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The effect of estrogens on hypothalamic structure and function
The effect of estrogens on hypothalamic structure and function F. NAFTOLlI\, M.D ., D . PIIIL.
J.
R. BRAWER, PII . D .*
M I/lltll'rt/, (!Ill'bl't, Cl/lwda Data accumulated from studies of several species indicate that sex steroids are metaboHzed by neuroendocrine tissues in a manner analogous to that of other target tissues. Evidence that androgens or their estrogenic metaboHtes affect the morphology and function of the nervous system in fetal, newborn, developing, and adult rats is presented . The destruction of neural processes subsequent to the administration of large doses of estrogen to intact rats can now. be added to the previously known effects on synaptogenesis, cell morphology, and function. We believe this destruction to be a form of chemical deafferentation and that it may underlie age-related hypothalamiC failure and the development of multifollicular ovaries in the rat. Implications for other species are not clear at present. (AM. J. OesTET. GVNECOL. 132: 758, 1978.)
RF. CE ~T l:-iTEIl.EST has been focused on the nature and actions of factors concerned with the developing and adult central nervous systems (C~S). The possibility of lIsing animals with short gestations and distinct reproductive patterns plus accessible biochemical processes and anatomy has drawll our attention to t.he laboratory rat as a model for the study of interact.ion of hormonal steroids, especially estrogens, in these tissues. The following article is intended to re\'iew and update our approach to study of the C1\ S. Proof of availabilit.y of estrogens to sensitive estrophilic eNS neurons has led to studies on effects of estrogens on developing and "developed" C:\"S neurons, the results of which encourage continued efforts along these lines as well as investigations in humans and other species.
Availability of estrogens to the eNS
Following the demonstration of in vitro aromatization of andmgens by the human fetal hvpothaiamus,1 From the Depm-tmmLI oj Obstetrics and Gynecolog)', ,\1c(;ill University and the Royd Victoria Hospital. This work was supported by M edim/ Research Council Grants M1'5823 (F.N.) and MA5 9 48 (J. R. B.) and Fmser Jlemoria/ Foundation Funds. Presented by invitatio/l at the One Hundred and First Annual Meeting of thi' American Gynecological Society, Coronado, Califonzin , April 26-29, 1978. R eprint requests: D1". Frederick Sajiolin, Department oj Obstetrics and Gynecology, Yale Medical Cmter, 3J3 Cednr SI., ,Vro' H avm, Connecticut 06510. *1)r. James R. B rawer is a M ediCilI R esearch Council Scholar.
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ill 197~, Ryan and collalx)rators~ re ported to this Society that in several species estrogell is t(>rmed from androgen by hypothalamic homogenates. The tmdertaking of this work was consonant with the idea ofex t ragonadal estrogen formation in male and female subjects. While this so-called " peripheral com er,ion" i~ of inte n's! as a general source of estrogens,a t.he question of it s occurrence in the brain had been raised because of the known ahility of estrogens to mimic mall Y of the ce ntral (C:\S and pituitary) influences previously ascribed directly to androgens . These include braind<> \'elopment and differentiation resulting in sexuallydi~ morphic control of gonadotropin secretion and sexual be havior, triggering precocious pubertv, and male and female behavior. Central to this cOllcept was the finding that. ring A-reduced androgens (surh as dihydrotestosterone) could not alwavs re place estrogens in mimicking such primary androgens as testosterolleand androstenedione' in these ,ictionsand tlte great potency of estrogens in this regard, :" 1\ Perinatal sex ual differentiation of the d eveloping rat brain was Ilsed as a model for testing t.his concept and the capstone of the work was the demo llstration that hypothalatllic homogenates from perinatal rats can aromatize a ndroge ns,7 that nonste r' ,idal estrogens, such as diethylstilbestrol, cause brai n differentiation with inordinate potency ," and that catecho l estrogens arc also able to "defeminize " the perinata l rat brain .n Although androgens appear to influe nce brain rlUferemiation in rats through in situ aromatization , there is considerable evidence of androgen influence on central -reproducU002-9378178/230758+08$OO.80 /0
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--------Ring A - - - - - - - Reduction
Aromatization
Hydroxylation
C21 Corticoids Progestins __
(~ Progestins)
C19 Androgens_
C 18 Estrogens -
Fig. 1. Hypothalamic metabolism of steroid hormones in vitro. (Reproduced, with permission, from 1\ aftolin. 13)
tive function via the Sa-reduced compounds,lO, II and several groups have shown the presence of ring A reduction 12 as well as aromatization and hydroxylation 1:1 in the brains of several species (see Fig. 1). ~ewly made estrogens have now been found to be bound in the nuclei of hypothalamic cells in androgentreated newborn rats. 14 In addition, activity of estrogens has been adduced for both the primary and catechol (2-hydroxylated) estrogens, using cytosol binding of steroids as a marker of C:'IIS biologic activity. 13, 16 Thus, the stage has been set to determine the modus operandi of estrogens in Cl\S tissues. To simplify this presentation we will focus upon the rodent brain as a model, while concentrating on brain morphology and reproductive patterns as markers of estrogen action. Estrogens as growth controlling factors in the developing rat brain
The difference between a reproductively cycling brain and a reproductively tonic (noncycling) brain in the pattern of reproduction in the adult rat depends upon the availability to the brain of estrogen or estrogen precursors (androgens) during a critical perinatal period. 7 That this may have an anatomic basis in neuronal differentiation is indicated by the undeveloped state of the hypothalamus in the neonate l7 ; subsequent development and differentiation occur during the critical period, i.e., at a time when steroids are known to influence sex-specific development of the
Fig. 2. Hypothalamic circuitry regulating gonadotropin secretion. This highly simplified diagram depicts some of the neuronal connections involved in gonadotropin regulation. .Neurons in the suprachiasmatic nucleus (SCH) and/or medial preoptic area (MPOA) project to and presumably drive hypophysiotropic cells in the medial basal hypothalamus (MBH), which includes the arcuate (ARC) and ventromedial (VM) nuclei. In addition, a direct connection from MPOA to the capillaries of the median eminence (ME) is indicated as a collateral branching off of an axon projecting to ARC nuclei. Extrahypothalamic (limbic and midbrain) influences are indicated by the heavy arrows. Ant Pit, Anterior pituitary; AHA, anterior hypothalamic area; CHO, optic chiasm; Srpt Campi, septal area and nuclei.
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PRESENCE OF ESTROGEN
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Fig. 3. A suggested scheme of the effeeLs of estrogen on the developing rat hypothalamus. In the presence of estrogens, the SeH /MPOA neurons connect with MBl! neurons, filling in a synaptic space and causing acyclic reproduction as seen in adult male rats. In the ahsence of estrogen, there is not sufficient growth from the estrophilic neurons in the sell lO fill the synaptic space on neurons in the MBll. Although the space is filled with synapses, they originate from other neurons. The latter arrangement allows cydk reproduction as seen in adult female rats (sec text for details and reference s). See legend to Fig. 2 for abbreviations. It has also been shown that exogenous estrogens can affect synaptogenesis when administered during this critical peri~e\. 20 Further, the recent demonstration of increased and estrogen-dependelllneulOnal growth in slices of mouse hypothalamus 21 conhnns the importance of estrogens in determining the rapidit.y and patterning of critical hypothalamic ci rcuitry in the rodent brain. 22 Probably the circuit responsible for driving (or permitting) sexual cyclicity, and therefore the one directl y affected by gonadal steroids during development, connects the medial preoptic (MPOA)-suprachiasmatic area (SCH) to the medial basal hypothalamus (MBH) .2:l Although the neuronal circuitry regulating gonadotropin secretion is extremely complex (see Fig. 2), neurons in the MPOA-SCH are generally thought to COIltrol or permit cyclic activity. These cells synapse 011 putative luteinizing hormone-releasing factor (LRF) containing neurosecretory cells in the MBH. They promote the release of LRF and. hence luteinizing horbrain.'~ ' 19
Fig. 4. Hasal-Iateral n:gion "r .In Il~ll(' Illl cil.'l" .-, 1 . ' II ulln;1 nclic adult kmale rat in di cstnh. This regiolll>.gt'linalh free of ~lial reaCtivity. An ast rO(')'lt" containing ; L ft:\\ g1iO:"O lllC'" appears ill tile upper left or the lidd (alTO" ., . ( )ngin;d mag ·
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1ll00IC (LH). This highly ovcrsimplified St heme don )lot take into account kllown L.RF fiber pJ'( ~ject iOIls from the !vIPOA 10 the median eminellce or tla · \" ide lariell of extrahvpotha lamic inputs thai influellce thi, system."" The action of estrogen l'l'Ccivcd by the Iwpothalarnu ~ durillg the neonatal period Illay he thal of differen tially efTectillg the filling ill of synaptic "spau'" OJ) recipient IlclIrons ill the MBH. t :llder the inflw 'Il{(:, of ('strogells tJlt' resulting sVllaptic paltcrll ill ;'I,fB H ell genders ac:ydicity in the adult. In the abscll[(' of estrogens or their aromatizable preclIrsor androgens, 1he circuitry dc\·e1ops that supports cvdici t; ul'rcprodLK· tjon and pa rticipates ill t.he mickyde sllrge of Ui ( Fig. 3). This concept does lIot require all effecl of estrogell Oil the "cyclic ('enrel'" of the S(~ H or \1 POA. Such all cHeet remains to lx' shown ,md the refl>IT canllot be assessed at present. [II aliI' el'e lll, surgical separatioll of the \IPOA froul the ;'I,IB H blocks t he receipt or ndic signals. alld )'esuifs . ill a tOllic secretion of gOlladotropins. independcllt of
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Fig. 5. Basal-lateral region of arcuate nucleus of a female rat treated with 2 mg. of estradiol valerate four months previously. This animal exhibits persistent cornified cells on vaginal smearing. Several clusters of astrocvtic gliosomes scattered throughout the regi(m are easily identified (arrows). (Magnificatioll x 1,300.)
any effect on more anterior structures such as the ~lP()A-SCH.2:'. 21<
Effects of estrogens on the hypothalamus in adult animals
In additjon to well-known crfects just described, estrogens can affect the function and anatomy of mature rat brains, i.e., large doses of administered estrogells can cause the development of acydicity and demonstrable lesions and gliosis in the adult rat hypothalamus, Estradiol valerate (E. V., 2 mg.) administered monthly has becn shown to cause severe hypothalamic degeneration and massive anterior pituitary gland enlargcmell1. 27 Single doses of 2 mg. of E.V.2s cause only llIodest (approximately 12 per cent) pituitary enlargement. Following the initial postinjection rise, circulating prolactin ami growth hormone levels return to the normal range by six weeks. Following the KV. injection the animals develop a random smearing pattern and then constant estrus smears at about four or five weeks. At that tjme there are high-normal levels of circulating LH and preovulatory levels of circulating estradiol (about 3() pg. per milliliter of serum). \\fhen removed after thc onset of constant estrus, the ovaries appear small and have multiple follicle cysts but 110 fresh corpora lutea. :Ylultifocal degeneration and gliosis occur and arc well localized to the lateral arcuate nHcleus
(Figs. 4 to 6). Since a similar picture is seen in aged, untreated female rats* the treatment with E.V. may be hastening the progress of a naturally occurring process. Further, this reaction is diminished or perhaps even eliminated in animals who have no ovaries at the time of E. V. ir~jection. * Since the occurrence of hypothalamic patholob'Y in treated female animals seemingly depends upon the presence of the ovary, two separate events may he occurring. The first is the effect of the large estrogen dose. At this point, we can show no cytologic distinction between the brains of E.V.-treated animals and those of controls. The second event is ovary dependent and it is the development of a lateral arcuate nucleus reaction (with acyclicity, etc.) (Fig. 7). A similar anovulatory syndrome also occurs after anterior MB II deafferen La tion. 2o, 26 In view of the degeneration of the hypothalamic arcuate nucleus seen after E.V. treatment in the adult rat, the result of the injection may be a functional chemical deafferentation of the MPOA-SCH from the :vrBH with the sequellae of acyclicity and multicystic ovaries. \Ye are presently investigating this possibility. In summary, estrogens are formed, bound, and biologically active in specific areas of the rat brain. De*Cnpublished data.
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Fig. 6. Basal-lateral region of a rcuate nuclell s of a female ra l p reviously treated with ~ mg. of estradiol valerate and in persistent vaginal estrus. (:\1agnification x 21 ,000.) This electron micrograph shows an astrocyte process filled with numerous pleomorphic inclusions. These correspond to the glioso mes seen at the light microscope level in Fig. 5. Other featu res of this area not shown in this micrograph include degenerating dendrit.ic processes.
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SCH
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Fig. 7. A schematic view of tl;e interruption of cyclicity by 2 mg. of estradiol valerate administered to the adult female rat. Under the influence of the anterior pituitary gland, apparently via the ovary, degeneration and gliosis appear in the area of the basal arcuate nucleus. This degeneration of neuron processes and reactive gliosis are thought to represent a chemical deafferentation or disconnection between the cyclic center and the LRF-delivering neurons that trigger ovulation (cyclicity). The tonic influences on gonadotropin release appear operative since the ovary becomes covered with multiple follicle cysts, the LH is at a high-normal level, and circulating estradiol is in the preovulatory range. The functional status of the so-called cyclic center (SCH-MPOA) is not clear under these circumstances since the deafferentation (broken lines) blocks the expression of the anterior hyperthalamic function . See legend to Fig. 2 for abbreviations.
pending on the timing and size of estrogen dose, a variety of functional and anatomic changes in neuronal wiring and reproductive activities can occur. The
REFERENCES l.
2. 3.
4.
5. 6. 7. 8. 9.
F., Ryan, K.]., and Petro, Z.: Aromatization of androstenedione by the diencephalon, ] . Clin . Endocrinol. Metab. 33: 368, 1971. Ryan, K. J., ~aftolin, "F., Reddy, V., Flores, F.,and Petro, Z.: Estrogen formation in the brain, AM. J. OBSTET. GYXECOL. 114: 4, 1972. Siiteri, P., and McDonald, P. C.: Role of extraglandular estrogen in human endocrinology, in Greep, R. 0., editor: Handbook of Physiology, Vol. 2, Female Reproductive System, Part I, Washington, 1973, American Physiological Society, pp. 615-629. Brown-Grant, K., ~unck, A. , Naftolin, F., and Sherwood, ~. R. C.: The effects of the administration of testosterone and related steroids to female rats during the neonatal period, Horm. Behav. 2: 173,1971. Wilson , J. G.: Reproductive capacity of adult female rats treated prepuberally with estrogenic hormone, Anat. Rec. 86: 341,1943. Gorski, R. A.: Modification of ovulatory mechanisms by postnatal administration of estrogen to the rat, Am. J. Physiol. 205: 842, 1963. Reddy, V. V. R., :\'aftolin, F., and Ryan, K.J.: Conversion of androstenedione to estrone by neural tissues from fetal and neonatal rats, Endocrinology 94: I, 1974. Slaughter, ~ . , Wilen, R., Ryan, K. J., and ~aftolin, F.: The effects of low dose diethylstilbestrol administration in neonatal female rats,]. Steroid Biochem. 8: 621, 1977. Parvizi, ~., and ~aftolin, F.: Effects of catechol estrogens on sexual differentiation in neonatal female rats, Psychoneuroendocrinology 2: 409-411, 1977. ~aftolin,
influence of estrogens in all of these models appears to be exerted at the synaptic level.
10. Swerdloff, R. S., Walsh, P. C., and Odell, W. D.: Control of LH and FSH secretion in the male : Evidence that aromatization of androgens to estradiol is not required for inhibition of gonadotrophin secretion , Steroids 20: 13, 1972. II. Naftolin, F., and Feder, H. H .: Suppression of LH secretion in male rats by 5 a androstan-17,B-OI-3-one (dihydrotestosterone) propionate. J. Endocrinol. 56: 155. 1973. 12. Jaffe. R. B.: Testosterone metabolism in target tissues: Hypothalamic and pituitary tissues of the adult rat and human fetus. and the immature rat epiph ysis. Steroids 14: 483 . 1969. 13. ~aftolin. F.: ~etabolism of steroids in the brain. in James. V. H. T ., editor: Endocrinology: Proceedings of the V International Congress of Eridocrinology. Hamburg,]uly 18-24 . 1976. Vol. I .Amsterdam, 1976. Excerpta ~edica. pp. 29-33 (International Congress Series 1'\0. 402). 14. Lieberburg. I.. and McEwen. B. S.: Estradiol-17,B: A metabolite of testosterone recovered in cell nuclei from limbic areas of neonatal rat brains. Brain Res. 85: 165. 1975. 15. Eisenfeld. A. J.: Hypothalamic estradiol binding macromolecules. ~ature 224: 1202. 1969. 16. Davies. I.J.. ~aftolin, F .• Ryan. K. J.. and Siu.].: Estradiol receptors in the pituitary and anterior hypothalamus of the rat: ~easurement by agar gel electrophoresis. Steroids 25: 5. 1975 . 17. Matsumoto, A .• and Arai. Y.: Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats. Cell Tissue Res. 169: 143. 1976.
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IH. Harris, (~ . W .. and Levine. S.: Sexual differentiation of the brain ,lTld its experimental control. .J. Physiol. 181:
19.
20.
21.
22. 2:1.
24 .
UtTcmhcr I. 197K Alii . J Obs'ct. C;YTl<"" (
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:179 . 196:;. Raisrnan . G .. and Fic:>ld. P. ;vI.: Sexual dimorphism in the preoptic a rea of the rat . Science 173: T\ I. 1971. Matsumoto, A., and Ara i. Y.: Effect of estroge n OIl earl y postnatal development of synaptic formation in the hypothalamic arcHate IludclIS of female rat s. NCHrosci. Lett. 2: 7~1. 1976. Toran-Allcrand. C. D.: Sex steroids and the development of the newhorn lIlouse hypothalamus and preoptic area in vitro: Implications for sexual differentiation. Brain Res. 106: 407 . 1970. :-';aftolin . F .. and Brawer . .J. R.: Sex horlIlones as growt.h promoting factors for the endocrine h ypothalamus, .I . Steroid Eiochem. 8: 339, 1977. Wiegand. S. J, Terasawa. E .• and Eridson. W. E.: Persistent estrus and blockage of progesterone-induced LH releaSt' follows lesions which do not damage the suprachiasmatic nucleus, Endocrinology 102: 164:;. 1978. Brawer, .J. R., and Van Houten, M.: Cellular organization of IU1einizing horm one-releasing factor delivery systems.
;11 :\aftolin. F .. Ryan , K. J..
Subcellulal' ~1ech an i sms in Reprod\lC1ive :\curo"",jocrinology. Amsterd am . E/7ti. Elsevier. '-:orth Holland Publishing Company. pp. 1-:\ I. 2:;. Halasz, E.: The endocrine etklls otisolatioti ,,t riwh ypo· tha lamus from the rest of 1he brain. ill Ganultg. W . L . and Martini. 1.. . editors: Frontiers in NI~lIr()endocr illol ogy. :\('w York. 1969. Ox\(ml l' ni ve rsit\· Pn·s,. I'p. :~ ()7 :H~.
26. Blake. c.. Scaramuzzi . R., Hilliard , .J.. altd SawHT. c. . Circulating It'vcls of piwilary gonad01rophiw, and o'ar· ian st e roids in rats aft.er hypothalamic deafkr('n1at.ion . "I'll rol'lldocrinology 12: HI). 197:{. 27 . Brawcr . .J. R .. and Sonnenschein. C.: Cvwpa1hnlogica l c ff('n s of estradiol on the ;ll'ClIat(· llucleus of th e f('male r~l: :\ possiblt' mechanism for pituit ary tllllloy·ig(,llesi,. :\111 . All at. 144: I, 197:;. 2H. BrawCt'. J. R .. :\aftolin . F.. ;vlartin . J. B .. and Sonnen· schein , C.: Effects of a single injection of estradiol vale ra te.· 011 t.he hypothal amic arcuate nudeu s and on rcprodunive funo ion ill the female rat , Elldocrinology 103: .' )0 I. 197H .
r.
Discussion DR. KENNETH J. RYAN, Boston, Massac husetts . I pmmised Dr. :\'aftolin I would not t.ell how long ago he gave me this paper for review, but it reminded me of a comment I heard Dr. Krevins, Dean a t Cniversity of' California at San Francisco. make just a short time ago. Dr. Krevins was asked about a year ahead of time to make a presentation, and he insisted on knowing his topic at that time . They asked, "'''' hy do you have to know now? It's a ycar away." He replied, "Well, if yo u want me 10 talk about fivc minutes, I'll need a year to prepare . If you want me to talk 15 minutes, I'll need aixlUt six months. If yo u t.ell me right bef(lrc the talk , I'll be spea king for more than an hour. " I willllot say when I received this paper, but if I talk for more than five minutes. you'll know what happened. I would like to congratulate the authors on this continuing work on the effects of steroids in the brain . I would like to point out some of the background [hat I think substantiates the significance of this line of study. In this regard, Bruce ~fcF.wen's work is terribly important. What he has done is adm inister testosterone to animals, remove specific areas of the brain, and show that the radioactive material ha s been converted to estrogen and is in the nuclei of the cells of the brain, suggesting not only that the transformation takes place, but 1hat the product is getting into the nucleus where it could. ill fact, have a biologic effect. I Othcr interesting studies from our laboratory have been performed by Dennis Vaccaro and Jack Cannick who demonstrated aromatization by hypothalamic cells in tissue culturc which points out tha1 thc neurons themselves are involved. 2 [n addition , the authors have raised the question of relevancy from species to species. One quest.ion of personal interest is where in the anima l kingdom does
aromatization take place- ill which srx:cics-am\ hllw far back in the evolutionary ,~cale G ill olle g()~ Dr. Gloria Callard has demonstrated braill ammatase in practically every species tesled, including reptiles. hsh. and Amphibia. It must be important since it is so wide!) dispersed in nature. " The question that arises with respect to the provocative new material presented by the authors concerns the effect of steroids on the an~ltomy of the brain a nd the steroids' mechanism of action. The question is still hcing raised whether or !lOI steroids ca ll have memhralle effects. since challges ill Ilent' firing ca n occur so quickl y after t heij' administratioll, or \dlcther they are ill fart always \"orking in the classic fashion of hormones, thal is, by transport into the nuclei and by cbanging new protein syllthe~js. One of the question s I would like to ask is whether the steroids are acting dircctly or whether the y arc, in fact, aC1ing- by affecting nellrotransm itter productioil or metabolism? It is known that manipulation of the Ilcuro1ransmit.tel' sysICm can provoke some anatomic cha nges ill i ht' central nervolls syst.em. I wonder whether steroids havc a direct cffect on ncr\'(: g-rowth factor. no VOtl have an y information on that possibility? Finally, can you tell liS wh\' the ovary is needed t.o observc the effects you <..iescribc? Have yo u thoug-ht of trying to manipulate this system so that the oyari es might be dispensed with? The presumption from this papeJ ' is that the O\'ary contributes something o ther than estrogen, or a pattern of estrogen administration that is different hom tha1 obtainable with the e xogenous estradiol valerate alone. The atlthors have bee n provocative -in t he best tradi ~ lioll by aski ng more questiolls than thcy have answered.
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REFERENCES I. Lieberburg, I.. and McEwen, B. S.: Estradiol 17f3: A
metabolite of testosterone recovered in cell nuclei from limbic areas of neonatal rat brains, Brain Res. 91: 171, 1975. 2. Canick,.J. A., Vaccaro, D. E., Ryan, K. .J., and Leeman, S. E.: The aromatization of androgens by primary monolayer cultures of fetal rat hypothalamus, Endocrinology 100: 250. 1977. 3. Callard, G. V., Petro, z., and Ryan, K. .J.: Phylogenetic distribution of aromatasc and other androgen-converting enzymes in the central nervous system. Submitted for publication.
DR. ROBERT B. JAFFE, San Francisco, California. I would like to further substantiate the comments of Drs. :\'aftolin, Brawer, and Ryan concerning the importance of central nervous svstem aromatization in the rodent, and to call this Society's attention to the work of my colleagues, Drs. Richard Wiener, Pentti Siiteri, and others. Dr. Wiener had been able to single out individual nuclear groups of the hypothalamus. Dr. Siiteri and co-workers have been able to demonstrate the greatest degree of aromatization in those nuclear groups responsible for production of gonadotropin-releasing hormone. DR. GEORGEANNA SEEGAR JONES, Baltimore, Maryland. This is a most interesting article on a subject I consider to be perhaps the most provocative and clinically promising area of research today. When Dr. Xaftolin and his colleagues first noted that neurons in the anterior hypothalamus had the capability of aromatizing androgens to estrogens, I found their work to be extremely helpful in the theoretical understanding of menstrual physiology and clinical pathology. This was particularly illustrated in the understanding of and treatment for so-called polycystic ovarian disease. According to the work of Barraclough, we had always assumed that the underlying pathology was androgen induced. It was, therefore, extremely difficult to understand and to explain to students how one might treat patients with this syndrome with an antiestrogen, Clomid. The demonstration that pre-estrogen androgens such as androstenedione and testerone might be aromatized to estrogens prior to their physiologic or pathologic effect made this understandable. Dr. :'\ aftolin and colleagues have now taken up the story, begun and beautifully expanded by Dr. Fishman over the years, of catechol estrogens. These peripherally inactive estrogen metabolites may well be the centrallv active ones. While talking with Dr. Pedro Cuatrocasa~ several years ago, he 'suggested that perhaps catechol estradiol might act at the membrane of the neuron by actually fitting into the catecholamine receptor. It might then either translate as a catecholamine or block the action of the catecholamine, thus acting as a competitive inhibitor. DR. N AFTOLIN (Closing). In response to the question
Effect of estrogens on hypothalamic structure and function
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concerning nerve growth factor, there has been some evidence that has caused Dr. K. Ruf to propound the theory that nerve growth factor may be important in sexual maturation. [ This is consistent with the continued growth of major brain tracts through the time of puberty. Regarding the role of the gonad, especiallv the ovary, it seems that the effect of estrogen on the development of the lesion is a two-part mechanism: one, the effect of estrogen; and two, the effect of some continuing inHuence. Since ovariectomy abolishes this progression the ovary is incriminated. Since male animals treated with estradiol valerate develop the same lesion,* estrogen seems the culprit. Presently, we are diminishing the dose of estradiol to refine the model. We can then use silicone rubber implants containing estrogen or other materials to dissect the model. With regard to Dr. Jones' comments, she raised these cogent question in 1972 and they were quite compatible with our own concerns. 2 It is possible that central conversion plays a role in polycystic ovary disease. However, as yet we have no direct evidence to bear on the matter. Some time ago, while working in our laboratory, Dr. George Merriam asked Dr. P. Greengard to study whether catechol estrogens displaced catecholamines from membrane-binding sites, and no displacement was found using an indirect method.* Finally, how does this estrogen effect operate? We do not know. We are extremelv interested in the dIect of catechol estrogens in this picture. They are formed in brain 3 and are biologically active; causing an elevation of gonadotropin in immature male rats. 4 They may function because of the peculiar catechol configuration in the ring A. These compounds could work in one of three ways-as estrogens with eHects on intracellular binding proteins and' the like, or as compounds that interfere with catecholamine metabolism because of their ability to competitively inhibit catechol-o-methyltransferase, or by forming their own metabolites such as the superoxides. In any case, we will be looking at these clements of metabolism and the future should be quite interesting. *Cnpublished data.
REFERENCES I. Ruf, K. B.: How does the brain control the process of puberty? Z. Neurol. 204: 95. 1973. 2. Editorial remarks, Obstet. Gynecol. Surv. 27: 267. 1972. 3. Fishman. J.: Estrogen metabolism by neuroendocrine tissues, in ~aftolin, F., Ryan, K. J., and Davies, I..J.: Subcellular Mechanisms in Reproductive Neuroendocrinology, Amsterdam, 1976, Elsevier Publishing Co., pp. 357-362. 4. ~aftolin, F., Morishita, H., Davies, I.J., Todd, R., Ryan, K. .1 .. and Fishman, J.: 2-Hydroxyestrone induced rise .in serum luteinizing hormone in the immature male rat, BIOchern. Biophys. Res. Comm. 64: 905, 1975.
J.
R. BRAWER, PII . D .*
M I/lltll'rt/, (!Ill'bl't, Cl/lwda Data accumulated from studies of several species indicate that sex steroids are metaboHzed by neuroendocrine tissues in a manner analogous to that of other target tissues. Evidence that androgens or their estrogenic metaboHtes affect the morphology and function of the nervous system in fetal, newborn, developing, and adult rats is presented . The destruction of neural processes subsequent to the administration of large doses of estrogen to intact rats can now. be added to the previously known effects on synaptogenesis, cell morphology, and function. We believe this destruction to be a form of chemical deafferentation and that it may underlie age-related hypothalamiC failure and the development of multifollicular ovaries in the rat. Implications for other species are not clear at present. (AM. J. OesTET. GVNECOL. 132: 758, 1978.)
RF. CE ~T l:-iTEIl.EST has been focused on the nature and actions of factors concerned with the developing and adult central nervous systems (C~S). The possibility of lIsing animals with short gestations and distinct reproductive patterns plus accessible biochemical processes and anatomy has drawll our attention to t.he laboratory rat as a model for the study of interact.ion of hormonal steroids, especially estrogens, in these tissues. The following article is intended to re\'iew and update our approach to study of the C1\ S. Proof of availabilit.y of estrogens to sensitive estrophilic eNS neurons has led to studies on effects of estrogens on developing and "developed" C:\"S neurons, the results of which encourage continued efforts along these lines as well as investigations in humans and other species.
Availability of estrogens to the eNS
Following the demonstration of in vitro aromatization of andmgens by the human fetal hvpothaiamus,1 From the Depm-tmmLI oj Obstetrics and Gynecolog)', ,\1c(;ill University and the Royd Victoria Hospital. This work was supported by M edim/ Research Council Grants M1'5823 (F.N.) and MA5 9 48 (J. R. B.) and Fmser Jlemoria/ Foundation Funds. Presented by invitatio/l at the One Hundred and First Annual Meeting of thi' American Gynecological Society, Coronado, Califonzin , April 26-29, 1978. R eprint requests: D1". Frederick Sajiolin, Department oj Obstetrics and Gynecology, Yale Medical Cmter, 3J3 Cednr SI., ,Vro' H avm, Connecticut 06510. *1)r. James R. B rawer is a M ediCilI R esearch Council Scholar.
758
ill 197~, Ryan and collalx)rators~ re ported to this Society that in several species estrogell is t(>rmed from androgen by hypothalamic homogenates. The tmdertaking of this work was consonant with the idea ofex t ragonadal estrogen formation in male and female subjects. While this so-called " peripheral com er,ion" i~ of inte n's! as a general source of estrogens,a t.he question of it s occurrence in the brain had been raised because of the known ahility of estrogens to mimic mall Y of the ce ntral (C:\S and pituitary) influences previously ascribed directly to androgens . These include braind<> \'elopment and differentiation resulting in sexuallydi~ morphic control of gonadotropin secretion and sexual be havior, triggering precocious pubertv, and male and female behavior. Central to this cOllcept was the finding that. ring A-reduced androgens (surh as dihydrotestosterone) could not alwavs re place estrogens in mimicking such primary androgens as testosterolleand androstenedione' in these ,ictionsand tlte great potency of estrogens in this regard, :" 1\ Perinatal sex ual differentiation of the d eveloping rat brain was Ilsed as a model for testing t.his concept and the capstone of the work was the demo llstration that hypothalatllic homogenates from perinatal rats can aromatize a ndroge ns,7 that nonste r' ,idal estrogens, such as diethylstilbestrol, cause brai n differentiation with inordinate potency ," and that catecho l estrogens arc also able to "defeminize " the perinata l rat brain .n Although androgens appear to influe nce brain rlUferemiation in rats through in situ aromatization , there is considerable evidence of androgen influence on central -reproducU002-9378178/230758+08$OO.80 /0
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--------Ring A - - - - - - - Reduction
Aromatization
Hydroxylation
C21 Corticoids Progestins __
(~ Progestins)
C19 Androgens_
C 18 Estrogens -
Fig. 1. Hypothalamic metabolism of steroid hormones in vitro. (Reproduced, with permission, from 1\ aftolin. 13)
tive function via the Sa-reduced compounds,lO, II and several groups have shown the presence of ring A reduction 12 as well as aromatization and hydroxylation 1:1 in the brains of several species (see Fig. 1). ~ewly made estrogens have now been found to be bound in the nuclei of hypothalamic cells in androgentreated newborn rats. 14 In addition, activity of estrogens has been adduced for both the primary and catechol (2-hydroxylated) estrogens, using cytosol binding of steroids as a marker of C:'IIS biologic activity. 13, 16 Thus, the stage has been set to determine the modus operandi of estrogens in Cl\S tissues. To simplify this presentation we will focus upon the rodent brain as a model, while concentrating on brain morphology and reproductive patterns as markers of estrogen action. Estrogens as growth controlling factors in the developing rat brain
The difference between a reproductively cycling brain and a reproductively tonic (noncycling) brain in the pattern of reproduction in the adult rat depends upon the availability to the brain of estrogen or estrogen precursors (androgens) during a critical perinatal period. 7 That this may have an anatomic basis in neuronal differentiation is indicated by the undeveloped state of the hypothalamus in the neonate l7 ; subsequent development and differentiation occur during the critical period, i.e., at a time when steroids are known to influence sex-specific development of the
Fig. 2. Hypothalamic circuitry regulating gonadotropin secretion. This highly simplified diagram depicts some of the neuronal connections involved in gonadotropin regulation. .Neurons in the suprachiasmatic nucleus (SCH) and/or medial preoptic area (MPOA) project to and presumably drive hypophysiotropic cells in the medial basal hypothalamus (MBH), which includes the arcuate (ARC) and ventromedial (VM) nuclei. In addition, a direct connection from MPOA to the capillaries of the median eminence (ME) is indicated as a collateral branching off of an axon projecting to ARC nuclei. Extrahypothalamic (limbic and midbrain) influences are indicated by the heavy arrows. Ant Pit, Anterior pituitary; AHA, anterior hypothalamic area; CHO, optic chiasm; Srpt Campi, septal area and nuclei.
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PRESENCE OF ESTROGEN
~
SCH
(MPOA)
MBH
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ABSENCE OF ESTROGEN
Fig. 3. A suggested scheme of the effeeLs of estrogen on the developing rat hypothalamus. In the presence of estrogens, the SeH /MPOA neurons connect with MBl! neurons, filling in a synaptic space and causing acyclic reproduction as seen in adult male rats. In the ahsence of estrogen, there is not sufficient growth from the estrophilic neurons in the sell lO fill the synaptic space on neurons in the MBll. Although the space is filled with synapses, they originate from other neurons. The latter arrangement allows cydk reproduction as seen in adult female rats (sec text for details and reference s). See legend to Fig. 2 for abbreviations. It has also been shown that exogenous estrogens can affect synaptogenesis when administered during this critical peri~e\. 20 Further, the recent demonstration of increased and estrogen-dependelllneulOnal growth in slices of mouse hypothalamus 21 conhnns the importance of estrogens in determining the rapidit.y and patterning of critical hypothalamic ci rcuitry in the rodent brain. 22 Probably the circuit responsible for driving (or permitting) sexual cyclicity, and therefore the one directl y affected by gonadal steroids during development, connects the medial preoptic (MPOA)-suprachiasmatic area (SCH) to the medial basal hypothalamus (MBH) .2:l Although the neuronal circuitry regulating gonadotropin secretion is extremely complex (see Fig. 2), neurons in the MPOA-SCH are generally thought to COIltrol or permit cyclic activity. These cells synapse 011 putative luteinizing hormone-releasing factor (LRF) containing neurosecretory cells in the MBH. They promote the release of LRF and. hence luteinizing horbrain.'~ ' 19
Fig. 4. Hasal-Iateral n:gion "r .In Il~ll(' Illl cil.'l" .-, 1 . ' II ulln;1 nclic adult kmale rat in di cstnh. This regiolll>.gt'linalh free of ~lial reaCtivity. An ast rO(')'lt" containing ; L ft:\\ g1iO:"O lllC'" appears ill tile upper left or the lidd (alTO" ., . ( )ngin;d mag ·
nific
1ll00IC (LH). This highly ovcrsimplified St heme don )lot take into account kllown L.RF fiber pJ'( ~ject iOIls from the !vIPOA 10 the median eminellce or tla · \" ide lariell of extrahvpotha lamic inputs thai influellce thi, system."" The action of estrogen l'l'Ccivcd by the Iwpothalarnu ~ durillg the neonatal period Illay he thal of differen tially efTectillg the filling ill of synaptic "spau'" OJ) recipient IlclIrons ill the MBH. t :llder the inflw 'Il{(:, of ('strogells tJlt' resulting sVllaptic paltcrll ill ;'I,fB H ell genders ac:ydicity in the adult. In the abscll[(' of estrogens or their aromatizable preclIrsor androgens, 1he circuitry dc\·e1ops that supports cvdici t; ul'rcprodLK· tjon and pa rticipates ill t.he mickyde sllrge of Ui ( Fig. 3). This concept does lIot require all effecl of estrogell Oil the "cyclic ('enrel'" of the S(~ H or \1 POA. Such all cHeet remains to lx' shown ,md the refl>IT canllot be assessed at present. [II aliI' el'e lll, surgical separatioll of the \IPOA froul the ;'I,IB H blocks t he receipt or ndic signals. alld )'esuifs . ill a tOllic secretion of gOlladotropins. independcllt of
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Fig. 5. Basal-lateral region of arcuate nucleus of a female rat treated with 2 mg. of estradiol valerate four months previously. This animal exhibits persistent cornified cells on vaginal smearing. Several clusters of astrocvtic gliosomes scattered throughout the regi(m are easily identified (arrows). (Magnificatioll x 1,300.)
any effect on more anterior structures such as the ~lP()A-SCH.2:'. 21<
Effects of estrogens on the hypothalamus in adult animals
In additjon to well-known crfects just described, estrogens can affect the function and anatomy of mature rat brains, i.e., large doses of administered estrogells can cause the development of acydicity and demonstrable lesions and gliosis in the adult rat hypothalamus, Estradiol valerate (E. V., 2 mg.) administered monthly has becn shown to cause severe hypothalamic degeneration and massive anterior pituitary gland enlargcmell1. 27 Single doses of 2 mg. of E.V.2s cause only llIodest (approximately 12 per cent) pituitary enlargement. Following the initial postinjection rise, circulating prolactin ami growth hormone levels return to the normal range by six weeks. Following the KV. injection the animals develop a random smearing pattern and then constant estrus smears at about four or five weeks. At that tjme there are high-normal levels of circulating LH and preovulatory levels of circulating estradiol (about 3() pg. per milliliter of serum). \\fhen removed after thc onset of constant estrus, the ovaries appear small and have multiple follicle cysts but 110 fresh corpora lutea. :Ylultifocal degeneration and gliosis occur and arc well localized to the lateral arcuate nHcleus
(Figs. 4 to 6). Since a similar picture is seen in aged, untreated female rats* the treatment with E.V. may be hastening the progress of a naturally occurring process. Further, this reaction is diminished or perhaps even eliminated in animals who have no ovaries at the time of E. V. ir~jection. * Since the occurrence of hypothalamic patholob'Y in treated female animals seemingly depends upon the presence of the ovary, two separate events may he occurring. The first is the effect of the large estrogen dose. At this point, we can show no cytologic distinction between the brains of E.V.-treated animals and those of controls. The second event is ovary dependent and it is the development of a lateral arcuate nucleus reaction (with acyclicity, etc.) (Fig. 7). A similar anovulatory syndrome also occurs after anterior MB II deafferen La tion. 2o, 26 In view of the degeneration of the hypothalamic arcuate nucleus seen after E.V. treatment in the adult rat, the result of the injection may be a functional chemical deafferentation of the MPOA-SCH from the :vrBH with the sequellae of acyclicity and multicystic ovaries. \Ye are presently investigating this possibility. In summary, estrogens are formed, bound, and biologically active in specific areas of the rat brain. De*Cnpublished data.
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Fig. 6. Basal-lateral region of a rcuate nuclell s of a female ra l p reviously treated with ~ mg. of estradiol valerate and in persistent vaginal estrus. (:\1agnification x 21 ,000.) This electron micrograph shows an astrocyte process filled with numerous pleomorphic inclusions. These correspond to the glioso mes seen at the light microscope level in Fig. 5. Other featu res of this area not shown in this micrograph include degenerating dendrit.ic processes.
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Fig. 7. A schematic view of tl;e interruption of cyclicity by 2 mg. of estradiol valerate administered to the adult female rat. Under the influence of the anterior pituitary gland, apparently via the ovary, degeneration and gliosis appear in the area of the basal arcuate nucleus. This degeneration of neuron processes and reactive gliosis are thought to represent a chemical deafferentation or disconnection between the cyclic center and the LRF-delivering neurons that trigger ovulation (cyclicity). The tonic influences on gonadotropin release appear operative since the ovary becomes covered with multiple follicle cysts, the LH is at a high-normal level, and circulating estradiol is in the preovulatory range. The functional status of the so-called cyclic center (SCH-MPOA) is not clear under these circumstances since the deafferentation (broken lines) blocks the expression of the anterior hyperthalamic function . See legend to Fig. 2 for abbreviations.
pending on the timing and size of estrogen dose, a variety of functional and anatomic changes in neuronal wiring and reproductive activities can occur. The
REFERENCES l.
2. 3.
4.
5. 6. 7. 8. 9.
F., Ryan, K.]., and Petro, Z.: Aromatization of androstenedione by the diencephalon, ] . Clin . Endocrinol. Metab. 33: 368, 1971. Ryan, K. J., ~aftolin, "F., Reddy, V., Flores, F.,and Petro, Z.: Estrogen formation in the brain, AM. J. OBSTET. GYXECOL. 114: 4, 1972. Siiteri, P., and McDonald, P. C.: Role of extraglandular estrogen in human endocrinology, in Greep, R. 0., editor: Handbook of Physiology, Vol. 2, Female Reproductive System, Part I, Washington, 1973, American Physiological Society, pp. 615-629. Brown-Grant, K., ~unck, A. , Naftolin, F., and Sherwood, ~. R. C.: The effects of the administration of testosterone and related steroids to female rats during the neonatal period, Horm. Behav. 2: 173,1971. Wilson , J. G.: Reproductive capacity of adult female rats treated prepuberally with estrogenic hormone, Anat. Rec. 86: 341,1943. Gorski, R. A.: Modification of ovulatory mechanisms by postnatal administration of estrogen to the rat, Am. J. Physiol. 205: 842, 1963. Reddy, V. V. R., :\'aftolin, F., and Ryan, K.J.: Conversion of androstenedione to estrone by neural tissues from fetal and neonatal rats, Endocrinology 94: I, 1974. Slaughter, ~ . , Wilen, R., Ryan, K. J., and ~aftolin, F.: The effects of low dose diethylstilbestrol administration in neonatal female rats,]. Steroid Biochem. 8: 621, 1977. Parvizi, ~., and ~aftolin, F.: Effects of catechol estrogens on sexual differentiation in neonatal female rats, Psychoneuroendocrinology 2: 409-411, 1977. ~aftolin,
influence of estrogens in all of these models appears to be exerted at the synaptic level.
10. Swerdloff, R. S., Walsh, P. C., and Odell, W. D.: Control of LH and FSH secretion in the male : Evidence that aromatization of androgens to estradiol is not required for inhibition of gonadotrophin secretion , Steroids 20: 13, 1972. II. Naftolin, F., and Feder, H. H .: Suppression of LH secretion in male rats by 5 a androstan-17,B-OI-3-one (dihydrotestosterone) propionate. J. Endocrinol. 56: 155. 1973. 12. Jaffe. R. B.: Testosterone metabolism in target tissues: Hypothalamic and pituitary tissues of the adult rat and human fetus. and the immature rat epiph ysis. Steroids 14: 483 . 1969. 13. ~aftolin. F.: ~etabolism of steroids in the brain. in James. V. H. T ., editor: Endocrinology: Proceedings of the V International Congress of Eridocrinology. Hamburg,]uly 18-24 . 1976. Vol. I .Amsterdam, 1976. Excerpta ~edica. pp. 29-33 (International Congress Series 1'\0. 402). 14. Lieberburg. I.. and McEwen. B. S.: Estradiol-17,B: A metabolite of testosterone recovered in cell nuclei from limbic areas of neonatal rat brains. Brain Res. 85: 165. 1975. 15. Eisenfeld. A. J.: Hypothalamic estradiol binding macromolecules. ~ature 224: 1202. 1969. 16. Davies. I.J.. ~aftolin, F .• Ryan. K. J.. and Siu.].: Estradiol receptors in the pituitary and anterior hypothalamus of the rat: ~easurement by agar gel electrophoresis. Steroids 25: 5. 1975 . 17. Matsumoto, A .• and Arai. Y.: Developmental changes in synaptic formation in the hypothalamic arcuate nucleus of female rats. Cell Tissue Res. 169: 143. 1976.
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IH. Harris, (~ . W .. and Levine. S.: Sexual differentiation of the brain ,lTld its experimental control. .J. Physiol. 181:
19.
20.
21.
22. 2:1.
24 .
UtTcmhcr I. 197K Alii . J Obs'ct. C;YTl<"" (
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:179 . 196:;. Raisrnan . G .. and Fic:>ld. P. ;vI.: Sexual dimorphism in the preoptic a rea of the rat . Science 173: T\ I. 1971. Matsumoto, A., and Ara i. Y.: Effect of estroge n OIl earl y postnatal development of synaptic formation in the hypothalamic arcHate IludclIS of female rat s. NCHrosci. Lett. 2: 7~1. 1976. Toran-Allcrand. C. D.: Sex steroids and the development of the newhorn lIlouse hypothalamus and preoptic area in vitro: Implications for sexual differentiation. Brain Res. 106: 407 . 1970. :-';aftolin . F .. and Brawer . .J. R.: Sex horlIlones as growt.h promoting factors for the endocrine h ypothalamus, .I . Steroid Eiochem. 8: 339, 1977. Wiegand. S. J, Terasawa. E .• and Eridson. W. E.: Persistent estrus and blockage of progesterone-induced LH releaSt' follows lesions which do not damage the suprachiasmatic nucleus, Endocrinology 102: 164:;. 1978. Brawer, .J. R., and Van Houten, M.: Cellular organization of IU1einizing horm one-releasing factor delivery systems.
;11 :\aftolin. F .. Ryan , K. J..
Subcellulal' ~1ech an i sms in Reprod\lC1ive :\curo"",jocrinology. Amsterd am . E/7ti. Elsevier. '-:orth Holland Publishing Company. pp. 1-:\ I. 2:;. Halasz, E.: The endocrine etklls otisolatioti ,,t riwh ypo· tha lamus from the rest of 1he brain. ill Ganultg. W . L . and Martini. 1.. . editors: Frontiers in NI~lIr()endocr illol ogy. :\('w York. 1969. Ox\(ml l' ni ve rsit\· Pn·s,. I'p. :~ ()7 :H~.
26. Blake. c.. Scaramuzzi . R., Hilliard , .J.. altd SawHT. c. . Circulating It'vcls of piwilary gonad01rophiw, and o'ar· ian st e roids in rats aft.er hypothalamic deafkr('n1at.ion . "I'll rol'lldocrinology 12: HI). 197:{. 27 . Brawcr . .J. R .. and Sonnenschein. C.: Cvwpa1hnlogica l c ff('n s of estradiol on the ;ll'ClIat(· llucleus of th e f('male r~l: :\ possiblt' mechanism for pituit ary tllllloy·ig(,llesi,. :\111 . All at. 144: I, 197:;. 2H. BrawCt'. J. R .. :\aftolin . F.. ;vlartin . J. B .. and Sonnen· schein , C.: Effects of a single injection of estradiol vale ra te.· 011 t.he hypothal amic arcuate nudeu s and on rcprodunive funo ion ill the female rat , Elldocrinology 103: .' )0 I. 197H .
r.
Discussion DR. KENNETH J. RYAN, Boston, Massac husetts . I pmmised Dr. :\'aftolin I would not t.ell how long ago he gave me this paper for review, but it reminded me of a comment I heard Dr. Krevins, Dean a t Cniversity of' California at San Francisco. make just a short time ago. Dr. Krevins was asked about a year ahead of time to make a presentation, and he insisted on knowing his topic at that time . They asked, "'''' hy do you have to know now? It's a ycar away." He replied, "Well, if yo u want me 10 talk about fivc minutes, I'll need a year to prepare . If you want me to talk 15 minutes, I'll need aixlUt six months. If yo u t.ell me right bef(lrc the talk , I'll be spea king for more than an hour. " I willllot say when I received this paper, but if I talk for more than five minutes. you'll know what happened. I would like to congratulate the authors on this continuing work on the effects of steroids in the brain . I would like to point out some of the background [hat I think substantiates the significance of this line of study. In this regard, Bruce ~fcF.wen's work is terribly important. What he has done is adm inister testosterone to animals, remove specific areas of the brain, and show that the radioactive material ha s been converted to estrogen and is in the nuclei of the cells of the brain, suggesting not only that the transformation takes place, but 1hat the product is getting into the nucleus where it could. ill fact, have a biologic effect. I Othcr interesting studies from our laboratory have been performed by Dennis Vaccaro and Jack Cannick who demonstrated aromatization by hypothalamic cells in tissue culturc which points out tha1 thc neurons themselves are involved. 2 [n addition , the authors have raised the question of relevancy from species to species. One quest.ion of personal interest is where in the anima l kingdom does
aromatization take place- ill which srx:cics-am\ hllw far back in the evolutionary ,~cale G ill olle g()~ Dr. Gloria Callard has demonstrated braill ammatase in practically every species tesled, including reptiles. hsh. and Amphibia. It must be important since it is so wide!) dispersed in nature. " The question that arises with respect to the provocative new material presented by the authors concerns the effect of steroids on the an~ltomy of the brain a nd the steroids' mechanism of action. The question is still hcing raised whether or !lOI steroids ca ll have memhralle effects. since challges ill Ilent' firing ca n occur so quickl y after t heij' administratioll, or \dlcther they are ill fart always \"orking in the classic fashion of hormones, thal is, by transport into the nuclei and by cbanging new protein syllthe~js. One of the question s I would like to ask is whether the steroids are acting dircctly or whether the y arc, in fact, aC1ing- by affecting nellrotransm itter productioil or metabolism? It is known that manipulation of the Ilcuro1ransmit.tel' sysICm can provoke some anatomic cha nges ill i ht' central nervolls syst.em. I wonder whether steroids havc a direct cffect on ncr\'(: g-rowth factor. no VOtl have an y information on that possibility? Finally, can you tell liS wh\' the ovary is needed t.o observc the effects you <..iescribc? Have yo u thoug-ht of trying to manipulate this system so that the oyari es might be dispensed with? The presumption from this papeJ ' is that the O\'ary contributes something o ther than estrogen, or a pattern of estrogen administration that is different hom tha1 obtainable with the e xogenous estradiol valerate alone. The atlthors have bee n provocative -in t he best tradi ~ lioll by aski ng more questiolls than thcy have answered.
Volume 132 :'I;umher 7
REFERENCES I. Lieberburg, I.. and McEwen, B. S.: Estradiol 17f3: A
metabolite of testosterone recovered in cell nuclei from limbic areas of neonatal rat brains, Brain Res. 91: 171, 1975. 2. Canick,.J. A., Vaccaro, D. E., Ryan, K. .J., and Leeman, S. E.: The aromatization of androgens by primary monolayer cultures of fetal rat hypothalamus, Endocrinology 100: 250. 1977. 3. Callard, G. V., Petro, z., and Ryan, K. .J.: Phylogenetic distribution of aromatasc and other androgen-converting enzymes in the central nervous system. Submitted for publication.
DR. ROBERT B. JAFFE, San Francisco, California. I would like to further substantiate the comments of Drs. :\'aftolin, Brawer, and Ryan concerning the importance of central nervous svstem aromatization in the rodent, and to call this Society's attention to the work of my colleagues, Drs. Richard Wiener, Pentti Siiteri, and others. Dr. Wiener had been able to single out individual nuclear groups of the hypothalamus. Dr. Siiteri and co-workers have been able to demonstrate the greatest degree of aromatization in those nuclear groups responsible for production of gonadotropin-releasing hormone. DR. GEORGEANNA SEEGAR JONES, Baltimore, Maryland. This is a most interesting article on a subject I consider to be perhaps the most provocative and clinically promising area of research today. When Dr. Xaftolin and his colleagues first noted that neurons in the anterior hypothalamus had the capability of aromatizing androgens to estrogens, I found their work to be extremely helpful in the theoretical understanding of menstrual physiology and clinical pathology. This was particularly illustrated in the understanding of and treatment for so-called polycystic ovarian disease. According to the work of Barraclough, we had always assumed that the underlying pathology was androgen induced. It was, therefore, extremely difficult to understand and to explain to students how one might treat patients with this syndrome with an antiestrogen, Clomid. The demonstration that pre-estrogen androgens such as androstenedione and testerone might be aromatized to estrogens prior to their physiologic or pathologic effect made this understandable. Dr. :'\ aftolin and colleagues have now taken up the story, begun and beautifully expanded by Dr. Fishman over the years, of catechol estrogens. These peripherally inactive estrogen metabolites may well be the centrallv active ones. While talking with Dr. Pedro Cuatrocasa~ several years ago, he 'suggested that perhaps catechol estradiol might act at the membrane of the neuron by actually fitting into the catecholamine receptor. It might then either translate as a catecholamine or block the action of the catecholamine, thus acting as a competitive inhibitor. DR. N AFTOLIN (Closing). In response to the question
Effect of estrogens on hypothalamic structure and function
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concerning nerve growth factor, there has been some evidence that has caused Dr. K. Ruf to propound the theory that nerve growth factor may be important in sexual maturation. [ This is consistent with the continued growth of major brain tracts through the time of puberty. Regarding the role of the gonad, especiallv the ovary, it seems that the effect of estrogen on the development of the lesion is a two-part mechanism: one, the effect of estrogen; and two, the effect of some continuing inHuence. Since ovariectomy abolishes this progression the ovary is incriminated. Since male animals treated with estradiol valerate develop the same lesion,* estrogen seems the culprit. Presently, we are diminishing the dose of estradiol to refine the model. We can then use silicone rubber implants containing estrogen or other materials to dissect the model. With regard to Dr. Jones' comments, she raised these cogent question in 1972 and they were quite compatible with our own concerns. 2 It is possible that central conversion plays a role in polycystic ovary disease. However, as yet we have no direct evidence to bear on the matter. Some time ago, while working in our laboratory, Dr. George Merriam asked Dr. P. Greengard to study whether catechol estrogens displaced catecholamines from membrane-binding sites, and no displacement was found using an indirect method.* Finally, how does this estrogen effect operate? We do not know. We are extremelv interested in the dIect of catechol estrogens in this picture. They are formed in brain 3 and are biologically active; causing an elevation of gonadotropin in immature male rats. 4 They may function because of the peculiar catechol configuration in the ring A. These compounds could work in one of three ways-as estrogens with eHects on intracellular binding proteins and' the like, or as compounds that interfere with catecholamine metabolism because of their ability to competitively inhibit catechol-o-methyltransferase, or by forming their own metabolites such as the superoxides. In any case, we will be looking at these clements of metabolism and the future should be quite interesting. *Cnpublished data.
REFERENCES I. Ruf, K. B.: How does the brain control the process of puberty? Z. Neurol. 204: 95. 1973. 2. Editorial remarks, Obstet. Gynecol. Surv. 27: 267. 1972. 3. Fishman. J.: Estrogen metabolism by neuroendocrine tissues, in ~aftolin, F., Ryan, K. J., and Davies, I..J.: Subcellular Mechanisms in Reproductive Neuroendocrinology, Amsterdam, 1976, Elsevier Publishing Co., pp. 357-362. 4. ~aftolin, F., Morishita, H., Davies, I.J., Todd, R., Ryan, K. .1 .. and Fishman, J.: 2-Hydroxyestrone induced rise .in serum luteinizing hormone in the immature male rat, BIOchern. Biophys. Res. Comm. 64: 905, 1975.