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Blood concentrations and biological effects of androstanediols at the onset of puberty in the female rat
J. steroid Biochem. Vol. 19, No. I, pp. 883-886, Printed in Great Britain. All rights reserved
1983 Copyright
0022-4731/83 $3.00+0.00 0 1983 Pergamon Press Ltd
31. Puberty
BLOOD CONCENTRATIONS AND BIOLOGICAL OF ANDROSTANEDIOLS AT THE ONSET PUBERTY IN THE FEMALE RAT BENJAMIN
EFFECTS OF
ECKSTEIN
Department of Zoology, The Institute of Life Sciences, Hebrew University of Jerusalem, Israel
SUMMARY Androstanediols are the major products of the immature rat ovary. About two-thirds are present in blood as sulfates. One of the sulfate conjugates was identified as 5a-androstane-3b,l7B-diol-3-monosulfate. This steroid effectively inhibits postcastrational LH elevation in a concentration in which it is present in blood. Other androstanediol sulfates examined were without this activity. In the immature rat, levels of 3a- and 3/Sdiol decrease sharply before the first ovulation. In Wistar rats, PMSG-treatment induced a similar decline in these steroids. When ovulation in PMSG-treated rats was blocked by pentobarbitone, the LH-surge and the decline of the 3a- and 3/?-diols was delayed. Thus, it seems that the decline in the blood concentration of the diols depends on the first LH surge.
In the female rat, puberty may be defined as the developmental stage at which cyclic ovulation is initiated. Accordingly, the first preovulatory surge of gonadotropins can be visualized as the final product of maturation of the CNS-pituitary-ovary interrelationship. The physiological mechanism leading to maturation are not yet understood. In the rat, serum levels of both gonadotropins remain low during the prepubertal period, to increase abruptly in a preovulatory surge on the afternoon of the first proestrus [l-3]. Recent observations have shown that LH release is pulsatile in peripubertal female rats. With approaching puberty, the magnitude of the afternoon peaks becomes significantly greater than that of the morning peaks [4]. Serum estradiol levels (E,) increase on the day of the first proestrus [S], or just before the first preovulatory surge of gonadotropins [6,7]. This increase in Ez titer seems to play a major role in inducing the first gonadotropin surge, thus leading to pubertal ovulation [S]. For earlier endocrine functions of ovarian steroids, as, for instance, maintaining the negative feedback of gonadotropin release in the infantile rat, steroids lacking uterotropic activity would be ideal, but they would have to be phased out around the time of the first ovulation. It was shown that in the immature rat ovaries the predominant products resulting from progesterone metabolism are See-reduced C 1,-steroids, mainly 5a-androstane-3q 17P-diol (3cc-diol ; [9, lo]). These compounds originate through biosynthetic pathways involving the formation of &-reduced pregnanes that serve as intermediates [IO, 1 I]. One of the reasons for the high levels of the See-reduced C,,-steroids is the high activity of 4-ene-steroid Sa-reductase in the immature ovary [ 121. The formation of See-reduced
C,g-steroids from labeled progesterone is dramatically reduced during the few hours preceding pregnant mare serum gonadotropin (PMSG)-induced ovulation [ 131. Direct measurements of Sa-reductase in the immature ovary induced to ovulate by PMSG showed that on the third day following gonadotropin administration, only one tenth of the high initial activity of the enzyme remained, which declined further to undetectable level [ 143. In the present paper we examine several biological effects of the androstanediols in controlling the endocrine balance up to the onset of puberty and describe serum concentrations of androgens during spontaneous sexual development and around first ovulation induced by PMSG-treatment. EFFECK OF 3&DIOL ON VAGINAL OPENING The only obvious phenotypical change associated with puberty in the rat and mouse is resorption of the vaginal membrane. It is recognized however, that vaginal opening is often dissociated from the first estrus, and that several steroids induce vaginal opening without advancing puberty; in most of the rats of the Hebrew University colony, these two events may show a time lag of 7.5 days [IS]. Similar observations have been reported by others [2, 161. Nevertheless, it was found that a dose of 50 pg 3/?-diol per day from days 22 to 26 induced precocious vaginal opening, while vaginal smears remained diestrous [ 151. Recently Kramer and Meijs-Roelofs in a paper devoted almost entirely to this point, confirmed our finding: “nine out of eleven rats showed a ‘pinhole’ type of vaginal opening some days before the day of first oestrus” [ 171. 883
X84
Bf >JAMfN
COVTROL
OF LH RELEASE
E(.ksit:ih BLOOD
CO%\ICE:1fRATIO\S
SPOIVTAUEOI’S
In the peripheral circulation of immature female rats of the Hebrew University strain (descendants of Wistar strain), the androstanediols are present conjugated to sulfates or glucuronates and only low levels are detected as free steroids [ 1S]. Since the major part of the androstanediol conjugates were sulfates, we determined the form of the conjugation. The 3/$diol were found to be present in blood as both mono(3/r’-A-MS) and disulfate. while the 3x-diol was found to be present only as disulfate (3x-A-DS). On further analysis it was shown that the sulfate moiety on 3/I-A-MS present in peripheral blood of immature rats is attached to the C-3 of the steroid nucleus. The androstanediol sulfates of interest were synthesized and their effects as well as those of the free 3cc-diol, 3/&diol and E, on LH suppression in ovariectomized immature rats were examined. In the immature rat, as well as in the adult, castration leads to a markedly increased blood concentration of LH. While in the adult E, seems to be the main suppressor of pituitary LH release, the predominance of this steroid as the negative feedback controller during earlier phases of life is less certain in view of its low concentration in blood from about 25 days onward [19] and strong binding to r-fetoprotein which is reported to circulate in high concentration up to 25 days [20] or up to 3G-35 days [2l]; binding to x-fetoprotein presumably prevents the E, from exerting its biological activity. Hence. it was of interest to see whether the 3fi-A-MS has an effect on tonic LH release. Quantitative analysis of the feedback relationship between LH release and the concentration of the peripheral steroid is not possible by any injection regimen, since the level of the injected steroid declines markedly during the time between injections. To establish the threshold value of the androstanediols which are effective as feedback signals and to eliminate the between-treatment fluctuations, we used controlled release (Silastic) capsules for the administration of the steroids. In a preliminary experiment it was found that a capsule of 3.0 cm released enough 3/I-A-MS (1.1 ng/ml serum) to establish a constant blood concentration similar or lower than those of intact rats. The results showed that 3/?-diol, 3a-dioldisulfate (6.0 cm) and 3P-diol-monosulfate ( 1.5cm) had no effect, while E, (l.Ocm), 3b-A-MS (3.0 and 6.0 cm) as well as 3a-diol (3.0 cm) were effective in suppressing LH release during the period of 31-45 days of age. Thus, it seems that a constant serum level of 1.1 ng/ml of 3fi-A-MS maintains a serum level of LH similar to that present in the intact rat. Extrapolating these findings to the normal physiological situation, it seems that this steroid by itself is capable of maintaining the negative feedback of LH release. Therefore, we propose that 3/j’-A-MS perhaps in conjunction with E, may be involved in the control of prepubertal LH release.
A’VD IYDlICED
Dl’RI\G SESl AL
\l.ATIJRATIOh
We compared serum concentrations of the nndrostanediols of two strains of rats. In the untreated Wistar rat, hydrolyzed 3cc-diol levels fell sharply between from weaning and the time of vaginal opening, 15.8 k I.5 to 3.1 f 0.X ngiml (mean + SE) [22], while in Sprague-Dawley rats (which develop at a faster rate) unhydrolyzed 3a-dial level fell between days 21-26 from 651 _t I28 to 198 + 31 pg/ml (mean + SEM)[23]. Thus, while the absolute values in the Sprague-Dawley rats were much lower, the decline in the active steroids were similar in both strains. These results are in accord with the recent finding of Advis er ~/.[24], who showed (using the Holtzman strain rat) that up to the first ovulation the rat ovary produces large quantities of 3x-diol when challenged iri vitro by hCG, while after ovulation the quantity produced by the ovary decreased to one tenth of the original value. In the Wistar strain, PMSG-treatment on day 25 induced a considerable decrease in hydrolyzed blood 3r- and 30-diol levels after 48 h. The level of 3x-diol reached a nadir 72 h following gonadotropin administration (I .O k 0.6 ng/ml). The 3/j’-diol fell below detection limit (80 pg/ml) 56 h following PMSG administration (Eckstein et trl. to be published). Comparable results were obtained by Armstrong[25] who measured metabolism of progesterone in intact ovaries irr virro following administration of 4 IU PMSG. Androsterone production gradually increased. reaching maximum levels on day I and remained elevated until 14:OO h on day 2 (the equivalent of proestrus). With the first detectable rise in serum LH levels on day 2. androsterone production declined abruptly reaching minimum levels at 22:00 h on day 2. In the PMSG model, the preovulatory LH surge. and hence ovulation, can be blocked by the administration of pentobarbitone during the early afternoon of the second day following the injection of the gonadotropin. This design was used to ascertain the possible relationship between the observed drop in diol concentrations and ovulation. To this end. rats received ;I dose of 10 ILJ PMSG at 08:OO h on day 25 and an i.p. injection of pentobarbitone (3Omgjkg b.wt.) at 13:45 h on day 27. The presence or absence of tubal ova was verified at 08:OO h on day 28. The results indicate that in unblocked animals high levels of 3a- and 3/i’-diol prevailed 52 h following PMSG (i.e. at l2:OO h on day 27). Six hours later, a sharp decline accompanied the LH surge. In pentobarbitoneblocked rats. no LH surge occurred at this time. and high levels of 3a- and 3[{-dial were present. The concentration of both diols was much lower. however. at 12:OO h on day 28, although the (pentobarbitonedelayed) LH surge occurred only at 16:OO h. Thus, it seems that the decrease in androstanediols is linked to
Effect of androstanediols
on puberty
the process published).
885
of first ovulation
CONTROL
(Eckstein
OF GONADOTROPIN
et al. to be
RELEASE
In Sprague-Dawley rats, chronic implants of 3cc-diol, but not 3/I-diol, prevented uterine and ovarian development as assessed on the basis of organ weights and by ovarian histology 30 days later. Anterior pituitary weights were also greatly reduced, and their LH content was less than 10% of blank implanted controls, or of rats bearing 3/Gdiol implants. When administered to prepubertal rats by single injection, 3a-diol was found to inhibit the gonadotropin surge triggered by sequential treatment with E, and progesterone and to prevent the El-induced sensitization of the pituitary gonadotrophs to LHRH [26]. It was our intent to see whether doses of 3fi-A-MS that are in the physiological range inhibit ovarian and uterine growth in immature rats. From Table 1 it is evident that even a very small dose of 1Opg 3/I-A-MS/100 g b.wt/day inhibited ovarian and adrenal weights. Uterine weight increase was inhibited only with a larger dose (25 pg/lOO g b.wt/day). The administration of 3~-A-DS in a dose IO times higher, had no effect on these parameters. The results show that the biologically effective androstanediols are potent inhibitors of reproductive organs in the female rat. The inhibitory effect of 3r-diol on the first ovulation has recently been confirmed [ 173. -OWN-WNO
dvi,-ci-+r;v(titi fl +I fl fl +I +I +I +I
-cavlmwcrv?* ti~rjr;drdr6ri
-N---N !2’=__----
*ww-
wwwt-
CONCLUSIONS
In the Wistar substrain of the Hebrew University, as well as the strain used at the Hormone Research Department of the Weizmann Institute in Rehovot, the androstanediols are conjugated as sulfates and, to a lesser extent, to glucuronates; only a very small fraction can be detected as free alcohols, Both androstanediols are present in peripheral blood of prepubertal females in the concentration range of several ng/ml. The values of both diols (conjugates) are high in the prepubertal animal and decline after the first ovulation. One of the androstanediol sulfates, 3P-A-MS, suppresses postcastrational LH elevation at a dose in which it is found in peripheral circulation. The same steroid inhibits ovarian, uterine and adrenal growth at a very low dose. The biological activity of 3fi-A-MS as opposed to the free 3b-diol is of interest. It is possible that the 3/I-A-MS is taken up directly by the tissue regulating the negative feedback signal, since large concentrations of the free 3/I-diol do not exert this effect. It has recently been demonstrated that in the rat and ram pituitary the 3/I-diol is bound with high affinity to specific proteins, most probably to estrogen receptors, where it is the most potent competitor of all the androgens tested [27]. Furthermore, 3fl-diol is able to
Bt \iJAhli'iEC
8X6
induce, in r:ir~~ and i,r rile some nuclear events similar to those produced by EZ. probably via its binding to the same receptor [2X]. At present, the role of the androstanediols in sexual maturation can be summarized as follows: ovarian steroid metabolism in the immature rat favors the k-reduced pathways producing mainly 3z-dial. This steroid is partially epimerized to .3/J-dial. In immature females of the Wistar strain, both steroids circulate in considerable concentrations up to puberty. Following the first ovulation. ovarian steroid metabolism shifts toward the production of 4-ene-3-oxosteroids. During the pre-pubertal period the a-diols inhibit the release of LH and reduce the pituitary LH!FSH ratio. They suppress the growth of uterus and ovary and of the pituitary and adrenal glands. It seems that they act in the pituitary via binding to Ez receptors. After the shift in ovarian progesterone metabolism, at the time of the first ovulation, their concentration in the circulation are low. REFERENCES
J. E.. Jameson H. E. and 1. Ojeda S. R., Wheaton McCann S. M.: The onset of puberty in the female rat: Changes in plasma prolactin. gonadotropins, futeinizing hormone-releasing hormone (LHRH). and hypothalamic LHRH content. ~}~~~~rj~~o~o~~ 98 (1976) 630-638. 2. Ramirez V. D.: In ~~~l~b~~~ t7f’Physiology {Edited by R. 0. Greep and E. B. Astwood). Eds, Williams & Wilkins, 3altimore, Vol. II, Section 7 (1974) pp. l-28. 3. Andrews W. W., Advis J. P. and Ojeda S. R.: The first proestrus in the female rat: Circulating steroid levels preceding and accompanying the preovulatory LH surge. PUIC. Sot. rsp. Bid. Med. 163 (1980) 3055309. 4. Andrews W. W. and Ojeda S. R. : A detailed analysis of the serum LH secretory profiles of conscious, freemoving female rats during the time of puberty. E&ocrino/ogr 109 (1981) 2032-2039. 5. Meijs-Roelofs H. M. A., IJilenbroek J. Th. J.. De Greef W. J.. de Jong F. D. and Kramer P.: Gon~ldotropin and steroid levels around the time of first ovulation in the rat. J. Otilotr. 67 (I 975) 275-2X7. events sur6. Parker C. R. and Mahesh V. B.: Hormonal rounding the natural onset of puberty in the female rats. Viol. Rrprod. 14 (1976) 347 353. I. Advis J. P.. Andrews W. W. and Ojeda S. R.: Changes in ovarian steroidal and prostnglandin E responsivcness to gonadotropins during the onset of puberty in the female rat. Endocriwlogy 104 (1979) 653-658. 8. Andrews W. W., Mizejewski G. J. and Ojeda S. R.: Development of estradiol positive feedback on LH release in the female rat: a quantitative study. Ertrlncri,roioqr 109 (1981) 1404. 1413. R. and Burstein S. H.: Identi9. Eckstein B., Mechoulam, fication of 5r-nndrost;~ne-3r.l7&diol as a principal metabolitc of pre~nenolone in rat ovary at onset of puberty. Nat& 2t8 (19701 X&X68. T.. Kurachi K., Aono 7. and Matsumoto 10 Karakawa K.: Formation of Sr-reduced C,&eroids from progesterone in eirro by pathways through 5x-reduced Cz ,-steroids in ovaries of late prepubertal rats. Et&crirrolo~qj, 98 (1976) 571-579.
ASTIIh
I I. Lerncr.
II?. 13.
14,
15.
16. 17,
IX.
19.
20.
21.
22.
23.
24.
N. and EcAstr~n B: lder~tilic~~ti~)i~ af two of prt)Sr-reduced prcgnance ah malor mctaboiites gestcrone in immature rat ot&cs (iOOO# supernatantl iri r.irr,,. Elrr(oc,rir~oioi/~ 98 (1976) 179-i XX. Mason N. R.: Steroid A-ring reduction by rat 01 arics. f:,lrl[lc,~i,ro/o~/~ 87 ( 1970) 350 355. Eckstcin B.. Lerner N. and Ychud S.: Pre-ovjulatory changes in steroidogenesis in ovaries from immature rats treated with pregnant mare serum gonadotropin. J. Endocr. 70 (I 976) 4X5-490. Eckstein B. and Lerner N.: Changes in ovartnn 5%~steroid reductase and 20x-hydroxysteroid dehydrogcnase activity produced by induction of first ovulation w’ith gonadotropin. Bir)c~itim. hictj~htx. ,4cttr 489 (I 977) 143- 149. Eckstein B.: Studies on the mechanisnl of the onset of puberty in the female rat. J, srcroid Biochm. 6 (1975) X73--878. Ojeda S. R., Andrews W. W.. Advis J. P. and Smith White S.: Recent advances in the endocrinology of puberty. Emfocr. Rev. 1 (1980) 228-257. Kramer P. and Meijs-Roelofs H. M. A.: Retardation of first ovulation in pubertal rats after treatment with 5r-androstane-3x. I7/&diol or its 3/?-epimer. J. Edocr. 92 (1982) 3 l-35. Ravid R. and Eckstein B.: Androstanediol sulphates in peripheral blood of immature rats and some of their biological effects. J. Em&m. 71 (1976) 299-304. Meijs-Roelofs H. M. A.. Uilenbroek J. Th. J., de Jong F. H. and Weischen R.: Plasma oestradio~-17~ and its rcl~~tionship to serum follicle-stimul~lting hormone in immature femaie rats. 1. Emfocr. 59 (1973) 295-304. Meijs-Roelofs H. M. A. and Kramer P.: Maturation of the inhibitory action of oestrogen on follicle stimulating hormone secretion in the immature female rat: a role for alphafoetoprotein. J. Endocr. 81 (1979) 199-208. Germain B. J.. Campbell P. S.. Anderson N. J.: Role of serum estrogen-binding protein in the control of tissue estradiol levels during post-natal development of the female rat. E,tt/ocri,foloM!, 103 ( 1978) 1401. Eckstcin B. and Ravid R.: Changes in pathways of steroid production taking place in the rat ovary around the time of the first ovulation, _I. srczroirl Bio&I??. 11 (1979) 593~597. Cohen J.. Dote Ch. Robaire B. and Ruf K. B.: Plasma c~~3~centr~ltlol~s of nndrogens during spontaneous and induced sexual maturation in the female rat. Ahsfr. Fcrr. Stwii. In press. Advis J. P.. Wiener S. L. and Ojeda S. R.: Changes in ovarian 3x-androstanediol response to human chorionit gonadotropin during puberty in the rat: Modulatorv role of nrolactin. Edocridoc/~ 109 (1981) -J?J __.
‘7X _-
‘5. Armstrong D. T.: Alterations of progesterone motaholirm in the immature rat ovary by luteinizing hormone. Bic~l. Rquwd. 21 (1979) 10251033. 26. Kruulis I., Naish S. J.. Gravenor D. and Ruf K. 8.: 5r-Androstnrte-3r’.I?fi-diol: Inhibitor of sexual maturation in the female rat. Bid. Rrproti. 24 (19X1)445 453. 77. ~rhielli~~nt M. L.. Samperez S. and Jouan P.: Evidence for Sz-androstnne-3P. 17/?-dial binding to the estrogen receptor in the cytosoi from malt rat pituitary. Errrk)c~~?itct~ogj.108 (19X I) 1552-i 560. 2%. Kreitmann B. and Bayard F.: Androgen inteaction with the estrogen receptor in human tissues, J. ,stomid Bioc~/lcwl.
I I ( 1979)
I589- 1594.
1983 Copyright
0022-4731/83 $3.00+0.00 0 1983 Pergamon Press Ltd
31. Puberty
BLOOD CONCENTRATIONS AND BIOLOGICAL OF ANDROSTANEDIOLS AT THE ONSET PUBERTY IN THE FEMALE RAT BENJAMIN
EFFECTS OF
ECKSTEIN
Department of Zoology, The Institute of Life Sciences, Hebrew University of Jerusalem, Israel
SUMMARY Androstanediols are the major products of the immature rat ovary. About two-thirds are present in blood as sulfates. One of the sulfate conjugates was identified as 5a-androstane-3b,l7B-diol-3-monosulfate. This steroid effectively inhibits postcastrational LH elevation in a concentration in which it is present in blood. Other androstanediol sulfates examined were without this activity. In the immature rat, levels of 3a- and 3/Sdiol decrease sharply before the first ovulation. In Wistar rats, PMSG-treatment induced a similar decline in these steroids. When ovulation in PMSG-treated rats was blocked by pentobarbitone, the LH-surge and the decline of the 3a- and 3/?-diols was delayed. Thus, it seems that the decline in the blood concentration of the diols depends on the first LH surge.
In the female rat, puberty may be defined as the developmental stage at which cyclic ovulation is initiated. Accordingly, the first preovulatory surge of gonadotropins can be visualized as the final product of maturation of the CNS-pituitary-ovary interrelationship. The physiological mechanism leading to maturation are not yet understood. In the rat, serum levels of both gonadotropins remain low during the prepubertal period, to increase abruptly in a preovulatory surge on the afternoon of the first proestrus [l-3]. Recent observations have shown that LH release is pulsatile in peripubertal female rats. With approaching puberty, the magnitude of the afternoon peaks becomes significantly greater than that of the morning peaks [4]. Serum estradiol levels (E,) increase on the day of the first proestrus [S], or just before the first preovulatory surge of gonadotropins [6,7]. This increase in Ez titer seems to play a major role in inducing the first gonadotropin surge, thus leading to pubertal ovulation [S]. For earlier endocrine functions of ovarian steroids, as, for instance, maintaining the negative feedback of gonadotropin release in the infantile rat, steroids lacking uterotropic activity would be ideal, but they would have to be phased out around the time of the first ovulation. It was shown that in the immature rat ovaries the predominant products resulting from progesterone metabolism are See-reduced C 1,-steroids, mainly 5a-androstane-3q 17P-diol (3cc-diol ; [9, lo]). These compounds originate through biosynthetic pathways involving the formation of &-reduced pregnanes that serve as intermediates [IO, 1 I]. One of the reasons for the high levels of the See-reduced C,,-steroids is the high activity of 4-ene-steroid Sa-reductase in the immature ovary [ 121. The formation of See-reduced
C,g-steroids from labeled progesterone is dramatically reduced during the few hours preceding pregnant mare serum gonadotropin (PMSG)-induced ovulation [ 131. Direct measurements of Sa-reductase in the immature ovary induced to ovulate by PMSG showed that on the third day following gonadotropin administration, only one tenth of the high initial activity of the enzyme remained, which declined further to undetectable level [ 143. In the present paper we examine several biological effects of the androstanediols in controlling the endocrine balance up to the onset of puberty and describe serum concentrations of androgens during spontaneous sexual development and around first ovulation induced by PMSG-treatment. EFFECK OF 3&DIOL ON VAGINAL OPENING The only obvious phenotypical change associated with puberty in the rat and mouse is resorption of the vaginal membrane. It is recognized however, that vaginal opening is often dissociated from the first estrus, and that several steroids induce vaginal opening without advancing puberty; in most of the rats of the Hebrew University colony, these two events may show a time lag of 7.5 days [IS]. Similar observations have been reported by others [2, 161. Nevertheless, it was found that a dose of 50 pg 3/?-diol per day from days 22 to 26 induced precocious vaginal opening, while vaginal smears remained diestrous [ 151. Recently Kramer and Meijs-Roelofs in a paper devoted almost entirely to this point, confirmed our finding: “nine out of eleven rats showed a ‘pinhole’ type of vaginal opening some days before the day of first oestrus” [ 171. 883
X84
Bf >JAMfN
COVTROL
OF LH RELEASE
E(.ksit:ih BLOOD
CO%\ICE:1fRATIO\S
SPOIVTAUEOI’S
In the peripheral circulation of immature female rats of the Hebrew University strain (descendants of Wistar strain), the androstanediols are present conjugated to sulfates or glucuronates and only low levels are detected as free steroids [ 1S]. Since the major part of the androstanediol conjugates were sulfates, we determined the form of the conjugation. The 3/$diol were found to be present in blood as both mono(3/r’-A-MS) and disulfate. while the 3x-diol was found to be present only as disulfate (3x-A-DS). On further analysis it was shown that the sulfate moiety on 3/I-A-MS present in peripheral blood of immature rats is attached to the C-3 of the steroid nucleus. The androstanediol sulfates of interest were synthesized and their effects as well as those of the free 3cc-diol, 3/&diol and E, on LH suppression in ovariectomized immature rats were examined. In the immature rat, as well as in the adult, castration leads to a markedly increased blood concentration of LH. While in the adult E, seems to be the main suppressor of pituitary LH release, the predominance of this steroid as the negative feedback controller during earlier phases of life is less certain in view of its low concentration in blood from about 25 days onward [19] and strong binding to r-fetoprotein which is reported to circulate in high concentration up to 25 days [20] or up to 3G-35 days [2l]; binding to x-fetoprotein presumably prevents the E, from exerting its biological activity. Hence. it was of interest to see whether the 3fi-A-MS has an effect on tonic LH release. Quantitative analysis of the feedback relationship between LH release and the concentration of the peripheral steroid is not possible by any injection regimen, since the level of the injected steroid declines markedly during the time between injections. To establish the threshold value of the androstanediols which are effective as feedback signals and to eliminate the between-treatment fluctuations, we used controlled release (Silastic) capsules for the administration of the steroids. In a preliminary experiment it was found that a capsule of 3.0 cm released enough 3/I-A-MS (1.1 ng/ml serum) to establish a constant blood concentration similar or lower than those of intact rats. The results showed that 3/?-diol, 3a-dioldisulfate (6.0 cm) and 3P-diol-monosulfate ( 1.5cm) had no effect, while E, (l.Ocm), 3b-A-MS (3.0 and 6.0 cm) as well as 3a-diol (3.0 cm) were effective in suppressing LH release during the period of 31-45 days of age. Thus, it seems that a constant serum level of 1.1 ng/ml of 3fi-A-MS maintains a serum level of LH similar to that present in the intact rat. Extrapolating these findings to the normal physiological situation, it seems that this steroid by itself is capable of maintaining the negative feedback of LH release. Therefore, we propose that 3/j’-A-MS perhaps in conjunction with E, may be involved in the control of prepubertal LH release.
A’VD IYDlICED
Dl’RI\G SESl AL
\l.ATIJRATIOh
We compared serum concentrations of the nndrostanediols of two strains of rats. In the untreated Wistar rat, hydrolyzed 3cc-diol levels fell sharply between from weaning and the time of vaginal opening, 15.8 k I.5 to 3.1 f 0.X ngiml (mean + SE) [22], while in Sprague-Dawley rats (which develop at a faster rate) unhydrolyzed 3a-dial level fell between days 21-26 from 651 _t I28 to 198 + 31 pg/ml (mean + SEM)[23]. Thus, while the absolute values in the Sprague-Dawley rats were much lower, the decline in the active steroids were similar in both strains. These results are in accord with the recent finding of Advis er ~/.[24], who showed (using the Holtzman strain rat) that up to the first ovulation the rat ovary produces large quantities of 3x-diol when challenged iri vitro by hCG, while after ovulation the quantity produced by the ovary decreased to one tenth of the original value. In the Wistar strain, PMSG-treatment on day 25 induced a considerable decrease in hydrolyzed blood 3r- and 30-diol levels after 48 h. The level of 3x-diol reached a nadir 72 h following gonadotropin administration (I .O k 0.6 ng/ml). The 3/j’-diol fell below detection limit (80 pg/ml) 56 h following PMSG administration (Eckstein et trl. to be published). Comparable results were obtained by Armstrong[25] who measured metabolism of progesterone in intact ovaries irr virro following administration of 4 IU PMSG. Androsterone production gradually increased. reaching maximum levels on day I and remained elevated until 14:OO h on day 2 (the equivalent of proestrus). With the first detectable rise in serum LH levels on day 2. androsterone production declined abruptly reaching minimum levels at 22:00 h on day 2. In the PMSG model, the preovulatory LH surge. and hence ovulation, can be blocked by the administration of pentobarbitone during the early afternoon of the second day following the injection of the gonadotropin. This design was used to ascertain the possible relationship between the observed drop in diol concentrations and ovulation. To this end. rats received ;I dose of 10 ILJ PMSG at 08:OO h on day 25 and an i.p. injection of pentobarbitone (3Omgjkg b.wt.) at 13:45 h on day 27. The presence or absence of tubal ova was verified at 08:OO h on day 28. The results indicate that in unblocked animals high levels of 3a- and 3/i’-diol prevailed 52 h following PMSG (i.e. at l2:OO h on day 27). Six hours later, a sharp decline accompanied the LH surge. In pentobarbitoneblocked rats. no LH surge occurred at this time. and high levels of 3a- and 3[{-dial were present. The concentration of both diols was much lower. however. at 12:OO h on day 28, although the (pentobarbitonedelayed) LH surge occurred only at 16:OO h. Thus, it seems that the decrease in androstanediols is linked to
Effect of androstanediols
on puberty
the process published).
885
of first ovulation
CONTROL
(Eckstein
OF GONADOTROPIN
et al. to be
RELEASE
In Sprague-Dawley rats, chronic implants of 3cc-diol, but not 3/I-diol, prevented uterine and ovarian development as assessed on the basis of organ weights and by ovarian histology 30 days later. Anterior pituitary weights were also greatly reduced, and their LH content was less than 10% of blank implanted controls, or of rats bearing 3/Gdiol implants. When administered to prepubertal rats by single injection, 3a-diol was found to inhibit the gonadotropin surge triggered by sequential treatment with E, and progesterone and to prevent the El-induced sensitization of the pituitary gonadotrophs to LHRH [26]. It was our intent to see whether doses of 3fi-A-MS that are in the physiological range inhibit ovarian and uterine growth in immature rats. From Table 1 it is evident that even a very small dose of 1Opg 3/I-A-MS/100 g b.wt/day inhibited ovarian and adrenal weights. Uterine weight increase was inhibited only with a larger dose (25 pg/lOO g b.wt/day). The administration of 3~-A-DS in a dose IO times higher, had no effect on these parameters. The results show that the biologically effective androstanediols are potent inhibitors of reproductive organs in the female rat. The inhibitory effect of 3r-diol on the first ovulation has recently been confirmed [ 173. -OWN-WNO
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CONCLUSIONS
In the Wistar substrain of the Hebrew University, as well as the strain used at the Hormone Research Department of the Weizmann Institute in Rehovot, the androstanediols are conjugated as sulfates and, to a lesser extent, to glucuronates; only a very small fraction can be detected as free alcohols, Both androstanediols are present in peripheral blood of prepubertal females in the concentration range of several ng/ml. The values of both diols (conjugates) are high in the prepubertal animal and decline after the first ovulation. One of the androstanediol sulfates, 3P-A-MS, suppresses postcastrational LH elevation at a dose in which it is found in peripheral circulation. The same steroid inhibits ovarian, uterine and adrenal growth at a very low dose. The biological activity of 3fi-A-MS as opposed to the free 3b-diol is of interest. It is possible that the 3/I-A-MS is taken up directly by the tissue regulating the negative feedback signal, since large concentrations of the free 3/I-diol do not exert this effect. It has recently been demonstrated that in the rat and ram pituitary the 3/I-diol is bound with high affinity to specific proteins, most probably to estrogen receptors, where it is the most potent competitor of all the androgens tested [27]. Furthermore, 3fl-diol is able to
Bt \iJAhli'iEC
8X6
induce, in r:ir~~ and i,r rile some nuclear events similar to those produced by EZ. probably via its binding to the same receptor [2X]. At present, the role of the androstanediols in sexual maturation can be summarized as follows: ovarian steroid metabolism in the immature rat favors the k-reduced pathways producing mainly 3z-dial. This steroid is partially epimerized to .3/J-dial. In immature females of the Wistar strain, both steroids circulate in considerable concentrations up to puberty. Following the first ovulation. ovarian steroid metabolism shifts toward the production of 4-ene-3-oxosteroids. During the pre-pubertal period the a-diols inhibit the release of LH and reduce the pituitary LH!FSH ratio. They suppress the growth of uterus and ovary and of the pituitary and adrenal glands. It seems that they act in the pituitary via binding to Ez receptors. After the shift in ovarian progesterone metabolism, at the time of the first ovulation, their concentration in the circulation are low. REFERENCES
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ASTIIh
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‘7X _-
‘5. Armstrong D. T.: Alterations of progesterone motaholirm in the immature rat ovary by luteinizing hormone. Bic~l. Rquwd. 21 (1979) 10251033. 26. Kruulis I., Naish S. J.. Gravenor D. and Ruf K. 8.: 5r-Androstnrte-3r’.I?fi-diol: Inhibitor of sexual maturation in the female rat. Bid. Rrproti. 24 (19X1)445 453. 77. ~rhielli~~nt M. L.. Samperez S. and Jouan P.: Evidence for Sz-androstnne-3P. 17/?-dial binding to the estrogen receptor in the cytosoi from malt rat pituitary. Errrk)c~~?itct~ogj.108 (19X I) 1552-i 560. 2%. Kreitmann B. and Bayard F.: Androgen inteaction with the estrogen receptor in human tissues, J. ,stomid Bioc~/lcwl.
I I ( 1979)
I589- 1594.