Brain Research, 178 (1979) 207-212 ©Elsevier/North-Holland Biomedical Press

207

Sex differences in serum testosterone and in exchangeable brain cell nuclear estradiol during the neonatal period in rats

IVAN LIEBERBURG*, LEWIS C. KREY and BRUCE S. McEWEN** The Rockefeller University, New York N.Y. 10021 (U.S.A.)

(Accepted March 1st, 1979) K e y words:

testosterone - - cell nuclei - - sexual differentiation - - male - - female - rat - - estradiol

Perinatal secretions of the testes permanently organize reproductive centers in the developing rat brain. Testosterone (T) administration to newborn females or neonatally-castrated males also produces the same permanent changes in reproductive function (see ref. 11 for review). Since T is the major steroid present in testes and blood of newborn male rats 14 and is also a n effective agent for evoking brain sexual differentiation n , it seems likely that T is the testicular factor responsible for producing these developmental changes. As a result, one would suspect that T levels in newborn females would be low. However, D6hler and Wuttke 4 have reported that serum T levels in newborn male rats overlap to some extent with levels in female rats. Female levels were on the average 40-70 ~o of those in males during the critical first 5 days of life. In this paper, we have reinvestigated temporal patterns of T in serum in neonates. We have employed a chromatographic step before radioimmunoassay in the event that D6hler and Wuttke's results could be due to immunoreactive steroids other than T. In addition, we have measured estradiol (E2) levels associated with cell nuclear receptor sites in the neonatal brain, because this steroid appears to arise in brain largely as a local metabolite of T 7. Furthermore, the conversion of T to E2 appears to be a key step in rat brain sexual differentiationl,10,19. Pregnant Sprague-Dawley (CD strain) albino rats were obtained from Charles River, Wilmington, Mass.) at least one week before parturition or were produced by matings in our laboratory animal facility. Mothers were housed individually at 23 °C under a 14:10 light-dark cycle (lights on at 05.00 h) and were fed Purina lab chow and tap water ad libitum. Newborn animals were found within 8 h of birth. Pups were decapitated between 15:00 and 19:00 h and trunk blood was collected with a clean 1 ml glass syringe and transferred immediately to a glass tube on ice. After clotting for 24 h at 4 °C, serum was stored at --20°C. Collections were made at the following ages: day 1 (6-24 h after birth); day 3; day 5; day 8. The blood from 8 animals was pooled on day 1 ; from 5 animals on day 3; from 3 animals on day 5; from 2 animals on day 8. These pools were formed across litters in order to control for inter-litter variability. * Present address: University of Miami, School of Medicine, Miami, Fla., U.S.A, ** To whom correspondence should be addressed.

208 Serum testosterone concentrations were determined by radioimmunoassay methods as described by Gay and Kerlan 5 using antiserum S 250 generated in sheep against a testosterone-11-bovine serum albumin conjugate. The binding properties of this antiserum (generously supplied by G.D. Niswender) have been characterized by Gay and Kerlan 5, and we have found a similar pattern of specificity. Since several androgens are recognized by this antiserum benzene-ethyl acetate (1:1) extracts of100-400 #1 of neonatal rat sera were subjected to chromatography on Sephadex L H 203. Using heptane-methanol-ethyl acetate (900:75:50) as eluent, it was possible to separate completely testosterone from dihydrotestosterone and other androgens. To account for procedural losses in the extraction and chromatography procedures, a small amount of 1,2,6,7-[3H]testosterone was added to each serum sample prior to extraction. Recoveries ranged from 40-80 ~ depending on the size of the eluate collected for assay. Chrornatographed and non-chromatographed extracts of sera from adrenalectomized-castrated rats were non-reactive in the assay, lntra-assay and inter-assay coefficients of variation were 12.1~ ( n ~ 6 ) and 14.9~ (n=16), respectively. Estrogen-receptor complexes which have translocated to cell nuclei were assayed in neonatal rat brains according to the procedure of Roy and McEwen 15. Following decapitation, brains were quickly removed from the skull and placed on ice; dissection was performed on an ice-cold frosted glass plate. A coronal cut was made at the level of the optic chiasm, and another at the anterior edge of the mammillary bodies. The resulting slab of tissue containing the hypothalamus was placed facing into the glass plate. A cut was made across the slab of tissue at the level of the rhinal fissures, and the portion below the cut (including hypothalamus and amygdala) constitutes the 'limbic' tissue which was analyzed for estradiol by exchange. A 'cortex' sample was prepared by pooling temporal, parietal, and cingulate cortex peeled away from underlying thalamus and caudate-putamen. The exchange assay was performed on 0.4 M KC1 extracts of purified cell nuclei obtained from cortical and limbic tissue from male end female rats killed at postnatal days 1, 3 and 5. At these ages, 4, 3 and 2 animals, respectively, were pooled for each determination; nuclear pellets in each determination contained between 300 and 700 #g DNA. D N A was analyzed by the method of BurtonL The 0.4 M KC1 extracts were incubated as described by Roy and McEwen 15 with 2.5-3.0 nM [ZH]estradiol with or without a 100-fold molar excess of the unlabeled estrogen, R 2858, in order to control for the non-specific binding and to minimize interference from alpha-fetoprotein which does not bind this synthetic estrogen 9,12. To estimate total cell nuclear estrogen binding capacity, 3 female rats on day 1 and 5 were given a saturating dose of unlabeled E2 (1 #mol/kg in 100 #1 of 25 ethanol in saline) 1 h before killing; cortical and limbic cell nuclear pellets were assayed by the exchange procedure. In another experiment, 18 male and 20 female pups at day 5 were subjected to analysis of limbic cell estrogen receptor content using exchange assays at various [3H]estradiol concentrations. This experiment is described in the legend to Fig. 1. Because of the possibility that steroids other than testosterone might give a positive reaction in the radioimmunoassay procedure, serum extracts from neonatal

209

15

females (E 2 Injected) 9-

9 0 x

0 × 7

7

LtJ LLI OE

LIJ L~J

~s

LL_5"

C3 Z

Z

0 3 CI3

0 3

I 4

12

20

BOUND (pM)

2_0

GO

I00

BOUND

(pM)

120

Fig. 1. Scatchard plots of exchange data on cell nuclei from limbic brain tissue of 18 5-day-old male and 20 5-day-old female rats. Note that females were given 1/~g/kg unlabeled estradiol (ED by sub-. cutaneous injection in 100/4 of 10 ~ ethanol-saline 1 h before killing. This dose of E2 saturates the estrogen receptor system and drives the maximum number of occupied receptors into the nucleus. Limbic tissue was obtained and processed as described in Methods in order to obtain salt extracts of cell nuclei which were incubated with a range of concentrations of [ZH]estradiol ± 0.3 #M unlabeled R2858 (ie. moxestrol, llfl-methoxy-17a-ethynyl-17/~-estradiol, obtained from Dr. J.-P. Raynaud, Roussel-Uclaf, Paris) according to the method of Roy and McEwenlL The resulting binding data, corrected for non-specific binding and analyzed according to the method of Scatchard 1~, gives an estimate of the actual degree of occupation of cell nuclei by estrogen-receptor complexes. The y-axis is the ratio of bound to free [ZH]estradiol and the x-axis is the concentration in pmol/1 (pM) ot bound receptor complexes. In this experiment, female limbic nuclei had a capacity (x-intercept) corresponding to 62.7 fmol/mg DNA; whereas male limbic nuclei had a degree of occupation corresponding to 9.2 fmol/mg DNA or approximately 14 ~ of the estimated capacity. These values are similar to those obtained by the single point exchange assay which are presented in Table I. The dissociation constant of binding obtained from the Scatchard plots for both males and females was 0.15 nM.

rats were subjected to c h r o m a t o g r a p h y prior to the assay. The results are s u m m a r i z e d in Table I. Testosterone (T) levels were significantly elevated in the male rats at all ages examined. Male levels averaged 420 pg/ml; female levels were about 25 pg/ml. There was no overlap o f T levels between males and females at any age. In an additional experiment n o t presented in Table I, the castration o f male rats o n day 4 reduced serum T measured 24 h later to 31 ~ 2 p g / m l ( n = 5 ; range 24-34). Levels o f exchangeable cell nuclear estradiol (E2) were estimated in limbic and cerebral cortical tissue from male and female rats at 1, 3 and 5 days o f age. These results are presented in Table I together with the estrogen binding capacity o f limbic and cortical nuclei at two postnatal ages f r o m female rat brains. (Irt this connection, we

210 TABLE I Serum testosterone (T) and exchangeable cell nuclear estradiol in neonatal male and female rat brains

Serum values are given as mean ± S.E.M. (number of determinations) with range of values indicated below. Cell nuclear estradiol and capacity are given as mean t S.E.M. - - 3 determinations were made at each age. Sex

Male

Age (days)

1 3 5

Serum T (pg/ml)

Cell nuclear estradiol (fmol/mg D N A )

Cell nuclear capacity (fmol/mg D N A )

Limbic

Cortex

Limbic

Cortex

0.26 -t- 0.14

--

--

0.36 :k 0.14

--

--

0.34 ~ 0.10

--

--

495 -E 82 (16) 4.83 + 1.53 (199-1188) 463 :k_ 60 (21) 6.60 i 0.68 (119-1102) 358 ± 31 (27) 6.04 ± 0.63

(118-674) Female

8

372 -4- 57 (19) .

1

31 -~ 6 (18-48) 25 ± 2 (21-31) 21 ± 1 (17-24) 22~2

3 5 8

.

.

.

(5)

1.49 ± 0.77

0.13 4- 0.1l

36.32-4- 0.31

(5)

0.94 £: 0.14

0.19 ± 0.09

--

--

(6)

0.54 ± 0.03 --0.23 ± 0.06

60.19 ± 3.14

37.33 ~ 0.72

(7)

.

.

.

8.21 ~ 0.37

.

have consistently failed to detect a sex difference in brain estrogen receptor levels during the neonatal period 8, although the occupation of estrogen receptors shows a sex difference.) Levels o f exchangeable cell nuclear E2 are elevated in male c o m p a r e d to female limbic tissues of 1-, 3- a n d 5-day-old rats; yet estrogen occupation in male limbic nuclei is only 10 ~ of the apparant capacity o f the receptor systems of 1- and 5-dayold rats. Cortical estrogen receptors do not appear to be significantly occupied in either sex at any o f the 3 ages. The numbers reported in Table I for cortex represent the limits o f sensitivity o f the exchange assay and do not necessarily indicate the presence o f any estradiol. The increase in E2 receptor capacity in cortex between days 1 and 5 reflects the delayed ontogeny o f this receptor system c o m p a r e d to that in limbic brain tissue (see ref. 8). As a further demonstration o f the extent of occupation o f limbic cell nuclear estrogen receptors in male rats during the neonatal period, we conducted a saturation analysis o f exchangeable estradiol in cell nuclear salt extracts f r o m 5-day-old males and f r o m 5-day-old females given 1/zmol/kg unlabeled E2 (see Methods). The results, presented in Fig. 1, indicate that the cell nuclear occupation is only a r o u n d 14 ~ of the capacity o f the system. Our findings support the long-held view that testosterone is the p r i m a r y testicular secretion responsible for the sexual differentiation of the neonatal rat brain. Circulating T levels were approximately 15-fold higher in n e w b o r n male t h a n in female rats. Furthermore, castration o f the neonatal male, a procedure which blocks further sexual differentiation of the male brain, reduced serum T levels to the range f o u n d in

2ll female sera. Although our male-female difference in serum T levels, 350-400 pg/ml, is comparable to the difference found by DShler and Wuttke 4, our absolute levels of T were significantly lower in both sexes. This difference in results may be due either to our chromatography step which effectively isolated T from other immunoreactive steroids or to strain differences in the rats studied. Using the exchange assay for estradiol, we found that E2 receptor levels in limbic cell nuclei were substantially higher in neonatal male brains compared to those of females. The lower limits of sensitivity of the exchange assay may well have precluded our seeing the same-fold sex difference in E2 receptor occupation as we did in the case of serum T levels. At the same time there appeared to be some exchangeable E2 in limbic brain nuclei of females at days 1 and 3. This low level of exchange activity, which is very hard to measure accurately and reproducibly, may reflect the residual effects of androgens present before parturition, since we have also found low levels of exchangeable E2 in limbic brain cell nuclei of 21~day-old female rat fetuses 8. The occupation of limbic estrogen receptors in male brains during the neonatal period is only 10-14 ~ of the estimated capacity of the system measured by the same exchange assay procedure. The relatively low level of receptor occupation might be due to an unequal distribution of aromatizing enzyme activity among estrogensensitive neurons, in which case the occupation of some estrogen-sensitive cells would be much higher than 10-14 ~ . This possibility is not given strong support by published autoradiographic studies of the neonatal rat brain which show similar patterns of neural uptake of [3HIT and [3H]E~ and cross-competition by the unlabeled form of both steroids17, is. However, such autoradiographic information does not provide quantitative data bearing on this point, and it is possible that some cells may aromatize more T than others. It has been reported that aromatization of T to E2 occurs in limbic tissue but not in cortex of newborn rat brains6, 7,13,z0, and our results for exchangeable E2, giving no indication of E2 on cortical estrogen receptors in untreated male or female rats, are in agreement with these observations and are also qualitatively similar to those of Westley and Salaman zl who used a different procedure for measuring estradiol receptor complexes. T conversion to E2 is recognized to be an obligatory step in at least some aspects of the organizational action of this androgen on the neonatal rat brain~, ~°,~9. Our findings that there are concurrent elevations in serum T and in limbic brain estrogen receptor occupation in the neonatal male rat provide important quantitative support for such a conclusion. This research was supported by an N I H Grant NS07080 to B. McE. and by an institutional grant, RF70095 from the RockefeUer Foundation for research in reproductive biology. We thank Ms. Winifred Berg Elton for graphics work and Mrs. Oksana Wengerchuk for editorial assistance.

212 1 Booth, J. E., Effects of the aromatization inhibitor, androst-4-ene-3,16,17-trione on sexual differentiation induced by testosterone in the neonatally castrated rat, J. Endocr., 72 (1977) 53P-54P. 2 Burton, K., A study of the conditions and mechanisms of the diphenylamine reaction for the colometric estimation of DNA, Biochem. J., 62 (1956) 315-323. 3 Carr, B. R., Mikhail, G. and Flickinger, G. L., Column chromatography of steroids on Sephadex LH-20, J. clin. Endocr., 33 (1971) 358-360. 4 D6hler, K. D. and Wuttke, W., Changes with age in levels of serum gonadotropins, prolactin, and gonadal steroids in prepubertal male and female rats, Endocrinology, 97 (1975) 898-907. 5 Gay, B. L. and Kerlan, J. T., Serum LH and FSH following passive immunization against circulating testosterone in the intact male rat and in orchidectomized rats bearing subcutaneous silastic implants of testosterone, Arch. AndroL, 1 (1978) 239-248. 6 Lieberburg, 1. and McEwen, B. S., Estradiol 17/3: a metabolite of testosterone recovered in cell nuclei from limbic areas of neonatal rat brain, Brain Research, 85 (1975) 165-170. 7 Lieberburg, I., Wallach, G. and McEwen, B. S., Theeffects of an inhibitor of aromatization (1,4,6androstatriene-3,17-dione) and anti-estrogen (CI 628) on in vivo formed testosterone metabolites recovered from neonatal rat brain tissues and purified cell nuclei. Implications for sexual differentiation of the rat brain, Brain Research, 128 (1977) 176-181. 8 Maclusky, N. J., Lieberburg, I. and McEwen, B. S., The development of estrogen receptor systems in the rat brain: perinatal development, Brain Research, 178 (1979) 129-142. 9 McEwen, B. S., Plapinger, L., Chaptal, C., Gerlach, J. and Wallach, G., Role of fetoneonatal estrogen binding proteins in the association of estrogen with neonatal brain cell nuclear receptors, Brain Research, 96 (1975) 2~00-406. 10 McEwen, B. S., Lieberburger, I., Chaptal, C. and Krey, L. C., Aromatization: important for sexual differentiation of the neonatal rat brain, Horm. Behav., 9 (1977) 249-263. 11 Plapinger, L. and McEwen, B. S., Gonadal steroid brain interactions in sexual differentiation. In J. Hutchinson (Ed.), Biological Determinants of Sexual Behavior, J. Wiley, New York, 1977, pp. 153-218. 12 Raynaud, J. P., Mercier-Bodard, C. and Baulie~ E. E., Rat estradiol binding plasma protein (EBP), Steroids, 18 (1971) 767-788. 13 Reddy, V. V. R., Naftolin, F. and Ryan, K. J., Conversion of androstenedione to estrone by neural tissues from fetal and neonatal rats, Endocrinology, 94 (1974) 117-121. 14 Resko, J. A., Feder, H. H. and Goy, R. W., Androgen concentrations in plasma and testes of developing rats, J. Endocr., 40 (1968) 485-491. 15 Roy, E. J. and McEwen, B. S., An exchange assay for estrogen receptors in cell nuclei of the adult rat brain, Steroids, 30 (1977) 657-669. 16 Scatchard, G., The attraction of protein for small molecules and ions, Ann. N.Y. Acad. Sei., 51 (1949) 660-672. 17 Sheridan, P. J., Sar, M. and Stumpf, W. E., Autoradiographic localization of 3H estradio! or its metabolites in the central nervous system of" the developing rat, Endocrinology, 94 (1974) 1386-1390. 18 Sheridan, P. J., Sar, M. and Stumpf, W. E., Interaction of exogenous steroids in the developing rat brain, Endocrinology, 95 (1974) 1749-1753. 19 Vreeburg, J. T. M., van der Vaart, P. D. M. and van der Schoot, P., Prevention of central defeminization but not masculinization in male rats by inhibition neonatally of oestrogen biosynthesis, J. Endocr., 74 (1977) 375-382. 20 Weisz, J. and Gibbs, C., Metabolites of testosterone in the brain of the newborn female rat after an injection of tritiated testosterone, Neuroendocrinology, 14 (1974) 72-86. 21 Westley, B. R. and Salaman, D. F., Role of oestrogen receptors in androgen-induced sexual differentiation of the brain, Nature (Lond.), 262 (1976) 407-408.

Sex differences in serum testosterone and in exchangeable brain cell nuclear estradiol during the neonatal period in rats.

Brain Research, 178 (1979) 207-212 ©Elsevier/North-Holland Biomedical Press 207 Sex differences in serum testosterone and in exchangeable brain cell...
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