0013-7227/78/1035-1822$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 5 Printed in U.S.A.
Steroid Induction of Gonadotropin Surges in the Immature Rat. I. Priming Effects of Androgens* ILZE KRAULIS, HELLI TRAIKOV, MANDY SHARPE, K. B. RUF, AND F. NAFTOLIN Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec H3A 1A1, Canada ABSTRACT. Sexually immature female rats were either primed with estradiol benzoate on day 23 or given daily injections of various androgens on days 23-25. Plasma for LH and FSH determinations was collected on day 26,5 h after an injection of progesterone. Massive gonadotropin surges were found after priming with estradiol benzoate or treatment with dehydroepiandrosterone (DHEA), A4-androstenedione, and testosterone, but not with ring A-reduced androgens (5a-dihydrotestosterone, 5a-androstane-3a,17/?-diol, its 3/8-epimer, and androsterone) or the nonaromatizable 11/J-hydroxy- and 11-ketoderivatives of A4-androstenedione. Rats bearing DHEA-containing Silastic implants also produced LH surges in response to progesterone. A single injection of
T
HE NEUROENDOCRINE mechanism which governs the first (pubertal) ovulation in the rat is remarkably similar to the one controlling recurrent ovulation during adult life (1-4). In both cases, the preovulatory surge of LH and FSH is preceded, and presumably triggered, by a rise in endogenous estrogen. In sexually immature female rats, precocious puberty can be induced by repeated administration of estrogen (5), and a premature surge of LH (6) and FSH (7) can be provoked with gonadal steroids provided that the animal has previously been exposed to estrogen. This steroid thus appears to play a crucial role in the process of sexual maturation, yet the nature of the physiological stimulus leading to its rise before first ovulation remains enigmatic. The immature ovary is capable of forming estrogen from androgens under the influence of FSH (8), but the induction of ovarian LH receptors, required for the biosynthesis of estrogen precursors from cholesterol (9), de-
an antiestrogen antiserum abolished gonadotropin surges in rats primed with estradiol benzoate or DHEA and greatly reduced the accompanying uterine hypertrophy. DHEA and A4-androstenedione were barely uterotrophic in ovariectomized rats but sustained progesterone-induced gonadotropin surges. The results indicate that certain (adrenal?) androgens are able to induce maturation of the steroid-sensitive surge system via extragonadal aromatization, whereas their uterotrophic effect is largely mediated by the ovaries. Coordinated increased conversion of androgens at central and peripheral sites may be of physiological importance for the triggering of puberty. (Endocrinology 103: 1822, 1978)
pends on the presence of both FSH and estrogen (10, 11). It seems possible, therefore, that the estrogen precursors required for the triggering of the pubertal gonadotropin surge are derived from an extraovarian source. Accordingly, we have tested a variety of androgens, some known to be synthesized by the rat adrenal cortex (12), for their capacity to induce precocious maturation of the gonadotropin surge system. A modification of the model of the estrogen-primed, progesterone (P)-challenged immature rat (6, 7) has been used for this purpose. Materials and Methods Hormonal priming of animals Twenty-one-day-old female rats of a SpragueDawley substrain weighing 50-55 g were obtained from Canadian Breeding Farms and Laboratories Ltd., Montreal, and housed in groups of five under conditions of controlled illumination (lights on 0500 h-1900 h). On day 23, at noon, they were either injected once with 10 /xg estradiol benzoateV0.2 ml
Received February 10,1978. Address reprint requests to: Dr. K. B. Ruf, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Que'The following trivial names and abbreviations for bec H3A 1A1, Canada. steroids were used: estradiol benzoate (EB) = 1,3,5(10)* This study was supported by MRC Grants MA-6235 estratrien -17/8 - ol - 3 - benzoate; dehydroepiandrosterone and MT-5823 and a contribution from the Royal Victoria sulfate (DHEA-S), 3/?-hydroxy-5-androsten-17-one sodium sulfate. Hospital Fraser Memorial Trust. 1822 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 17:44 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN PRIMING AND PUBERTY sesame oil sc, or treatment with one of the following steroid hormones was begun: dehydroepiandrosterone (DHEA) DHEA-S, A4-androstenedione (A4), 11-keto-A4, ll/?-hydroxy-A4-androstenedione, testosterone (T), 5a-dihydrotestosterone (DHT), 5aandrostane-3/?,17/?-diol, 5a-androstane-3a, 1 lfi-dio\, or androsterone. All androgens were used as received from Steraloids Inc., Wilton, NH. Unless otherwise stated, they were administered in 0.2 ml propylene glycol sc once daily for 3 days; the doses used were initially based on the report by Knudsen and Mahesh (13) and are reported under Results. Where indicated, animals were additionally ovariectomized under light ether anesthesia on day 22 of life. In one experiment, the daily steroid injections were replaced by steroid implants made from medical grade Silastic tubing (Dow Corning Corp., MI) inserted under the dorsal skin on day 23.
1823
ric acid as catalyst according to the method of Aso et al. (15); 70% was recovered. The free and solvolyzed DHEA fractions were purified on Celite microcolumns as described by Buster and Abraham (16) and their concentrations were determined in one single assay using antiserum S-1502 no. 7 purchased from Dr. G. E. Abraham. The unpaired Student's t test was used for the statistical analysis of results, a P value of < 0.05 being considered significant. Results P-induced LH surges after priming with various androgens
As shown in Fig. 1, the higher dose (6 mg/ 100 g BW) of both DHEA and A4 efficiently primed immature rats to respond with a masTriggering of LH and FSH surges sive LH surge 5 h after the P injection (P < On day 26, at noon, all animals were given 1 mg 0.005 as compared with unprimed controls). P in 0.2 ml propylene glycol sc, and trunk blood The lower dose (0.6 mg/100 g BW) was inefwas obtained by decapitation 5 h later. Collection fective. In contrast, this lower dose efficiently tubes were heparinized and plasma was stored at primed the animals when the androgen used —20° before assay. At the time of sacrifice, uteri for this purpose was T (P < 0.01). Dose rewere quickly dissected on blotting paper and quirements for T appear well defined, since weighed to the nearest milligram. either a 10-fold reduction or increase in dose abolished this priming effect (P < 0.05). None Estrogen Antiserum of the ring A-reduced androgens given at In one series, animals received injection of an either dose level were effective. In addition, it antiserum raised in sheep against a bovine serum was found that the 11-keto- or 11-hydroxyalbumin-hemisuccinate-17/?-estradiol conjugate analogs of A4 did not possess the priming (14); this serum was administered (0.2 ml ip) at the ability of the parent compound. time of first steroid priming. At a 1:100 dilution, its cross reactivity, measured at 50% displacement of 17/?-[3H]estradiol (32 ng for 17/?-estradiol) was as follows: estrone, 59%; 17a-estradiol, 34%; estriol, 5%. None of the aromatizable androgens used in this study (T, A4, DHEA) showed any significant cross reactivity in concentrations up to 1 jug. RIA for gonadotropins Plasma LH and FSH were assayed in duplicate _£*! and. at two different dose levels using NIAMDD A kits. Results are expressed in nanograms per ml of the respective RP-1 standards. Variability of 0.05 as compared with JT nonprimed controls) and uterine weights were significantly (P < 0.02) reduced, although FIG. 3. Uterine weights; same design as in Figs. 1 and 2. they still reflected some residual estrogenic DHEA
DOSE: -
6
6
A4
T
11-0 11-OH
DHT
3t»diol
6 6
06 6 6
6 6
6 6
6 6
3j9diol
A
.6 6
6
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1825
ANDROGEN PRIMING AND PUBERTY
effect (P < 0.02 as compared with nonprimed controls). In the case of T, a similar trend was observed, but because of the lesser magnitude of changes induced in intact animals of this series, neither decrement reached statistical significance. The theoretical possibility that constituents of the estrogen antiserum administered earner might interfere with gonadotropin assays and lead to spuriously low results was not tested in these experiments but should be borne in mind.
crease in uterine weights was observed in the case of A4. Both androgens, and A4 in particular, prevented the castration-induced LH rise as efficiently as EB when compared to solventinjected ovariectomized controls (P < 0.001). Ovariectomy did not affect body weights in any of the treatment groups. Priming ability of the DHEA-containing Silastic implants
In order to avoid the stress and inconvenience of daily androgen injections and to Studies on the site of aromatization ofandroachieve more stable circulating concentrations gens of androgens, DHE A was administered chronIn view of the demonstrated ability of the ically in an additional experiment (Table 2). immature ovary to synthesize estrogens from Silastic implants (id 0.058 inch, od 0.077 inch) androgens (8) it was of interest to assess the of 2 cm length released insufficient steroid to influence of ovariectomy on the priming abil- prime the animals for P-induced LH release it}r of some of the aromatizable androgens or to affect uterine weights, but significant tested. As expected, the priming ability of EB increments in both parameters were achieved was unaffected by ovariectomy (Table 1), both by the simultaneous implantation of two 2-cm in terms of the P-induced LH surge and uter- capsules. A proportional increase in both free ine hypertrophy. In DHEA-primed ovariec- and conjugated DHEA was found in plasma tomized rats, the P-induced LH surge was taken at the time of sacrifice. somewhat reduced, but the difference did not Discussion reach statistical significance, whereas the uterotrophic effect was nearly abolished. No All three aromatizable androgens tested in difference in the LH surge but a similar de- this study, i.e. DHEA, A4, and T, were found TABLE 1. Influence of ovariectomy (day 22) on steroid-primed immature rats (days 23-25) challenged with P at 1200 h on day 26
Solvent/solvent Solvent/solvent Solvent/P
Intact ovx ovx
6 11 6
Plasma LH (ng/ ml) 227 ± 49 1211 ± 247 980 ± 202
EB/solvent EB-solvent EB/P EB/P
Intact ovx Intact ovx
6 6 13 6
142 ± 47 418 ± 110 3388 ± 326 3788 ± 148°
134 ± 14 135 ± 13 132 ± 9 136 ± 7°
72 70 71 69
±2 ±1 ±1 ±1
DHEA/solvent DHEA/solvent DHEA/P DHEA/P
Intact ovx Intact ovx
7 6 12 17
164 ±46 454 ± 277 2800 ± 432 1929 ± 290"
133 ± 17 47 ± 4 190 ± 4 53 ±1"
73 63 73 69
±1 ±3 ±3 ±1
Treatment
Uterine wt (mg)
Final BW (g)
32 ± 1 28 ± 2 20 ± 1
72 ±2 73 ±2 69 ±3
137 ± 58 66 ± 4 68 ± 5 A4/solvent ovx 5 69 ± 2 11 2002 ± 272 164 ± 8 Intact A4/P 68 ± 2 2520 ± 466a ovx 10 67 ±3* A4/P Animals were sacrificed 5 h after P injection. Mean ± SEM. n = number of animals in each group. EB was injected sc at a dose of 10 jtxg/0.2 ml oil/rat at 1200 h on day 23. DHEA and A4 were given sc at a dose of 6 mg/0.2 ml propylene glycol/100 g BW at 1200 h on days 23-25. ." Not significantly {P > 0.05) different from corresponding intact controls. 6 Significantly (P < 0.001) lower than in corresponding intact controls.
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KRAULIS ET AL.
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Endo • 1978 Vol 103 • No 5
TABLE 2. Priming efficiency of DHEA containing capsules (in situ days 23-26) and plasma steroid concentrations achieved Implant
n
Plasma LH (ng/ ml)
Plasma DHEA (pg/ml) Uterine wt (mg)
Sulfate Free 9 ± 4 (2) 7 ± 6 (2) 49 43 ± 6 ± 5 3 Blank 113 ± 26 (3)* 53 ± 5 (3)" 62 ± 7 48 ± 3 4 DHEA, 2 cm 510 ± 80 (4)* 108 ± 6 (4)e 675 ± 106c 142 ± 18" 5 DHEA, 2 x 2 cm All rats were injected with P at 1200 h on day 26 and sacrificed 5 h later. Mean ± SEM. n = number of animals; numbers in parentheses refer to number of animals yielding sufficient plasma for steroid determinations. " Significantly greater than blank-implanted controls: P < 0.01. 6 Significantly greater than blank-implanted controls: P < 0.05. c Significantly greater than blank-implanted controls: P < 0.005.
to promote the maturation of the steroid-sensitive surge system for gonadotropins in prepubertal rats and to increase uterine weights to within the adult range (Figs. 1-3). When any of these steroids were administered in the
surge system remained unaffected by ovariectomy (Table 1). Because of the discrepancy between the uterotropic and gonadotropic effects in ovariectomized rats, it seems likely that the conversion of androgens to estrogen
form of single repeated daily injections, the
required for this process takes place within
dose requirements were in the milligram range and might thus be considered pharmacological. However, comparable results were obtained with Silastic implants of DHEA (Table 2) that maintained near physiological concentrations as judged by the results of Parker and Mahesh [(17) basal DHEA levels in rats of comparable age: 160-270 pg/ml)]. Experiments with estrogen antiserum (Fig. 4) suggest that conversion of DHEA, A4, and T to estrogen is essential for the observed priming effect on the gonadotropin surge system. In contrast, the uterotropic effect of these androgens is only partially neutralized by antibodies, an observation which has previously been reported for estradiol itself (18). In the case of T, the maturation of the surge system was found to require lower doses than the induction of uterine growth; it is possible that the latter effect is partially due to the formation of ring A-reduced products rather than aromatization to estrogen, since several of these nonaromatizable compounds also exhibited significant uterotrophic effects (Fig. 3). The uterotropic activity of DHEA and A4 was nearly abolished by ovariectomy (Table 1). This observation implicates the ovary as a major site of peripheral aromatization of exogenous androgens. In remarkable contrast, the efficiency of these steroids to induce maturation of the steroid-sensitive gonadotropin
the brain itself, which, in the rat, displays demonstrable aromatase activity from fetal life onwards (19). In addition, the discrepancy in dose requirements observed for T suggests that, at least for this androgen, the brain might have a lower threshold than the uterus. It is noteworthy that only androgens which fulfill the structural requirements for aromatization (20) were effective in priming immature rats for subsequent P-induced LH release; neither the ring A-reduced androgens tested nor the 11/Miydroxy- and 11-keto-derivatives of A4, both of which sterically interfere with aromatizing enzymes, led to a LH surge (Fig. 1). In contrast, the ring A-reduced androgen 5a-androstane-3a,17/?-diol also led to a small but significant increment in the plasma concentration of FSH 5 h after the administration of P (Fig. 2). The priming ability of the 3/8epimer was below statistical significance in this particular design, but it is conceivable that a similar FSH-stimulating effect accounts for the observation that 5a-androstane-3/?,17/?-diol advances puberty in the rat if given over prolonged periods of time (21); slight but chronic elevation of FSH achieved by other means [hippocampal stimulation (22)] has likewise been reported to result in precocious vaginal opening. Knudsen and Mahesh (13) previously described that repeated injections of DHEA induce precocious ovulation in immature rats,
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ANDROGEN PRIMING AND PUBERTY
and Parker and Mahesh (17) demonstrated increased systemic concentrations of estrogen as early as 2 h after the administration of the androgen. During spontaneous sexual maturation in the female rat, T concentrations and nonspecified "androgens" have been measured by Dohler and Wuttke (3), but, to our knowledge, no systematic studies are available for weaker androgens such as DHEA and A4. In order to explain the process of puberty, the hypothesis which is most frequently advanced is the so-called "gonadostat" theory, according to which sex steroids become progressively less efficient in inhibiting pituitary gonadotropin secretion by negative feedback (23). However, some recent observations have cast; doubt on the applicability of this concept to the female rat. For instance, during spontaneous sexual maturation as well as during precocious puberty induced by brain lesions, circulating FSH concentrations do not display the steady rise anticipated if the level of negative feedback control were "reset" to a lower level of sensitivity (24). Similarly, small unilateral lesions which spare the majority of demonstrated steroid uptake sites within the basal hypothalamus are sufficient to cause precocious maturation of the gonadotrophin surge system in a manner similar to exogenous estrogen (25). Such lesions, therefore, can no longer be considered to advance puberty by eliminating the sites of negative steroid feedback. Clinicians have long been aware that increased adrenal secretion of androgens ("adrenarche") may precede the activation of the gonads by several years (6, 7) and might be causally related to the onset of puberty. In the female, these androgens would be ideally suited to circumvent the initial block in gonadal steroid pathways caused by the absence of LH receptors (10,11) and could allow estrogen synthesis to proceed before these receptors are induced. The existence of a separate pituitary hormone controlling the secretion of adrenal androgens has been proposed (8, 9). Our studies show that, if confirmed, such a hormone might have to be considered as a prime candidate for the role of the elusive trigger of puberty in the female rat.
1827
Acknowledgments We thank Dr. J. R. G. ChaUis for advice on the DHEA assay and for cross-reactivity studies on the antiserum used and Dr. B. Robaire for helpful suggestions on the preparation of Silastic implants. RIAs were carried out in collaboration with Dr. D. Lee and Ms. Patricia Wong and were based on materials provided by the NIAMDD rat pituitary hormone distribution program. The antiestrogen antiserum used was a gift from Dr. B. V. Caldwell.
References 1. Meijs-Roelofs, H. M. A., J. T. J. Uilenbroek, J. W. de Greef, F. H. de Jong, and P. Kramer, Gonadotrophin and steroid levels around the time of first ovulation in the rat, J Endocrinol 67: 275, 1975. 2. Ojeda, S. R., J. E. Wheaton, H. E. Jameson, and S. M. McCann, The onset of puberty in the female rat; changes in plasma prolactin, gonadotropins, luteinizing hormone-releasing hormone (LHRH), and hypothalamic LHRH content, Endocrinology 98: 630, 1976. 3. Dohler, K. D., and W. Wuttke, Changes with age in levels of serum gonadotropins, prolactin, and gonadal steroids in prepubertal male and female rats, Endocrinology 97: 898, 1975. 4. Parker, C. R., Jr., and V. B. Mahesh, Hormonal events surrounding the natural onset of puberty in female rats, Biol Reprod 14: 347, 1976. 5. Ramirez, V. D., and C. H. Sawyer, Advancement of puberty in the female rat by estrogen, Endocrinology 76: 1158, 1965. 6. Caligaris, L., J. J. Astrada, and S. Taleisnik, Influence of age on the release of luteinizing hormone induced by oestrogen and progesterone in immature rats, J. Endocrinol 55: 97, 1972. 7. Caligaris, L., J. J. Astrada, and S. Taleisnik, Development of the mechanisms involved in the facilitatory and inhibitory effects of ovarian steroids on the release of follicle-stimulating hormone in the immature rat, J Endocrinol 58: 547, 1973. 8. Dorrington, J. H., Y. S. Moon, and D. T. Armstrong, Estradiol-17/? biosynthesis in cultured granulosa cells from hypophysectomized immature rats; stimulation by follicle-stimulating hormone, Endocrinology 97: 1328, 1975. 9. Hall, P. F., and D. K. Young, Site of action of trophic hormones upon the biosynthetic pathways to steroid hormones, Endocrinology 82: 559, 1968. 10. Channing, C. P., and A. Tsafriri, Mechanism of action of luteinizing hormone and follicle-stimulating hormone on the ovary in vitro, Metabolism 26: 413,1977. 11. Lindner, H. R., A. Amsterdam, Y. Salomon, A. Tsafriri, A. Nimrod, S. A. Lamprecht, U. For, and Y. Koch, Intraovarian factors in ovulation: determinants of follicular response to gonadotrophins, J Reprod Fertil 51: 215, 1977. 12. Askari, H. A., Sexual differences in the biogenesis of the androgens by the adrenal cortex in rat, Endocri-
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1828
KRAULIS ET AL.
Endo • 1978 Vol 103 • No 5
nplogyST: 1377, 1970. 21. Eckstein, B., R. Golan, and J. Shani (Mishinsky), 13. Knudsen, J. F., and V. B. Mahesh, Initiation of preOnset of puberty in the immature female rat induced cocious sexual maturation in the immature rat treated by 5a-androstane-3/?,17/?-diol, Endocrinology 92: 941, with dehydroepiandrosterone, Endocrinology 97: 1973. 458, 1975. 22. Kawakami, M., K. Seto, F. Kimura, and E. Terasawa, 14. Thorneycroft, I. S., S. A. Tillson, G. E. Abraham, R. Nature of the hippocampal function in relation to J. Scaramuzzi, and B. V. Caldwell, Preparation and gonadotropin secretion, In Yagi, K., and S. Yoshida purification of antibodies to steroids. In Peron, F. G., (eds.), Neuroendocrine Control, John Wiley & Sons, and B. V. Caldwell (eds), Immunologic Methods in New York, 1973, p. 229. Steroid Determination, Plenum Press, New York, 23. Ramirez, D. V., and S. M. McCann, Comparison of 1970, p. 63. the regulation of luteinizing hormone (LH) secretion 15. Aso, T., A-R. Aedo, and S. Z. Cekan, Simultaneous in immature and adult rats, Endocrinology 72: 452, determination of the sulphates of dehydroepiandros1963. terone and pregnenolone in plasma by radioimmu- 24. Advis, J. P., and V. D. Ramirez, Plasma levels of LH noassay following a rapid solvolysis, J Steroid Bioand FSH in female rats with precocious puberty inchem 8: 1105, 1977. duced by hypothalamic lesions, Biol Reprod 17: 313, 1977. 16. Buster, J. E., and G. E. Abraham, Radioimmunoassay of plasma dehydroepiandrosterone sulfate, Anal Lett 25. Ruf, K. B., M. Wilkinson, and D. de Ziegler, Brain lesions and precocious puberty in rats, Nature 257: 5: 543, 1972. 404, 1975. 17. Parker, C. R., and V. B. Mahesh, Dehydroepiandrosterone (DHA) induced precocious ovulation: correla- 26. Talbot, N. B., E. H. Sobel, J. W. McArthur, and J. D. Crawford, Functional Endocrinology from Birth to tive changes in blood steroids, gonadotropins and Adolescence, Harvard University Press, Cambridge, cytosol estradiol receptors of anterior pituitary gland MA, 1952. and hypothalamus, J Steroid Biochem 8: 173, 1977. 18. Ferin, M., J. Raziano, A. Tempone, and R. Vande 27. Korth-Schutz, S., L. S. Levine, and M. I. New, Evidence for the adrenal source of androgens in precoWiele, The use of antibodies as a tool in studies in cious adrenarche, Acta Endocrinol 82: 342, 1976. reproductive physiology, In Peron, F. G., and B. V. Caldwell (eds.), Immunologic Methods in Steroid De- 28. Mills, I. H., R. V. Brooks, and F. T. G. Prunty, The relationship between the production of cortisol and of termination, Plenum Press, New York, 1970, p. 199. androgen by the human adrenal, In Currie, A. R., T. 19. Reddy, V. V. R., F. Naftolin, and K. J. Ryan, ConverSymington, and J. K. Grant (eds.), The Human Adsion of androstenedione to estrone by neural tissues renal Cortex, E. & S. Livingstone, Ltd., London, 1962, from fetal and neonatal rats, Endocrinology 94: 117, p. 204. 1974. 20. Engel, L. L., The biosynthesis of estrogens, In Greep, 29. Parker, L., and W. Odell, Control of adrenal androgen secretion by a new pituitary factor: cortical androgen R. O., and E. B. Astwood (eds.), Handbook of Physistimulating hormone (CASH), Clin Res 25: 299A, ology, Williams & Wilkins, Baltimore, 1973, Section 7, 1977 (Abstract). vol. 2, part 1, p. 467.
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Vol. 103, No. 5 Printed in U.S.A.
Steroid Induction of Gonadotropin Surges in the Immature Rat. I. Priming Effects of Androgens* ILZE KRAULIS, HELLI TRAIKOV, MANDY SHARPE, K. B. RUF, AND F. NAFTOLIN Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec H3A 1A1, Canada ABSTRACT. Sexually immature female rats were either primed with estradiol benzoate on day 23 or given daily injections of various androgens on days 23-25. Plasma for LH and FSH determinations was collected on day 26,5 h after an injection of progesterone. Massive gonadotropin surges were found after priming with estradiol benzoate or treatment with dehydroepiandrosterone (DHEA), A4-androstenedione, and testosterone, but not with ring A-reduced androgens (5a-dihydrotestosterone, 5a-androstane-3a,17/?-diol, its 3/8-epimer, and androsterone) or the nonaromatizable 11/J-hydroxy- and 11-ketoderivatives of A4-androstenedione. Rats bearing DHEA-containing Silastic implants also produced LH surges in response to progesterone. A single injection of
T
HE NEUROENDOCRINE mechanism which governs the first (pubertal) ovulation in the rat is remarkably similar to the one controlling recurrent ovulation during adult life (1-4). In both cases, the preovulatory surge of LH and FSH is preceded, and presumably triggered, by a rise in endogenous estrogen. In sexually immature female rats, precocious puberty can be induced by repeated administration of estrogen (5), and a premature surge of LH (6) and FSH (7) can be provoked with gonadal steroids provided that the animal has previously been exposed to estrogen. This steroid thus appears to play a crucial role in the process of sexual maturation, yet the nature of the physiological stimulus leading to its rise before first ovulation remains enigmatic. The immature ovary is capable of forming estrogen from androgens under the influence of FSH (8), but the induction of ovarian LH receptors, required for the biosynthesis of estrogen precursors from cholesterol (9), de-
an antiestrogen antiserum abolished gonadotropin surges in rats primed with estradiol benzoate or DHEA and greatly reduced the accompanying uterine hypertrophy. DHEA and A4-androstenedione were barely uterotrophic in ovariectomized rats but sustained progesterone-induced gonadotropin surges. The results indicate that certain (adrenal?) androgens are able to induce maturation of the steroid-sensitive surge system via extragonadal aromatization, whereas their uterotrophic effect is largely mediated by the ovaries. Coordinated increased conversion of androgens at central and peripheral sites may be of physiological importance for the triggering of puberty. (Endocrinology 103: 1822, 1978)
pends on the presence of both FSH and estrogen (10, 11). It seems possible, therefore, that the estrogen precursors required for the triggering of the pubertal gonadotropin surge are derived from an extraovarian source. Accordingly, we have tested a variety of androgens, some known to be synthesized by the rat adrenal cortex (12), for their capacity to induce precocious maturation of the gonadotropin surge system. A modification of the model of the estrogen-primed, progesterone (P)-challenged immature rat (6, 7) has been used for this purpose. Materials and Methods Hormonal priming of animals Twenty-one-day-old female rats of a SpragueDawley substrain weighing 50-55 g were obtained from Canadian Breeding Farms and Laboratories Ltd., Montreal, and housed in groups of five under conditions of controlled illumination (lights on 0500 h-1900 h). On day 23, at noon, they were either injected once with 10 /xg estradiol benzoateV0.2 ml
Received February 10,1978. Address reprint requests to: Dr. K. B. Ruf, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Que'The following trivial names and abbreviations for bec H3A 1A1, Canada. steroids were used: estradiol benzoate (EB) = 1,3,5(10)* This study was supported by MRC Grants MA-6235 estratrien -17/8 - ol - 3 - benzoate; dehydroepiandrosterone and MT-5823 and a contribution from the Royal Victoria sulfate (DHEA-S), 3/?-hydroxy-5-androsten-17-one sodium sulfate. Hospital Fraser Memorial Trust. 1822 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 17:44 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN PRIMING AND PUBERTY sesame oil sc, or treatment with one of the following steroid hormones was begun: dehydroepiandrosterone (DHEA) DHEA-S, A4-androstenedione (A4), 11-keto-A4, ll/?-hydroxy-A4-androstenedione, testosterone (T), 5a-dihydrotestosterone (DHT), 5aandrostane-3/?,17/?-diol, 5a-androstane-3a, 1 lfi-dio\, or androsterone. All androgens were used as received from Steraloids Inc., Wilton, NH. Unless otherwise stated, they were administered in 0.2 ml propylene glycol sc once daily for 3 days; the doses used were initially based on the report by Knudsen and Mahesh (13) and are reported under Results. Where indicated, animals were additionally ovariectomized under light ether anesthesia on day 22 of life. In one experiment, the daily steroid injections were replaced by steroid implants made from medical grade Silastic tubing (Dow Corning Corp., MI) inserted under the dorsal skin on day 23.
1823
ric acid as catalyst according to the method of Aso et al. (15); 70% was recovered. The free and solvolyzed DHEA fractions were purified on Celite microcolumns as described by Buster and Abraham (16) and their concentrations were determined in one single assay using antiserum S-1502 no. 7 purchased from Dr. G. E. Abraham. The unpaired Student's t test was used for the statistical analysis of results, a P value of < 0.05 being considered significant. Results P-induced LH surges after priming with various androgens
As shown in Fig. 1, the higher dose (6 mg/ 100 g BW) of both DHEA and A4 efficiently primed immature rats to respond with a masTriggering of LH and FSH surges sive LH surge 5 h after the P injection (P < On day 26, at noon, all animals were given 1 mg 0.005 as compared with unprimed controls). P in 0.2 ml propylene glycol sc, and trunk blood The lower dose (0.6 mg/100 g BW) was inefwas obtained by decapitation 5 h later. Collection fective. In contrast, this lower dose efficiently tubes were heparinized and plasma was stored at primed the animals when the androgen used —20° before assay. At the time of sacrifice, uteri for this purpose was T (P < 0.01). Dose rewere quickly dissected on blotting paper and quirements for T appear well defined, since weighed to the nearest milligram. either a 10-fold reduction or increase in dose abolished this priming effect (P < 0.05). None Estrogen Antiserum of the ring A-reduced androgens given at In one series, animals received injection of an either dose level were effective. In addition, it antiserum raised in sheep against a bovine serum was found that the 11-keto- or 11-hydroxyalbumin-hemisuccinate-17/?-estradiol conjugate analogs of A4 did not possess the priming (14); this serum was administered (0.2 ml ip) at the ability of the parent compound. time of first steroid priming. At a 1:100 dilution, its cross reactivity, measured at 50% displacement of 17/?-[3H]estradiol (32 ng for 17/?-estradiol) was as follows: estrone, 59%; 17a-estradiol, 34%; estriol, 5%. None of the aromatizable androgens used in this study (T, A4, DHEA) showed any significant cross reactivity in concentrations up to 1 jug. RIA for gonadotropins Plasma LH and FSH were assayed in duplicate _£*! and. at two different dose levels using NIAMDD A kits. Results are expressed in nanograms per ml of the respective RP-1 standards. Variability of 0.05 as compared with JT nonprimed controls) and uterine weights were significantly (P < 0.02) reduced, although FIG. 3. Uterine weights; same design as in Figs. 1 and 2. they still reflected some residual estrogenic DHEA
DOSE: -
6
6
A4
T
11-0 11-OH
DHT
3t»diol
6 6
06 6 6
6 6
6 6
6 6
3j9diol
A
.6 6
6
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1825
ANDROGEN PRIMING AND PUBERTY
effect (P < 0.02 as compared with nonprimed controls). In the case of T, a similar trend was observed, but because of the lesser magnitude of changes induced in intact animals of this series, neither decrement reached statistical significance. The theoretical possibility that constituents of the estrogen antiserum administered earner might interfere with gonadotropin assays and lead to spuriously low results was not tested in these experiments but should be borne in mind.
crease in uterine weights was observed in the case of A4. Both androgens, and A4 in particular, prevented the castration-induced LH rise as efficiently as EB when compared to solventinjected ovariectomized controls (P < 0.001). Ovariectomy did not affect body weights in any of the treatment groups. Priming ability of the DHEA-containing Silastic implants
In order to avoid the stress and inconvenience of daily androgen injections and to Studies on the site of aromatization ofandroachieve more stable circulating concentrations gens of androgens, DHE A was administered chronIn view of the demonstrated ability of the ically in an additional experiment (Table 2). immature ovary to synthesize estrogens from Silastic implants (id 0.058 inch, od 0.077 inch) androgens (8) it was of interest to assess the of 2 cm length released insufficient steroid to influence of ovariectomy on the priming abil- prime the animals for P-induced LH release it}r of some of the aromatizable androgens or to affect uterine weights, but significant tested. As expected, the priming ability of EB increments in both parameters were achieved was unaffected by ovariectomy (Table 1), both by the simultaneous implantation of two 2-cm in terms of the P-induced LH surge and uter- capsules. A proportional increase in both free ine hypertrophy. In DHEA-primed ovariec- and conjugated DHEA was found in plasma tomized rats, the P-induced LH surge was taken at the time of sacrifice. somewhat reduced, but the difference did not Discussion reach statistical significance, whereas the uterotrophic effect was nearly abolished. No All three aromatizable androgens tested in difference in the LH surge but a similar de- this study, i.e. DHEA, A4, and T, were found TABLE 1. Influence of ovariectomy (day 22) on steroid-primed immature rats (days 23-25) challenged with P at 1200 h on day 26
Solvent/solvent Solvent/solvent Solvent/P
Intact ovx ovx
6 11 6
Plasma LH (ng/ ml) 227 ± 49 1211 ± 247 980 ± 202
EB/solvent EB-solvent EB/P EB/P
Intact ovx Intact ovx
6 6 13 6
142 ± 47 418 ± 110 3388 ± 326 3788 ± 148°
134 ± 14 135 ± 13 132 ± 9 136 ± 7°
72 70 71 69
±2 ±1 ±1 ±1
DHEA/solvent DHEA/solvent DHEA/P DHEA/P
Intact ovx Intact ovx
7 6 12 17
164 ±46 454 ± 277 2800 ± 432 1929 ± 290"
133 ± 17 47 ± 4 190 ± 4 53 ±1"
73 63 73 69
±1 ±3 ±3 ±1
Treatment
Uterine wt (mg)
Final BW (g)
32 ± 1 28 ± 2 20 ± 1
72 ±2 73 ±2 69 ±3
137 ± 58 66 ± 4 68 ± 5 A4/solvent ovx 5 69 ± 2 11 2002 ± 272 164 ± 8 Intact A4/P 68 ± 2 2520 ± 466a ovx 10 67 ±3* A4/P Animals were sacrificed 5 h after P injection. Mean ± SEM. n = number of animals in each group. EB was injected sc at a dose of 10 jtxg/0.2 ml oil/rat at 1200 h on day 23. DHEA and A4 were given sc at a dose of 6 mg/0.2 ml propylene glycol/100 g BW at 1200 h on days 23-25. ." Not significantly {P > 0.05) different from corresponding intact controls. 6 Significantly (P < 0.001) lower than in corresponding intact controls.
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KRAULIS ET AL.
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Endo • 1978 Vol 103 • No 5
TABLE 2. Priming efficiency of DHEA containing capsules (in situ days 23-26) and plasma steroid concentrations achieved Implant
n
Plasma LH (ng/ ml)
Plasma DHEA (pg/ml) Uterine wt (mg)
Sulfate Free 9 ± 4 (2) 7 ± 6 (2) 49 43 ± 6 ± 5 3 Blank 113 ± 26 (3)* 53 ± 5 (3)" 62 ± 7 48 ± 3 4 DHEA, 2 cm 510 ± 80 (4)* 108 ± 6 (4)e 675 ± 106c 142 ± 18" 5 DHEA, 2 x 2 cm All rats were injected with P at 1200 h on day 26 and sacrificed 5 h later. Mean ± SEM. n = number of animals; numbers in parentheses refer to number of animals yielding sufficient plasma for steroid determinations. " Significantly greater than blank-implanted controls: P < 0.01. 6 Significantly greater than blank-implanted controls: P < 0.05. c Significantly greater than blank-implanted controls: P < 0.005.
to promote the maturation of the steroid-sensitive surge system for gonadotropins in prepubertal rats and to increase uterine weights to within the adult range (Figs. 1-3). When any of these steroids were administered in the
surge system remained unaffected by ovariectomy (Table 1). Because of the discrepancy between the uterotropic and gonadotropic effects in ovariectomized rats, it seems likely that the conversion of androgens to estrogen
form of single repeated daily injections, the
required for this process takes place within
dose requirements were in the milligram range and might thus be considered pharmacological. However, comparable results were obtained with Silastic implants of DHEA (Table 2) that maintained near physiological concentrations as judged by the results of Parker and Mahesh [(17) basal DHEA levels in rats of comparable age: 160-270 pg/ml)]. Experiments with estrogen antiserum (Fig. 4) suggest that conversion of DHEA, A4, and T to estrogen is essential for the observed priming effect on the gonadotropin surge system. In contrast, the uterotropic effect of these androgens is only partially neutralized by antibodies, an observation which has previously been reported for estradiol itself (18). In the case of T, the maturation of the surge system was found to require lower doses than the induction of uterine growth; it is possible that the latter effect is partially due to the formation of ring A-reduced products rather than aromatization to estrogen, since several of these nonaromatizable compounds also exhibited significant uterotrophic effects (Fig. 3). The uterotropic activity of DHEA and A4 was nearly abolished by ovariectomy (Table 1). This observation implicates the ovary as a major site of peripheral aromatization of exogenous androgens. In remarkable contrast, the efficiency of these steroids to induce maturation of the steroid-sensitive gonadotropin
the brain itself, which, in the rat, displays demonstrable aromatase activity from fetal life onwards (19). In addition, the discrepancy in dose requirements observed for T suggests that, at least for this androgen, the brain might have a lower threshold than the uterus. It is noteworthy that only androgens which fulfill the structural requirements for aromatization (20) were effective in priming immature rats for subsequent P-induced LH release; neither the ring A-reduced androgens tested nor the 11/Miydroxy- and 11-keto-derivatives of A4, both of which sterically interfere with aromatizing enzymes, led to a LH surge (Fig. 1). In contrast, the ring A-reduced androgen 5a-androstane-3a,17/?-diol also led to a small but significant increment in the plasma concentration of FSH 5 h after the administration of P (Fig. 2). The priming ability of the 3/8epimer was below statistical significance in this particular design, but it is conceivable that a similar FSH-stimulating effect accounts for the observation that 5a-androstane-3/?,17/?-diol advances puberty in the rat if given over prolonged periods of time (21); slight but chronic elevation of FSH achieved by other means [hippocampal stimulation (22)] has likewise been reported to result in precocious vaginal opening. Knudsen and Mahesh (13) previously described that repeated injections of DHEA induce precocious ovulation in immature rats,
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ANDROGEN PRIMING AND PUBERTY
and Parker and Mahesh (17) demonstrated increased systemic concentrations of estrogen as early as 2 h after the administration of the androgen. During spontaneous sexual maturation in the female rat, T concentrations and nonspecified "androgens" have been measured by Dohler and Wuttke (3), but, to our knowledge, no systematic studies are available for weaker androgens such as DHEA and A4. In order to explain the process of puberty, the hypothesis which is most frequently advanced is the so-called "gonadostat" theory, according to which sex steroids become progressively less efficient in inhibiting pituitary gonadotropin secretion by negative feedback (23). However, some recent observations have cast; doubt on the applicability of this concept to the female rat. For instance, during spontaneous sexual maturation as well as during precocious puberty induced by brain lesions, circulating FSH concentrations do not display the steady rise anticipated if the level of negative feedback control were "reset" to a lower level of sensitivity (24). Similarly, small unilateral lesions which spare the majority of demonstrated steroid uptake sites within the basal hypothalamus are sufficient to cause precocious maturation of the gonadotrophin surge system in a manner similar to exogenous estrogen (25). Such lesions, therefore, can no longer be considered to advance puberty by eliminating the sites of negative steroid feedback. Clinicians have long been aware that increased adrenal secretion of androgens ("adrenarche") may precede the activation of the gonads by several years (6, 7) and might be causally related to the onset of puberty. In the female, these androgens would be ideally suited to circumvent the initial block in gonadal steroid pathways caused by the absence of LH receptors (10,11) and could allow estrogen synthesis to proceed before these receptors are induced. The existence of a separate pituitary hormone controlling the secretion of adrenal androgens has been proposed (8, 9). Our studies show that, if confirmed, such a hormone might have to be considered as a prime candidate for the role of the elusive trigger of puberty in the female rat.
1827
Acknowledgments We thank Dr. J. R. G. ChaUis for advice on the DHEA assay and for cross-reactivity studies on the antiserum used and Dr. B. Robaire for helpful suggestions on the preparation of Silastic implants. RIAs were carried out in collaboration with Dr. D. Lee and Ms. Patricia Wong and were based on materials provided by the NIAMDD rat pituitary hormone distribution program. The antiestrogen antiserum used was a gift from Dr. B. V. Caldwell.
References 1. Meijs-Roelofs, H. M. A., J. T. J. Uilenbroek, J. W. de Greef, F. H. de Jong, and P. Kramer, Gonadotrophin and steroid levels around the time of first ovulation in the rat, J Endocrinol 67: 275, 1975. 2. Ojeda, S. R., J. E. Wheaton, H. E. Jameson, and S. M. McCann, The onset of puberty in the female rat; changes in plasma prolactin, gonadotropins, luteinizing hormone-releasing hormone (LHRH), and hypothalamic LHRH content, Endocrinology 98: 630, 1976. 3. Dohler, K. D., and W. Wuttke, Changes with age in levels of serum gonadotropins, prolactin, and gonadal steroids in prepubertal male and female rats, Endocrinology 97: 898, 1975. 4. Parker, C. R., Jr., and V. B. Mahesh, Hormonal events surrounding the natural onset of puberty in female rats, Biol Reprod 14: 347, 1976. 5. Ramirez, V. D., and C. H. Sawyer, Advancement of puberty in the female rat by estrogen, Endocrinology 76: 1158, 1965. 6. Caligaris, L., J. J. Astrada, and S. Taleisnik, Influence of age on the release of luteinizing hormone induced by oestrogen and progesterone in immature rats, J. Endocrinol 55: 97, 1972. 7. Caligaris, L., J. J. Astrada, and S. Taleisnik, Development of the mechanisms involved in the facilitatory and inhibitory effects of ovarian steroids on the release of follicle-stimulating hormone in the immature rat, J Endocrinol 58: 547, 1973. 8. Dorrington, J. H., Y. S. Moon, and D. T. Armstrong, Estradiol-17/? biosynthesis in cultured granulosa cells from hypophysectomized immature rats; stimulation by follicle-stimulating hormone, Endocrinology 97: 1328, 1975. 9. Hall, P. F., and D. K. Young, Site of action of trophic hormones upon the biosynthetic pathways to steroid hormones, Endocrinology 82: 559, 1968. 10. Channing, C. P., and A. Tsafriri, Mechanism of action of luteinizing hormone and follicle-stimulating hormone on the ovary in vitro, Metabolism 26: 413,1977. 11. Lindner, H. R., A. Amsterdam, Y. Salomon, A. Tsafriri, A. Nimrod, S. A. Lamprecht, U. For, and Y. Koch, Intraovarian factors in ovulation: determinants of follicular response to gonadotrophins, J Reprod Fertil 51: 215, 1977. 12. Askari, H. A., Sexual differences in the biogenesis of the androgens by the adrenal cortex in rat, Endocri-
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Endo • 1978 Vol 103 • No 5
nplogyST: 1377, 1970. 21. Eckstein, B., R. Golan, and J. Shani (Mishinsky), 13. Knudsen, J. F., and V. B. Mahesh, Initiation of preOnset of puberty in the immature female rat induced cocious sexual maturation in the immature rat treated by 5a-androstane-3/?,17/?-diol, Endocrinology 92: 941, with dehydroepiandrosterone, Endocrinology 97: 1973. 458, 1975. 22. Kawakami, M., K. Seto, F. Kimura, and E. Terasawa, 14. Thorneycroft, I. S., S. A. Tillson, G. E. Abraham, R. Nature of the hippocampal function in relation to J. Scaramuzzi, and B. V. Caldwell, Preparation and gonadotropin secretion, In Yagi, K., and S. Yoshida purification of antibodies to steroids. In Peron, F. G., (eds.), Neuroendocrine Control, John Wiley & Sons, and B. V. Caldwell (eds), Immunologic Methods in New York, 1973, p. 229. Steroid Determination, Plenum Press, New York, 23. Ramirez, D. V., and S. M. McCann, Comparison of 1970, p. 63. the regulation of luteinizing hormone (LH) secretion 15. Aso, T., A-R. Aedo, and S. Z. Cekan, Simultaneous in immature and adult rats, Endocrinology 72: 452, determination of the sulphates of dehydroepiandros1963. terone and pregnenolone in plasma by radioimmu- 24. Advis, J. P., and V. D. Ramirez, Plasma levels of LH noassay following a rapid solvolysis, J Steroid Bioand FSH in female rats with precocious puberty inchem 8: 1105, 1977. duced by hypothalamic lesions, Biol Reprod 17: 313, 1977. 16. Buster, J. E., and G. E. Abraham, Radioimmunoassay of plasma dehydroepiandrosterone sulfate, Anal Lett 25. Ruf, K. B., M. Wilkinson, and D. de Ziegler, Brain lesions and precocious puberty in rats, Nature 257: 5: 543, 1972. 404, 1975. 17. Parker, C. R., and V. B. Mahesh, Dehydroepiandrosterone (DHA) induced precocious ovulation: correla- 26. Talbot, N. B., E. H. Sobel, J. W. McArthur, and J. D. Crawford, Functional Endocrinology from Birth to tive changes in blood steroids, gonadotropins and Adolescence, Harvard University Press, Cambridge, cytosol estradiol receptors of anterior pituitary gland MA, 1952. and hypothalamus, J Steroid Biochem 8: 173, 1977. 18. Ferin, M., J. Raziano, A. Tempone, and R. Vande 27. Korth-Schutz, S., L. S. Levine, and M. I. New, Evidence for the adrenal source of androgens in precoWiele, The use of antibodies as a tool in studies in cious adrenarche, Acta Endocrinol 82: 342, 1976. reproductive physiology, In Peron, F. G., and B. V. Caldwell (eds.), Immunologic Methods in Steroid De- 28. Mills, I. H., R. V. Brooks, and F. T. G. Prunty, The relationship between the production of cortisol and of termination, Plenum Press, New York, 1970, p. 199. androgen by the human adrenal, In Currie, A. R., T. 19. Reddy, V. V. R., F. Naftolin, and K. J. Ryan, ConverSymington, and J. K. Grant (eds.), The Human Adsion of androstenedione to estrone by neural tissues renal Cortex, E. & S. Livingstone, Ltd., London, 1962, from fetal and neonatal rats, Endocrinology 94: 117, p. 204. 1974. 20. Engel, L. L., The biosynthesis of estrogens, In Greep, 29. Parker, L., and W. Odell, Control of adrenal androgen secretion by a new pituitary factor: cortical androgen R. O., and E. B. Astwood (eds.), Handbook of Physistimulating hormone (CASH), Clin Res 25: 299A, ology, Williams & Wilkins, Baltimore, 1973, Section 7, 1977 (Abstract). vol. 2, part 1, p. 467.
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