hypothesis (Swerdloff, Walsh, Jacobs & Odell, 1971; Odell, Swerdloff, Jacobs & Hescox,

Transcription

1 LEVELS OF LUTEINIZING HORMONE, FOLLICLE-STIMULTING HORMONE, TESTOSTERONE N IHROTESTOSTERONE IN THE CIRCULTION OF SEXULL MTURING INTCT MLE RTS N FTER ORCHIECTOM N EXPERIMENTL BILTERL CRPTORCHIISM. GUPT, K. RGER, J. ZRZCKI N M. EICHNER epartment of iagnostic Endocrinology, University Children's Hospital, University of Tübingen, Germany (Received 2 September 1974) SUMMR Plasma concentrations of LH, FSH, testosterone and dihydrotestosterone (HT) have been measured in normal sexually maturing male rats from the age of 16\p=n-\90days. Between 16 and 25 days plasma testosterone levels were low, but rose suddenly on day 26. similar increment occurred at the same time in plasma HT levels, but this steroid reached its peak concentration later than testosterone. Plasma LH levels rose steadily from day 25 onwards, reaching their highest values on day 30. marked increase in FSH levels was found on day 16, and a peak was reached on day 33 followed by a decline to a level characteristic of the adult. In addition, plasma levels of all these hormones were estimated in the male animals at various stages of development after orchidectomy and cryptorchidism. Four days after operation, the plasma levels of LH and FSH in the orchidectomized animals reached higher levels than those found in the intact animals, indicating the existence of a dynamic feedback relationship before puberty between gonadal steroids and pituitary gonadotrophic secretion. However, results from the experimental bilaterally cryptorchid animals, suggested that the gonadal steroid\p=n-\gonadotrophicfeedback relationship could not be the only factor initiating puberty. INTROUCTION There is considerable evidence to suggest that there is a change in the feedback sensitivity between gonadal steroids and gonadotrophins during sexual maturation (Ramirez & Mc Cann, 1965; Critchlow & Bar-Sela, 1967; Smith & avidson, 1967; Negro-Vilar, Krulich & McCann, 1973; Gupta, 1974). There are, however, other views which disagree with this hypothesis (Swerdloff, Walsh, Jacobs & Odell, 1971; Odell, Swerdloff, Jacobs & Hescox, 1973; Swerdloff, Jacobs & Odell, 1973). In spite of the large number of reports on the changes in profile of plasma testosterone (Resko, Feder & Goy, 1968; Knorr, Vanha- Perttula & Lipsett, 1970; Grota, 1971; Coyotupa, Parlow & Kovacic, 1973; Miyachi, Nieschlag & Lipsett, 1973) and serum gonadotrophins (Ramirez & McCann, 1965; Gay & Midgley, 1969; amamoto, iebel & Bogdanove, 1970; matayakul, Ryan, Uozumi & lbert, 1971 ; Goldman, Grazies, Kamberi & Porter, 1971 ; Swerdloff et al ; Root & * Present address : II Paediatric Clinic, Lodz, Poland.

2 Russ, 1972; Walsh & Swerdloff, 1973; Negro-Vilar et al. 1973) there have been no systematic studies of the levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone and the more potent androgen, dihydrotestosterone (HT), in the circulation of sexually maturing male rats. There are no reports which have correlated, in the same animal, the changes in both plasma gonadotrophins and androgens occurring during development, after orchidectomy or after experimental bilateral cryptorchidism at various stages of sexual development. The present study was therefore undertaken. MTERILS N METHOS Experimental design The animals used in these experiments were male rats of the Ivanovac strain (llgäu, Germany), maintained in communal cages (5 to 6 adult, 10 to 14 immature rats) under artificial light (lights on from to h). Water and food were freely available. ll surgical operations were performed under ether anaesthesia. Plasma gonadotrophins and androgens from early prepubertal stage to full maturity Normal prepubertal, pubertal and adult rats were used. Ten animals were killed for each day of life up to 40 days of age, and the blood collected by cardiac puncture, always at noon, was pooled. fter 40 days, only six animals were killed each day and blood was col lected as before. Their plasma androgen levels were measured daily between days 16 and 40. This value represents the mean of four determinations from pooled samples obtained from ten or six animals. In the plasma sample four to six determinations of LH and FSH were performed and the average values recorded. Effect of castration on plasma gonadotrophins and androgens and mature rats at various ages in immature Five groups of animals aged 16, 36, 56, 66, 86 days were orchidectomized. The animals were killed 4 days after the operation and blood was collected by cardiac puncture. There were six rats/group except in the case of the groups killed on days 20 and 40 which contained ten rats each. Effect of surgical bilateral cryptorchidism in immature and mature rats at various ages Experimental bilateral cryptorchidism was carried out on five groups of animals at similar stages of development and of similar population size as above. For surgical cryptorchidism, both testes were drawn through the inguinal canal into the abdomen and sutured to the abdominal wall after severing the gubernaculum. This method was found to be reliable in keeping the testes in the abdomen. The animals were killed 4 days after the operation as in the previous experiment and blood was collected by cardiac puncture. The number of animals per group was as described in the previous section. Blood samples were pooled in groups, centrifuged and stored at 20 C. Four estimations for each pooled sample were carried out for testosterone, HT, LH and FSH using radioimmunoassay techniques. Radioimmunoassay of testosterone and HT modification of the radioimmunoassay method of ttanasio, McCafferty, Raaf, Rager & Gupta (1973) and Gupta, ttanasio & Raaf (1975) was used for the measurement of testosterone and HT. In brief, plasma samples were extracted with ether, and the dried

3 extract was chromatographed on a Sephadex LH-20 column. On the standard curve 10 pg were significantly different from 0 pg at a probability of 99 % for both steroids assayed. The recoveries ranged from % for both testosterone and HT. s determined by assaying ten 1 ml samples of plasma, the intra-assay coefficient of variation was less than 8 % for both steroids. For each time-point in a single assay for testosterone and another assay for HT, the hormone concentrations of the test samples were measured and the intra-assay coefficients of variation were 8-6 and 9-3 % respectively. Radioimmunoassay for LH and FSH Plasma LH and FSH radioimmunoassays were performed as described in the radio immunoassay kit supplied by the Rat Pituitary istribution Program, NIM. The second antibody used was anti-rabbit gamma globulin, purchased from Wellcome Labora tories (England). Pure rat LH and FSH were iodinated using the chloramine-t method of Greenwood, Hunter & Glover (1963). ll samples from each experiment were measured within a single assay. The intra-assay coefficient of variation was 7 % of the values deter mined. LH values are expressed in terms of NIM-Rat-LH-RP-1 and FSH values are given in terms of NIM-Rat-FSH-RP-1 as ng/ml. RESULTS Plasma hormone concentration Testosterone Figure 1 a shows the mean plasma testosterone concentration in male rats from the age of 16 days onwards. Between 16 and 25 days the plasma testosterone concentration was low (never exceeding 50 ng/100 ml) and constant. sudden increment was seen on day 26 (125 ng/100 ml) and the peak (260 ng/100 ml) was reached on the 29th day, i.e. plasma testosterone concentration undergoes a more than fivefold increment during the course of 4 days. The highest concentration (294-8 ng/100 ml) was seen at 70 days of age. ihydrotestosterone Figure 1 b shows the pattern of plasma HT concentration in the rat during various stages of sexual maturation. The concentration of HT like that of testosterone was low (below 5 ng/100 ml) until day 25, and on day 26 a sudden increment was recorded. But unlike testosterone, it took longer (i.e. until day 33) to reach a peak (16-3 ng/100 ml). On day 35 the concentration decreased to half the value seen on day 33, then rose steeply to a new peak value of about 20 ng/100 ml on day 38. fter this the level decreased a little to plateau at approximately 15 ng/100 ml. Luteinizing hormone Figure 2 a shows the plasma LH concentration in developing male rats. Between days 16 and 24 plasma LH concentrations were low, the minimum being 20 ng/ml on day 21, and the average value never exceeding 50 ng/ml. steady increment was recorded from day 25 onwards reaching a peak (84 ng/ml) on day 30, before decreasing gradually until day 40. The levels remained low until the 60th day, whereupon they again rose to 60 ng/ml on day 70 and remained constant thereafter.

4 Follicle-stimulating hormone Figure 2b shows the profile of the mean plasma FSH concentration in male rats from the age of 16 days onwards. The pattern of FSH concentration showed a marked rise from day 16 (180 ng/ml) onwards with a peak on day 33 (770 ng/ml). The values started decreasing after day 33, reached adult levels at day 40 and remained constant thereafter. Castrated animals Figure 1 a shows the effect of orchidectomy on plasma testosterone levels at various times after birth. On day 20 the mean plasma testosterone value was 38-4 ng/100 ml which was ^_,-1, [-f l'i ge of animals (days) Fig. 1. Mean levels of (a) plasma testosterone and (b) plasma dihydrotestosterone (HT) as a function of age in 16- to 90-day-old male rats. The levels for these steroids in castrated and cryptorchid animals at various ages are shown as solid squares and solid circles respectively. Number of animals/group : ten up to 40 days and six after 40 days. The vertical lines denote ±s.d.

5 - not significantly different from the level seen in the intact animal. t day 40 the castrated animals had a mean plasma testosterone concentration which was not significantly different from the level seen on day 20, but was only 15 % of the value seen in the intact animal of the same age. On days 60, 70 and 90 castrated animals showed very similar plasma testo sterone levels (65-0, 66-0 and 87-4 ng/100 ml, respectively) which were about 23 % of the values found in the intact animals at the same ages («) g (b) \- -\ ge of the animals (days) Fig. 2. Mean levels of (a) plasma LH and (b) plasma FSH as a function of age in 16- to 90-day-old male rats. The levels of plasma LH in castrated and cryptorchid animals at various ages are shown as solid squares and solid circles respectively. Number of animals/group: ten up to 40 days and six after 40 days. The vertical lines denote ±s.d.

6 Figure 1 b shows the changes in plasma levels of HT at various stages of life. fter orchidectomy plasma HT levels decreased below the sensitivity threshold of our method and have been reported as zero. There was a clear increment in LH secretion (Fig. 2d) in response to castration. On day 20, the mean plasma LH concentration in the orchidectomized animal was 166 ng/ml, a value 4-7 times greater than the mean value in the intact animal. There were further incre ments on days 40 and 60 when the differences in the values between castrated and intact 70 this difference fell and animals became and 141-fold greater, respectively. On day by 90 days the level was 3-4 times that of the intact animals. Castration also resulted in a prompt increase in FSH (Fig. 2b) similar to that seen for LH. The level at 20 days was about four times that of the intact animals. However, the relationship between the levels seen in the castrated and in the age-matched controls did not change a great deal with maturation. Experimental bilateral cryptorchid animals t 20 days of life cryptorchid animals showed a significantly (P < 0-01) higher con centration (65-5 ng/100 ml) of plasma testosterone than that seen in the intact animals (44-1 ng/100 ml). From day 40 onwards the levels were not significantly different from those of the intact animals (Fig. 1 a). Figure 1 b shows the plasma concentration of HT in the same animals. t day 20 of life the level of HT was virtually the same for intact and cryptorchid animals. The levels in the cryptorchid animals were, however, found to be significantly less (P < 0-01) than those seen in the intact animals at other stages studied. In the cryptorchid animals, mean plasma LH rose but not as promptly as seen in the castrated animals. The level at 20 days of age was 171 % of that in the 20-day-old intact animals, and rose to 600 % at 40 days. t 60, 70 and 90 days the values had reached levels of 533, 183 and 185 %, respectively. The pattern of FSH concentrations in the cryptorchid animals was strikingly different from that of LH; as in the immature animals aged 20 days, no increment was seen (only 70 % of the control value; Fig. 2b). By day 40 this level was 157 % of the control value and then it increased to a new, fairly consistent level between 200 and 300% of normal. Feedback mechanism in vivo The increase of plasma LH and FSH concentrations after castration of the male rat at various ages clearly demonstrated the existence of a dynamic hypothalamic-pituitarygonadal relationship. t the same time, this response, when closely examined, indicated different levels of sensitivity of the pituitary during the prepubertal stage compared with late puberty and adulthood. Figure 3 shows the changes in plasma levels of LH (LH) and testosterone (testosterone) after castration. When the incremental rise in LH is related to unit decrease of testosterone (LH : testosterone) a similar age-related dif ference is shown. On day 20 the ratio is 16-6 which decreases to 1-23 at the onset of puberty (day 40) and then remains fairly constant ( TO). This difference in sensitivity between LH and testosterone secretion with castration in adult and immature animals was also reflected in the FSH profiles. Figure 4 shows that whereas the increment in plasma FSH concentration, per 1 ng/100 ml decrement of testo sterone, was ng/ml in the immature 20-day-old animal, all the pubertal and postpubertal groups showed much lower values which fell between 7-40 and 8-71 ng/ml.

7 + LH (ng/ml) LH: testosterone -r o o -Testosterone fcvivivtvtvtvi's m-..imi iiiiiiiroarra (ng/100 ml) " ' 11 f I " " " " 60 IIIÌHIIIM1II 70 -"-' iffl IIH. 1.' rzza +LH I-1 Testosterone LH: testosterone Fig. 3. Levels of change ( ) in plasma LH and testosterone after castration in male rats at various ages. On the left side of the vertical age bars the positive and negative changes are depicted whereas on the right is given the ratio LH : testosterone, which reflects the positive change in LH due to unit negative change in plasma concentration of testosterone. ISCUSSION Intact rat We are unaware of any previous study that has recorded the simultaneous levels of plasma LH, FSH, testosterone and HT at daily intervals during puberty in the rat. number of investigations have, however, measured testosterone (Resko et al. 1968; Knorr et al. 1970; Grota, 1971; Coyotupa et al. 1973; Miyachi et al. 1973) or LH and FSH (Ramirez & McCann, 1965; Gay & Midgley, 1969; amamoto et al. 1970; matayakul et al. 1971; Goldman et al. 1971; Swerdloff et al. 1971; Root & Russ, 1972; Negro-Vilar et al. 1973; Walsh & Swerdloff, 1973) at various intervals during pubertal growth in this species. The results recorded here show that testosterone levels rise dramatically after postnatal

8 . + FSH (ng/ml) I _0 -Testosterone ruttiitniiniitf " (ng/100 ml) 20 FSH: testosterone UM I llllllllii. > EJ H+l II I I I I I I I I I I I!. *»»»»«60 ' I*«*«*.'«*.*»'«*-*.*»* *.*.* * *» *** *»*»* *.* **' *. 70.-J. 90 :::-ii;-n-^-m4fflffl r^vn +FSH Testosterone m il FSH: testosterone Fig. 4. Levels of change ( ) in plasma FSH and testosterone due to castration in male rats at various ages. On the left side of the vertical age columns the positive and negative changes are depicted whereas on the right is given the ratio FSH : testosterone, which reflects the positive change in FSH due to unit negative change in plasma concentration of testosterone. day 25 and achieve a fivefold increase in the 3 subsequent days. Over the next 40 days, although some minor fluctuations occur, the level only rises another 11 %. Knorr et al. (1970) measured testosterone at 5- and 10-day intervals and found the first discernible rise at 35 days of age and a maximal level (770 ng/100 ml) on day 60. This level then fell by 46 % by the time the animals were fully mature. Much better agreement is found between our findings and those of Miyachi et al. (1973), who showed a peak (300 ng/100 ml) level of testosterone after day 25. No comparative data are available for HT. The results show that the levels begin increasing at the same time as testosterone but achieve peak values later (days 33 and 38). Our findings on the profiles of plasma LH and FSH for intact male rats correspond to those recently reported by Negro-Vilar et al. (1973). These investigators stated that the

9 pattern of LH contrasted sharply with that of FSH in that there is no peak at days but a gradual increase during sexual development. Careful examination of the results, how ever, reveals that their LH value (NIH-LH-S-1) at 30 days was 200 % higher than that seen at day 20. Similarly, between 60 and 70 days of age there was another twofold increment. lthough this peak was not as sharp as that of FSH, other investigators observed the existence of a plasma LH peak in male rats between 23 and 30 days of age (Ojeda & Ramirez, 1972 ; Miyachi et al ; Bloch, Masken, Kragt & Ganong, 1974). Our results demonstrate that plasma LH had a clear peak between age 25 and 30 days after which levels fell only to rise again at 70 days. Plasma FSH concentration reached a clearly discernible peak after which values fell. This finding agrees well with the reports by matayakul et al. (1971), Negro-Vilar et al. (1973), Swerdloff et al, (1973) and Odell et al. (1973). The results show that the FSH concentrations almost doubled by day 33 and a sharp decline started by day 35, i.e. just before mature sperm appears in the tubule. The events associated with higher hormonal concentrations seen during days 25 to 33 in the male rat can perhaps be related to the initiation of the Leydig cell differentiation noted at about the same time (Resko et al ; Knorr et al. 1970). It is, however, surprising that by day 40, when the growth rates of the sex accessories such as the seminal vesicle and ventral prostate glands are the greatest (Bloch et al. 1974), the levels of these hormones show a sharp decline. Castrated rat fter castration, plasma levels of LH and FSH increased within 4 days in both juvenile and adult male rats, confirming previous work (amamoto et al. 1970; matayakul et al ; Goldman et al ; Swerdloff et al ; Odell et al. 1973). These results indicate the existence in the prepubertal male rats of an intact feedback relationship between the gonad and pituitary gonadotrophic secretion. In the present study, however, stress has been laid more on the magnitude of the gonadotrophic response observed at the various ages studied. While amamoto et al. (1970) observed that the rise in LH after castration was equally large in neonatal, pubertal and adult life, our results demonstrate that the minimum incre ment in LH occurred by day 60. The response of FSH to castration was, however, found to be fourfold at all the ages studied, although Ojeda & Ramirez (1972) found much higher plasma levels after castration between days 10 and 20 than after castration between days 30 and 40. matayakul et al. (1971) found that the levels of testosterone decrease by half within 21 days of castration. Other investigators (Coyotupa et al. 1973) found a rapid and total disappearance of testosterone within hours of orchidectomy. In the present study, the level of testosterone declined only 13 % in the 20-day-old animals but 85 % in the 40-day-old animals; 4 days after castration in both cases. Plasma HT concentration in all the castrated animals, however, decreased below the level of sensitivity of the method. One of the striking findings in this study was the large increase in plasma gonadotrophins accompanied by a pronounced decrease in plasma testosterone in the 20-day-old animals. This did not occur at later stages. This observation strongly indicates a decline in the thres hold of sensitivity with sexual maturation, although the cause for this remains unclear. Whether the decreased ability of the hypothalamic or pituitary tissue to metabolize testo sterone to its 5a-reduced forms (Massa, Stupnicka, Kniewald & Martini, 1972; enef, Magnus & McEwen, 1974; Klemm & Gupta, 1975), which is more pronounced in adult rats than in immature animals, has anything to do with this change is a matter of speculation.

10 Cryptorchid rats In contrast to the results seen in the castrated animals, those of the cryptorchid groups demonstrate that the gonadal steroid-gonadotrophin feedback relationship cannot be the only factor in the initiation of puberty. lthough mean testosterone levels significantly increased in the immature cryptorchid animals, they did not differ significantly from the control animals at later stages. However, HT values indicated a mean trend of decrement in relation to the intact values at each stage of development except in the 20-day-old animals. The mean level of FSH in plasma during the early stages of development was not different from that of the age-matched controls, but continued to rise up to day 60 as compared with the fall in the intact animals. In contrast, LH levels rose in the cryptorchid animals much earlier before the onset of puberty. This finding does not agree with the observations of Swerdloff et al. (1971) who reported no difference between cryptorchid and intact animals until day 49. The great disparity in the levels of the gonadotrophins in the castrated and bilaterally cryptorchid animals indicate that some other factors are possibly involved in the feedback process. In contrast to the changes in testosterone levels seen in the intact pre- and postpubertal animals, FSH secretion correlates more uniformly with the HT levels. It has also been re ported that HT and 5<x-androstane-3a,17/?-diol suppress plasma FSH levels in intact as well as in orchidectomized rats (Swerdloff, Walsh & Odell, 1972; Zanisi, Motta & Martini, 1973; Rager, Eichner, Willmann & Gupta, 1974). part from androgenic inhibition of FSH secretion it has also been postulated that the germinal epithelium exerts a negative feedback effect (Swerdloff et al. 1971). These postulations, taken together with the earlier observations of Schally, rimura, Kastin, Matsuo, Baba, Redding, Nair & ebeljuk (1971), Steinberger & Chowdhury (1974) and Gupta (1974) on the apparent difference in the regulatory mech anism of FSH secretion to that of LH, add strong support to the view that the steady levels of plasma FSH in the cryptorchid immature rats are perhaps a result of the absence of the FSH-inhibiting factor(s). It would seem that two factors have to be considered in this the presence or absence of active respect when examining results from immature animals : germinal epithelium and the circulating levels of HT. The present observations indicate that one important and still unknown element in the initiation of puberty may be the neural mechanism that regulates the secretion of FSH. The authors would like to thank the Humboldt-Stiftung for a stipend to J. Z., the eutsche Forschungsgemeinschaft for generous grants, the National Institutes of Health, Bethesda, Maryland, for the gifts of materials for the rat gonadotrophin radioimmunoassay, Professor Klyne of the Steroid Reference Collection (London) for authentic steroids, and Professor J. R. Bierich, the irector of the Universitäts-Kinderklinik Tübingen, for encouragement. REFERENCES matayakul, K., Ryan, R. T., Uozumi, T. & lbert,. (1971). reinvestigation of testicular-anterior pituitary relationship in the rat: I. Effect of castration and cryptorchidism. Endocrinology 88, ttanasio,., McCafferty, E., Raaf, S., Rager,. & Gupta,. (1973). The inter-relationship of plasma oestrogens with the androgens in sexually maturing children. cta Endocrinologica Suppl. 177, 307. Bloch, G. T., Masken, T., Kragt, C. L. & Ganong, W. F. (1974). Effect of testosterone on plasma LH in male rats of various ages. Endocrinology 94, Coyotupa, T., Parlow,. F. & Kovacic, N. (1973). Serum testosterone and dihydrotestosterone levels fol lowing orchidectomy in the adult rat. Endocrinology 92, Critchlow, V. & Bar-Sela, M. E. (1967). Control of the onset of puberty. In Neuroendocrinology, vol. 2, pp Eds L. Martini & W. P. Ganong. New ork and London: cademic Press.

11 enef, C, Magnus, C. & McEwen, B. S. (1974). Sex-dependent changes in pituitary 5-alpha dihydro testosterone and 3-alpha androstanediol formation during postnatal development and puberty in the rat. Endocrinology 94, Gay, V. L. & Midgley,. R., Jr (1969). Response of the adult rat to orchidectomy and ovariectomy as de termined by LH radioimmunoassay. Endocrinology 84, Goldman, B.., Grazies,. R., Kamberi, I.. & Porter, J. C. (1971). Serum gonadotropin concentrations in intact and castrated neonatal rats. Endocrinology 88, Greenwood, F. C, Hunter, W. M. & Glover, J. S. (1963). The preparation of labelled human growth hormone of high specific radioactivity. Biochemical Journal 89, Grota, L. J. (1971). Effects of age and experience on plasma testosterone. Neuroendocrinology 8, Gupta,. (1974). Gonadal steroids and gonadotrophin feedback regulation of mammalian sexual maturation. In Recent progress in reproductive endocrinology. Eds P. G. Crossignani & V. H. T. James. London: cademic Press. Gupta,., ttanasio,. & Raaf, S. (1975). Plasma estrogen and androgen concentrations in children during adolescence. Journal of Clinical Endocrinology and Metabolism 40, Klemm, W. & Gupta,. (1975). Pituitary biotransformation of testosterone to its 5-<x-reduced metabolites in sexually maturing male rats and after orchidectomy and experimental bilateral cryptorchidism. Journal of Endocrinology. 65, 12P. Knorr,. W., Vanha-Perttula, T. & Lipsett, M. B. (1970). Structure and function of rat testis through pubescence. Endocrinology 86, Massa, R., Stupnicka, E., Kniewald,. & Martini L. (1972). The transformation of testosterone into di hydrotestosterone by the brain and the anterior pituitary. /. Steroid Biochemistry 3, Miyachi,., Nieschlag, E. & Lipsett,.. (1973). The secretion of gonadotropins and testosterone by neonatal male rat. Endocrinology 92, 1-5. Negro-Vilar,., Krulich, L. & McCann, S. M. (1973). Changes in serum prolactin and gonadotropins during sexual development of the male rat. Endocrinology 93, Odell, W.., Swerdloff, R. S., Jacobs, M. S. & Hescox, M.. (1973). FSH induction of sensitivity to LH: one cause of sexual maturation in the male rat. Endocrinology 92, Ojeda, S. R. & Ramirez, V.. (1972). Plasma levels of LH and FSH in maturing rats: response to hemigonadectomy. Endocrinology 90, Rager, K, Eichner, M., Willmann, W. & Gupta,. (1974). Studies on the relative sensitivity for androgen feedback inhibition of gonadotropins in rats during sexual maturation. Communication to the 13th annual meeting of the European Society for Paediatric Endocrinology, Paris. Pediatrie Research, (In Press). Ramirez, V.. & McCann, S. M. (1965). Inhibition effect of testosterone on luteinizing hormone secretion in immature and adult rats. Endocrinology 76, Resko, J.., Feder,.. & Goy, R. W. (1968). ndrogen concentrations in plasma and testis of developing rats. Journal of Endocrinology 40, Root,. W. & Russ, R.. (1972). Short-term effect of castration upon pituitary and serum levels of luteiniz ing hormone and follicle stimulating hormone in male rats. cta Endocrinologica 70, Schally,. V., rimura,., Kastin,. J., Matsuo, H., Baba,., Redding, T. W., Nair, R. M. G. & ebeljuk, L. (1971). Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle stimulating hormone. Science 173, Smith, E. R. & avidson, J. M. (1967). ifferential responses to hypothalamic testosterone in relation to male puberty. merican Journal ofpsychology 212, Steinberger, E. & Chowdhury, M. (1974). Control of pituitary FSH in male rats. cta Endocrinologica 76, Swerdloff, R. S., Jacobs, H. S. & Odell, W.. (1973). Hypothalamic-pituitary-gonadal interrelationship in the rat during sexual maturation. In Gonadotropins, pp Eds B. B. Saxena, C. G. Beling & H. M. Gandy. New ork: Wiley-Interscience. Swerdloff, R. S., Walsh, P. C, Jacobs, H. S. & Odell, W.. (1971). Serum LH and FSH during sexual maturation in the male rat: effect of castration and cryptorchidism. Endocrinology 88, Swerdloff, R. S., Walsh, P. C. & Odell, W.. (1972). Control of FSH and LH secretion in the male: evi dence that aromatization of androgens to estradiol is not required for inhibition of gonadotropin secretion. Steroids 20, Walsh, P. C. & Swerdloff, R. S. (1973). Experimental cryptorchidism: effect on serum LH and FSH in the rat. Urological Research 1, amamoto, M., iebel, N.. & Bogdanove,. M. (1970). nalysis of initial and delayed effects of orchi dectomy and ovariectomy on pituitary and serum LH levels in adult and immature rats. Endocrinology 86, Zanisi, M., Motta, M. & Martini, L. (1973). Inhibitory effect of 5-alpha-reduced metabolites of testosterone on gonadotrophin secretion. Journal ofendocrinology 56,