum P rhythm persisted following gonadal ablation in male rats (Table 1). Surprisingly, however, bilateral ~ren~~orny also failed to modify thin rhythm, whereas it appeared to abolish the daily hypersecretion of serum T even in the presence of normal serum LH levels [3]. Following both adrenalectomy and castration, as expected, the serum P and T levels were non-detectable. From these interesting observations it was apparent that in male rats the adrenals and the testes actively secrete progesterone in a rhythmic fashion although the relative contribution of each gland in the co~guration of the normal rhythm is presently obscure. Importantly, however, the alterations of circadian fluctuations in serum T levels following adrenalectomy clearly

984

P. S.

KALRA

and S. P.

KALRA

Table 1. Effects of anterior hypothalamic deatferentation (AHD), adrenalectomy or castration on circadian variations in serum testosterone and progesterone Testosterone (ng/ml)

Progesterone (pg/ml)

Experiment

08.00 h

22.00 h

08.00 h

Control (sham)

3.88 k 0.79 (4) 4.71 + 1.27 (5) 4.67 k 1.14 (6) 1.32 f 0.23 (1OB

0.83 f 0.20 (5) 2.24 k 0.28

92.1 f 10.48 (4) 106.7 + 20.97 (5) 86.9 + 17.8 (6) 82.7 f 23.3 (10) 74.0 + 21 (5)

AHD Control (sham) Adrenalectomy

ON 1.26 + 0.263 (9) 1.49 * 0.30 (14) -

Castration

22.00 h 343.8 f 66.7* (5) 258.5 f 69.5 (5) 438.5 k 135.51 (9) 388.5 + lll.l$ (14) 316.0 f 106* (5)

Numbers in parentheses indicate the number of rats. * P < 0.01 vs 08.00 h values. t P c 0.02 vs 22.00 h control values. 1 P < 0.02 vs 08.00 h values. $ P < 0.02 vs 08.00 h control values.

demonstrated that the adrenals may secrete in circadian fashion either T itself or some key product(s) which modulates testicular T production. Our current view of the interrelationship between the hypothalamo-pituitaryry-adrenal and the hypotha-

It is well recognized that hypothalamic LHRH regulates the pituitary release of LH which, in turn,

larno-pituitary-gonadal

controls

axes is represented

in Fig. 4.

CONADAL HYPOTHALAMIC

gonadal

ENVIRONMENTAL

__._ .-. .-

steroid

STEROIDS

AND

LHRH ACTIVITY

hormone

production.

FACTORS

- _...._ -.--

SECRETION

/ FLYI

AtTH \

I

J TESTIS ADfWUAL d -Pm PROGESTERONE ( ? 1

Fig. 4. Diagrammatic representation of our hypotheses regarding the interrelationship between the hypothalamo-pituitary-adrenal axis and the hypothalamepituitary-gonadal axis. The neural links between the preoptic-anterior hypothalamic area (POA-AHA) and the medial basal hypothalamus (MBH) comprise the final common neural pathway that incorporates various inputs (corticoid feedback, limbic system, environmental factors, etc.) which regulate the circadian rhythmicity of adrenal corticoid secretions. Disruption of the hypothafam*pituitary-adrenal axis either by transection of these neural links or by adrenalectomy abolished the daily rhythmicity in serum gonadal steroids. This led us to postulate that the circadian adrenal secretion(s) (progesterone?) cause rhythmic variations in gonadal hormone secretion. (ACTH-adrenocorticotropic hormone; cbllm-cerebellum; hipp-hippocampus; pin-pineal; pit-pituitary; poa-preoptic area; sch-suprachiasmatic nucleus; vmh-ventro-medial nucleus). From Kalra and Kalra[4].

Our

Gonadal steroid rhythms

earlier results in male rats showed that whereas fluctuations in the POA LHRH were random, there was a distinct daily rhythm in the MBH LHRH contents [3]. Levels were lowest between 11.00-16.30 h, and these were preceeded and followed by significantly higher levels at 08.30 and 19.3Oh. However, there was no evidence of associated changes in pituitary LH secretion. The fascinating aspect of these observations is related to the elucidation of the mechanisms controlling these MBH LHRH rhythms. It is not known whether the hypothal~i~ LHRH rhythms are endogenously controlled within the neurons that synthesize this hormone or whether other neural or hormonal inputs to the peptidergic neurons influence their secretory activity. We have systematically studied the effects of gonadal steroids on the activity of the hypothalamic LHRH neurons. Castration caused a marked depletion of LHRH in the MBH while replacement with T or oestrogen in both sexes restored LHRH levels to those found in intact rats [l l-143. Further investigations were undertaken to (a) determine the time course of T action

ESTROGEN

985

in raising the MBH LHRH concentrations, (b) identify the active metabolite of T and (c) identify the hypothalamic site(s) at which the steroid effect may be exerted. (i) E$ecFs of subcutaneous imp~a~Farion of gonadni steroids on the ~ypo~~~~rn~ LHRH ~eue~s In castrate rats, restoration of serum T to intact levels by the subcutaneous (s.c.) implantation of T-filled silastic capsules promptly suppressed LH levels within 24 h while the MBH LHRH stores were restored after 7 days [14]. Similarly, in a converse experiment, withdrawal of these T-implants raised LH and FSH levels within 8 and 24h, respectively, whereas significant decrease in the MBH LHRH contents occurred 4-7 days later. Apparently, a period of 4-7 days is required for the changes in the hypothalamic contents of LHRH to occur in response to either treatment with T or following the withdraws of this feedback action. Several possibilities suggest themselves to explain this long latency in the MBH LHRH response to T.

ui-RH (ii%iAN

ANDROGEN@AR and SruMPF 1975) _,-

(NAIK 1975) at d MB)

STEROlD-IMPLANT SITES WHICH RAISE MBH !HRH LEVELS . c--*\

Fig. 5. Diagrammatic comparison of sites containing oestrogen [201 and androgen [21 J concentrating neurons with the localization of LHRH in the hy~thalamus [22 231. Although the LHRH containing peptidergic neurons are congruent with oestrogen and androgen containing neurons we were consistently unable to alter the POA LHRH content (including the LHRH activity in the OVLT) by any steroid treatment. E2 implants (triangles) in the POA as well as the MBH were equally effective in raising the MBH LHRH stores whereas androgens (open circles) were effective only when placed in the MBH. These results suggest the existence of a discrete population of LHRH neurons which are steroid-sensitive and terminate in the median eminence. (AHA-anterior hypothalamic area; ARHarcuate region; CA anterior commissure; CO optic chiasm; ME-median eminence; MM-mammillary body; OVLT-organum vasculosum of the lamina terminalis; POA-preoptic area; SC-suprachiasmatic area; Tb-thalamus; VMH-ventro-medial nucleus).

986

P. S. KALRAand S. P. KALRA

It is possible that physiological T replacement in castrate rats stimulated the de nouo synthesis of LHRH and a period of 3-7 days was necessary to activate and synchronize the cellular events associated with the elaboration, packaging and transport of LHRH to the MBH at a rate higher than in castrates. Similarly, after the withdrawal of T stimulus, the return to castrate levels of these cellular processes, leading to a depletion of LHRH stores may require longer than 4 days. Variations in the rate of intracellular degradation or release of LHRH may also account for the observed changes in MBH LHRH stores. Our ongoing studies are directed towards the investigation of these possibilities. (ii) E$ects

of T metabolites

on hypothalamic

LHRH

neurons in the diencephalon may represent two anatomically different subgroups. Indeed, the highest concentrations of E2 receptors has been detected in the POA-septal region [17, IS], whereas DHT receptors were abundant in the MBH [19]. Alternatively, it is plausible that the receptor sites involved in the regulation of LHRH secretion for both classes of steroid are predominant within the boundaries of the MBH. The effectiveness of the POA E2 implants may be due simply to the relative ease of diffusion of E2 in threshold amounts LHRH storage there.

to the

MBH

to

stimulate

Acknowledgemenrs-This work was supported by grants from the NIH (HD 08634 and HD 11362). Secretarial help of Ms. Lyn Thomas is gratefully acknowledged.

levels

Considerable evidence shows that the central action of T, the predominant circulating androgen in male rats, is manifested following either its aromatization to oestrogen or reduction to Sa-DHT [15,16]. Our comparative study of the effects of different gonadal steroids revealed that DHT and oestradiol were as potent as T in elevating. the MBH LHRH activity in castrate rats. However, the anti-androgen, Flutamide, but not the antioestrogen, Nafoxidine hydrochloride, blocked the stimulatory effects of T and DHT on the MBH LHRH activity. This led us to suggest that in male rats central metabolism of T to DHT may be a physiological requisite in regulating the hypothalamic LHRH activity [14]. (iii) Eflects of intrahypothalamic steroids on hypothalamic LHRH

implants of gonadal levels

The androgens and E2 concentrating neurons and the LHRH neurons display remarkable congruency in the septal-preoptic area and the MBH [S]. Despite this anatomical relationship the S.C. implants of T, DHT or E2 promoted storage of LHRH in the MBH whereas the LHRH content in the rostra1 regions including OVLT was not altered by any of these treatments. This differential action of steroids on the hypothalamic LHRH levels was also apparent following the direct application of small amounts of these steroids in the POA and the MBH [14]. None of the steroids ‘modified LHRH activity locally when implanted in the POA. Androgens were effective only when placed in the MBH whereas implants of E2 in the POA and the MBH promoted storage of LHRH in the MBH. The anatomical localization of LHRH and of androgen and oestrogen receptors along with the identification of hypothalamic sites at which the steroids are effective in raising the MBH LHRH stores are depicted in Fig. 5. There are two possible explanations for this differential site of action of androgen and E2. It is likely that the neural field of E2 is more extensive than that of androgens and probably extends rostrally into the POA. In that case it raises the intriguing possibility that in male rats, the E2 and T concentrating

REFERENCES Kalra S. P. and Kalra P. S.: Temporal interrelationships among circulating levels of estradiol, progesterone and LH during the rat estrous cycle: effects of exogenous progesterone. Endocrinology 95 (1974) 1711-1718. Kalra P. S. and Kalra S. P.: Temporal changes in the hypothalamic and serum luteinizmg hormone-releasiti hormone (LH-RH) levels and the circulating ovarian steroids during the rat oestrous cycle. ACM Endocr., Copenh. (1977) 449-455. Kalra P. S. and Kalra S. P.: Circadian periodicities of serum androgens, progesterone, gonadotropins and luteinizing hormone-releasing hormone in male rats: the effects of hypothalamic deatferentation, castration and adrenalectomy. Endocrinology 101 (1977) 18211827. Kalra S. P. and Kalra P. S.: Central control of endocrine rhythms in the rat. In Enoironmental Endocrinology (Edited by I. Assenmacher and D. S. Farner). Springer-Verlag (1978) pp. 153-162. . . Stumpf W. E. and Sar M.: Steroid hormone target cells in the periventricular brain: relationship to -peptide hormone producing cells. Fed. Proc. 36 (1977) 1973-1977. 6. Brown-Grant K.: The role of steroid hormones in the control of gonadotropin secretion in adult female mammals. In Steroid Hormones and Brain Function (Edited bv C. H. Sawver and R. A. Gorski). Univ. Calif. Press, Los Angeles (i971) pp. 269-288. 7. Hess D. L. and Resko J. A.: The effects of progesterone on the patterns of testosterone and estrabiol concentrations in the systemic plasma of the female rhesus monkey during the intermenstrual period. Endocrinology 92 (1973) 446-453. 8. Yates F. E.: Modeling periodicities in reproductive, adrenocortical and metabolic systems. In Biosynthesis and Human Reproduction. (Edited by M. Ferin, F. Halberg, R. M. Richart and R. L. VandeWiele). Wiley, New York (1974) pp. 133-144. 9. Critchlow V., Liebolt R. A., Barsela M., Mountcastle W. and Lipscomb H. S.: Sex differences in resting pituitary-adrenal function in the rat. Am. J. Physiol. 205 (1963) 807-815. 10. Halasz B.: The endocrine effects of isolation of the hypothalamus from the rest of the brain. In Frontiers in Neuroendocrinology. (Edited by W. F. Ganong and ,L. Martini). Oxford University Press, New York (1969) pp. 307-342. 11. Kalra S. P.: Tissue levels of luteinizing hormonereleasing hormone in the preoptic area and hypothalamus, and serum concentrations of gonadotropins fol-

Gonadal steroid rhythms

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lowing anterior hypothalamic deafferentation and estrogen treatment of the female rat. Endocrinology 99 (1976) 101-107. Kalra S. P., Kalra P. S. and Mitchell E. 0.: Differential response of luteinizing hormone releasing hormone in the basal hypothalamus and the preoptic area following anterior hypothalamic deafferentation and/or castration in male rats. Endocrinology 100 (1977) 201-204. Kalra P. S. and Kalra S. P.: Effects of intrahypothalamic testosterone implants on LHRH levels in the preoptic area and the medial basal hypothalamus. ti@ Sci. 23 (1978) 65-68. Kalra P. S. and Kalra S. P.: Modulation of hypothalamic luteinizing hormone-releasing hormone levels by intracranial and subcutaneous implants of gonadal steroids in castrate rats: effects of androgen and estrogen antagonists. Endocrinology. Submitted@ publication (1978). Naftolin F., Ryan K. J., Davies T. J., Reddy V. V., Flores F., Petro Z., Kuhr M., White R. J., Takaoka Y. and Wolin L.: The formation of estrogens by central neuroendocrine tissue. Recent Progr. Hormone Res. 31 (1975) 295-319. Massa R., Stupnicka E., Kniewald Z. and Martini L.: The transformation of testosterone into dihydrotestosterone by the brain and the anterior pituitary. J. steroid Biochem. 3 (1972) 385-399.

17. Kato J.: Brain receptors for sex steroid hormones and their characterization and functional implications. In

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Neural Hormones and Reproduction (Edited by D. E. Scott, G. P. Kozlowski and A. Weindle). S. Karger, Base1 (1978) pp. 286-301. 18. McKewen B.: Steroid receptors in neuroendocrine tissues: topography, subcellular distribution and functional implications. In Subcellular Mechanisms in Reproductive Neuroendocrinoiogy (Edited by F. Naftolin, K. J. Ryan and J. Davies). Elsevier, Amsterdam (1976) pp. 277-304. 19. Kato J. J.: The role of hypothalamic and hypophyseal Sadihydrotestosterone, estradiol and progesterone receptors in the mechanism of feedback action. J. steroid Biochem. 6 (1975) 979-987. 20. Stumpf W. E.: Autoradiographic techniques and the localization of estrogen, androgen and glucocorticoid in the pituitary and brain. Am. Zoo/. 11, (1971) 72s739. 21. Sar M. and Stumpf W. E.: Distribution of androgenconcentrating neurons in rat brain. In Anaromical Neuroendocrinology (Edited by W. E. Stumpf and L. D. Grant). S. Karger, Basel(1975) pp. 12ci33. 22. Naik D. V.: Immunoreactive LH-RH neurons in the hypothalamus identified by light and fluorescent microscopy. Cell Tiss. Res. 157 (1975) 423436. 23. Hoffman G. E., Melnyk V., Hayes T., Bennett-Clarke C. and Fowler E.: Immunocytology of LHRH neurons. In Neural Hormones and Reproduction (Edited by D. E. Scott, G. P. Kozlowski and L. Weindle). S. Barger, Base1 (1978) pp. 67-82.