2 Changes in the gonadal and adrenal steroid patterns during puberty

2

Changes in the Gonadal and Adrenal Steroid Patterns during Puberty DEREK GUPTA

Puberty is the stage in life when the development of secondary sexual characteristics occurs, followed by the attainment of the reproductive capacity. Although the mechanism initiating the maturation of the reproductive tract has become the subject matter of numerous reports, the endocrinological explanations of these multi-phase pubertal events, their sequence and timing are still hard to find. However, during the last few years we have witnessed spectacular advances in the measurement of minute quantities of hormones in biological fluids and tissues, for which ten years ago a litre of plasma would have been required. Such methodological developments are perhaps nowhere more fruitful than in advancing our knowledge of the regulatory mechanism which initiates pubertal processes in the human as well as in experimental animals. For the sake of brevity and to simplify the presentation I assume that the following can be agreed upon. I. The levels of sex steroid hormones in boys and girls rise during puberty. 2. I n normal men, testosterone is the single important plasma androgen. 3. The synthesis and secretion of the sex steroid hormones are under the control of the pituitary gonadotropins 4. The old hypothesis which postulated that gonadotropins were first secreted at the time of puberty and the qualitative changes were responsible for the onset of pu berry, no longer holds true. A fairly substantial body of knowledge concerning the steroid concentrations in body fluids in relation to sexual maturity has now become available through the recent large-scale studies performed in normal children by groups in San Francisco, Baltimore, Los Angeles, Winnipeg, London, Lyon, Munich and Tubingen, Moreover, some comprehensive reviews on the endocrinological findings during puberty have recently been published (Root, 1973; Visser, 1973). The purpose of this chapter is to review the state of our present knowledge with respect to the following questions: I. At what chronological age and sexual maturation stages do the adrenal and gonadal steroid hormones begin to rise toward adult levels? Clinics in Endocrinology and Metaholisrn---Vol. 4. No. I, March 1975.

27

28

DEREK GUPTA

2. What is the subsequent time-course during puberty once the increments in the sex steroid hormones have begun? 3. How do the results of assays of the circulating steroid hormones compare with those of the excreted ones? 4. Can the hormonal data obtained in experimental an imals during puberty be compared with those of children?

PUBERTAL DEVELOPMENTAL CHANGES The attainment of reproductive capacity is a complex proce ss which requires the maturation and interaction not only of the gonads and reproductive tract, but also of the pituitary, and most importantly, of the neuro-endocrine mechanism which ultimately controls gonadotropin secretion. In a normal child this development proceeds through multi-phase stages which are generally divided into five broad groups (Tanner, 1962). Although various modifications of the Tanner schemata for somatic changes have been suggested by a number of investigators, in the present study we have related our endocrinological findings throughout to the genitalia or breast-maturational ratings as described by Tanner (1962, 1969). The recent investigations (Tanner, 1974) on the somatic changes occurnng during adolescence have revealed that the relationship between various areas of development varies considerably. It is perhaps possible to note the existence of constancy of sequence within a given development but inconstancy between different areas of development. It is also possible that all children may not pass through all the individual stages of sexual ratings. Marshall and Tanner (1969) in an investigation of 192 British girls observed that seven per cent did not pass through breast stage 4 prior to entering stage 5. Tanner (1974) has demonstrated that within genitalia maturation stage 3 boys may be in pubic hair stages of I, 2, 3 or 4 in proportions of about 45, 40, 10 and 5 per cent respectively. Another intere sting variation in the sequence of events during male sexual maturation is the rapidity with which the stages are passed. The average boy takes a little more than a year to go from genitalia stage 2 to 3, while some take as long as 2.5 years. Girls also demonstrate considerable variation in relation to breast and pubic hair stages. Girls in stage B2 comprised 55 per cent still in pubic hair stage I, 20 per cent in PH2, 20 per cent in PH3 and five per cent in PH4 or 5. By the time they reach B4, few girls still remain in PHI. In the male, pubic hair stage 6 is achieved when sexual hair extends up the linea alba. In English boys, axillary hair appears during genital stages 3 and 4, while full beard growth is not achieved until many years after the attainment of genital stage 5 (Marshall and Tanner, 1970). In the girls, although the correlation between breast development and pubic hair growth is high, there is a significant individual variation. Marshall and Tanner (1969) observed that adult-type pubic hair was present in 11 per cent of girls examined who had B3 breast development, and in certain cases B4 breast development occurred before the appearance of pubic hair. For this reason the investigators who wish to relate their endocrinological findings should be careful in

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

29

defining the ratings with which they have associated their findings. Because of the absence of constancy in the parallel development of the sexual characteristics it would be misleading just to say that hormonal findings have been associated with pubertal stages.

SECRETION AND PERIPHERAL CONVERSION OF STEROID HORMONES Before we come to the question of the levels of steroid hormones in body fluids, it should be made clear that the demonstration of a particular steroid in the body fluid, or its concentration, does not give any information about its secretion. The rigorous criterion to prove the secretion of a steroid should be a demonstration that the venous effluent from the gland has a higher concentration of that particular steroid than is seen in the peripheral blood. Understandably, therefore, the complex problems associated with the secretion of steroid hormones in normally sexually maturing children are justly handicapped on ethical grounds. Data from animal experiments (Lindner, 1961a and b), however, demonstrate that the testes of the lamb, calf and piglet secrete testosterone at a rate of 1/10 to 1/50 of those of the adult animals. Similarly Resko, Feder and Goy (1968) and the present author and colleagues (Gupta et al, in press) found that the testes of the 20-day-old rat secrete testosterone. However, significant amounts of plasma testosterone in normal subjects can be derived from many different steroid precursors, e.g. androstenedione (Horton and Tait, 1966), 17-0H-progesterone (Camacho and Migeon, 1964), dehydroepiandrosterone (Horton and Tait, 1967), and its sulphate (Slaunwhite, Burgett and Sandburg, 1967). The problem regarding the source of the oestrogens in men is not very clear either. MacDonald, Rombart and Siiteri (1967) calculated, on the basis of extensive infusion studies with labelled steroid hormones, that 1.8 per cent of blood androstenedione was converted to blood oestrone, thereby accounting for about 18 J.lg or a major portion of the daily oestrone production rate in man. These investigators also showed that 0.5 per cent of plasma testosterone was converted to oestradiol in the male, whereas in the female these values were 0.8 and 0.2 per cent respectively. Single determinations of plasma testosterone and androstenedione levels reflect only the momentary balance between the entry and removal of that substance from the blood pool. If the plasma pool contains only one hormone secreted by the gonads or the adrenals, then a true 'secretion rate' can be measured with a tracer dilution study. However, if a significant amount of this steroid is derived from the peripheral conversion of a precursor, then only the 'production rate' is measured. The production rate of testosterone is usually calculated from the total specific activity of the urinary testosterone glucuronide. Testosterone production rates calculated in this manner have been found to be 6.5 mg/day for the adult male, and 1.7 mg/day for the adult female (Van de Wiele et al, 1963; New, Pitt and Peterson, 1963; Camacho and Migeon, 1966). New, Pitt and Peterson (1963) found a production rate in children of less than 0.5 mg/day.

30

DEREK GUPTA

The discussion on the androgen sources and production rates is seriously limited, however, by the scarcity of information on younger subjects. Whether the gonads of pre-pubertal children are active enough in secreting gonadal steroids and metabolising them efficiently is still a subject of speculation.

STEROID HORMONES DURING PUBERTAL DEVELOPMENT In the early stages of pubertal development there are distinct differences from the normal adult profiles of steroid hormones, whether of adrenal or of gonadal origin. Adrenal Origin As puberty approaches there is a gradual increase in the level of the adrenal steroids in the urine and plasma in both sexes. Corticosteroids There are few published figures for individual corticosteroid metabolites in the urine or in plasma during adolescent growth. Guignard de Meyer, Crigler and Gold (1963) demonstrated that normal children have a higher proportion of ll-oxo metabolites in urine than do adults. It was demonstrated by Gupta (1965) that the proportion of cortisol excreted as tetrahydrocortisone (THE) and cortolones was greater before puberty and the excretion of THE was lower. The proportion of allotetrahydrocortisol was approximately the same in the pre-adolescent children as in the pre-school group (Gupta, 1970). The ratio of THE/THF was near unity in the adolescent and pre-adolescent. This was significantly higher (P < 0.01) than the ratio found in the pre-school children (Gupta and Marshall, 1971). There was no difference in the ratio between sexes for this ratio at pre-adolescent and adolescent stages. Little has been published about the excretion of the two other major metabolites of cortisol, i.e., the cortols and cortolones. Fukushima et al (1960) and Romanoff et al (1961) have given values for adults, but there is no c ocumentation for the excretion of these steroids by children at different stages of maturation. The percentages of the cortols and cortolones within the total cortisol metabolites was 5.2 and 17 per cent, respectively for the adolescents. No sex difference was observed in the excretion patterns of these steroids. Eagle (1956) showed that there was a much greater secretion of corticosterone than of cortisol in neonates and premature infants. Buus et al (1962) drew attention to the observation that in infants, during the first days of life, the plasma contains considerable quantities of corticosterone and dehydroepiandrosterone. Dohan, Bulaschenko and Richardson (1962), Peterson and Pierce (1960), Cost and Vegter (1962) and Van der Straeten, Vermeulen and Orie (1963) reported a mean cortisol/corticosterone ratio in normal adults of 6 to 7. Gupta (1970) and Gupta and Marshall (1971) showed that although the ratio of the excreted cortisol metabolites to the corticosterone metabolites in the pre-school children was significantly higher (P < 0.01) than in the pre-adolescent and adolescent groups, the magnitude

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

31

of the difference was, however, not as great as that reported by previous investigators. Also, in contrast to the observation of Cost and Vegter (1962), Gupta (1970) demonstrated that THA (tetrahydro-Il-dehydrocorticosterone) was the most preponderant steroid among the corticosterone metabolites in all age groups of children studied. The l l-deoxycortisol metabolites, although these increased gradually with age in children, did not show any discernible increments when body weight or height were allowed for. Kenny et al (1966) examined the cortisol production rate in children and concluded that, except for the first five days of life, these metabolites are similar for all ages when the values are corrected for body surface area. Although there is no quantitative difference in the production rate or urinary excretion values between an infant and a mature child, there are certain qualitative differences in corticosteroid metabolism related to the growth and maturation of the child. Green (1965) has summarised some of these explicit differences: an increase in the half-life of injected radioactive cortisol; diminished conversion of 3~-hydroxy-115-steroids to M-3-ketones; diminished A-ring reduction and glucuronoside formation; an increase in sulphate conjugation; and a greater conversion of cortisol to the more polar 6-oxygenated metabolites. Androgens The principal sources of androgens in the adult are the gonads and the adrenal cortex. In the adult male, testosterone is mainly of testicular origin, androstenedione is secreted by both testes and adrenal, while dehydroepiandrosterone and its sulphate are products of the adrenal cortex (Chapdelaine et al, 1965; Prunty, 1966). The female adrenal secretes the same steroids as the male adrenal, but the ovary secretes mostly androstenedione, and a small amount of testosterone (Horton, Romanoff and Walker, 1966). Over half the amount of testosterone seen in the female plasma is the product of a peripheral conversion and not of a direct secretion.

Urinary excretions Investigations in children have already established that during puberty, there is an increased urinary excretion of 17-oxosteroids in both sexes. Until the age of around 12 years, there is no sex difference, but afterwards excretion in the male becomes higher than in the female (Gupta, 1965; Knorr, 1965; Teller, 1967; Blunck, 1968; Tanner and Gupta, 1968; Gupta, 1970). This increase in the urinary 17-oxosteroid excretion in both sexes is mainly due to increased production of the adrenal androgens. Dehydroepiandrosterone can be taken as a typical adrenal androgen since isotopic studies have already indicated that the testis secretes only a negligible amount of this hormone and little or no DHA.sulphate (Chapdelaine et al, 1965). During the early stages of puberty most of the urinary androsterone is derived from DHA. Gupta (1970) demonstrated the increments in the excretion of androsterone in boys and girls at successive chronological ages (Figures l a and b). Although most of the individuals give a rather regularly increasing curve, there is great variation among individuals. At age II years, the age at which the study had the greatest number of children, the values ranged from about 0.2 to 2.6

32

DEREK GUPTA

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mg/24 hours. At age 14, the range for the boys was 1.4 to about 5.2 mg/24 hours , whereas for the girls it was about 1.0 to 1.5 mg/24 hours . The boys start showing a higher excretion of androsterone from around the age of 12 years.

33

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Some of the variation in excretion is due to differences in the developmental age of the children. Some children are skeletally more mature than others at the sa me chronological age. When the same da ta from Figures la and lb were plotted against skeletal ages, the va riation among the ind ividuals was considera bly reduced (Figures 2a and b). The high excretors, being also early developer s, had their cur ves shifted to wa rd the higher skeletal ages. Also among the girls high excretors had shifted to the right as being early maturers.

34

DEREK GUPTA

The individual curves for DHA excretion, plotted against chronological age, demonstrate similar patterns to those found earlier. This steroid is definitely present in all the subjects studied, but at a very low level (often less than 0.1 mg/24 hours) in most of them, until close to II years. Some children are again consistently high excretors while others are low excretors, When plotted against skeletal age, the curves show the same patterns of variability as had been found earlier for androsterone. One of the major findings in the urinary steroid studies published was the lack of any significant sex difference before the age of 16 years. Yet in the l l-deoxy-U-oxosteroid excretion of the adult there is a considerable difference, as men produced substantially more. Evidently, the boys' excretion continues to rise during the later part of their adolescent growth spurt, at a time when the girls' growth spurt and their increase in Il-deoxy-17-oxosteroid excretion is over. But this is mainly in terms of chronological age, and is perhaps physiologically and auxologically misleading. The boys' adolescent spurt occurs two years later than that of the girls. Thus a more informative comparison could be achieved by plotting steroid excretion in relation to physiological or developmental age, as represented by skeletal maturity. In Figure 3 the mean trend in the increment of androsterone excretion with 4 ~

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skeletal maturity score is given. This is a measure common to both boys and girls to the degree that skeletal maturity has been achieved. The score of 800, for example, can be regarded as representing a skeleton that is 80 per cent mature. Plotted this way, boys clearly demonstrate a higher rate of excretion of androsterone over girls for the same skeletal maturation. Plots of cortisol metabolites (not shown here) did not show a sex difference in the patterns of excretion.

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

35

Circulating levels A rise in the plasma concentration of OHA and androsterone in both sexes during puberty has been reported (Migeon, 1960; Rosenfield and Eberlein, 1969). As observed by Rosenfield and Eberlein (1969) OHA was detected in the pooled plasma from children aged between one and five years to be 2.7 ~g/IOO ml. This level rose to an average of 13.5 Jlg/100 ml in the children between the ages of six and nine years. Androsterone sulphate was undetectable in the lower age group, whereas the level was 8.6 Jlg/100 ml in the higher age group of the children examined. In 21 boys and nine girls of PH stages 1 to 5 (chronological age 8.5 to 17.5 years), Rosenfield and Eberlein (1969) detected OHA-sulphate in all subjects. Androsterone sulphate was detected in 87 per cent of the population. Gandy and Peterson (1968) in a study on 18 pre-adolescent boys (aged four to nine years) found a OHA plasma level of 59 1 53 ng/100 ml in the boys and 74 ± 26 ng/IOO ml in the girls. Saez and Bertrand (I 968) observed a OHA plasma concentration of 101 ng/IOO ml in 10 boys aged between two and 7.6 years. Forest and Migeon (1970), however, found the level to be 142 ng/100 ml in a group of 10 girls aged from 1.8 to 12 years. In the one to eight-year-old range the DHa-sulphate values found by another group of investigators (Boon et ai, 1972) agreed with those reported by Saez and Bertrand (1968). About 50 per cent of the boys and girls below 8 years of age demonstrated the level of OHA and DHA-sulphate to be in the non-detectable range. Androsterone sulphate was virtually below the detection limit in the majority of cases in this age group. As puberty approached, adrenal androgen concentrations in all ages increased considerably in the male plasma approximately two years before physical manifestations became evident. In the case of the females, the principal increase occurred in the conjugated adrenal androgens, with only a small increase in DHA. The nine to 18-year-old males exhibited quite a wide range of DHA concentration in the plasma. The adrenal component of adolescence is almost certainly under the control of the pituitary gland, since subjects suffering from hypopituitarism do not show it. It is possible that the increased production of adrenal androgens at the onset of puberty is the result of stimulation by a specific pituitary hormone which is synergistic to ACTH (Mills, 1968). There is no adolescent spurt in the secretion of cortisol when calculated on the basis of body surface area. This in fact argues against an increase of ACTH stimulation during pubertal development as being the major cause for the sudden increment in the adrenal androgen production. It is possible that a certain factor or factors may modify the response of the adrenal glands to ACTH action during the onset of puberty.

Gonadal Origin During recent years several studies have contained reported values for androgens and oestrogens in urine (Vestergaard, Raabe and Vedso, 1966; Loras et aI, 1966; Gupta, 1967; Knorr, 1967; Gupta and Butler, 1969; Steeno et al, 1967; MacRoberts, Olson and Herrmann, 1968; Pennington

36

DEREK GUPTA

and Dewhurst, 1969) and in plasma (August, Tkachuk and Grumbach, 1972; Frasier and Horton, 1966; Saez and Bertrand, 1968; Frasier, Gafford and Horton, 1969; Gandy and Peterson, 1968; Winter and Grant, 1971 ; Rivarola, Bergada and Cullen, 1970; Rosenfield and Eberlein, 1969; Gupta, MacCaffertyand Rager, 1972; Wieland, Yen and Pohlman, 1970; Boon et ai, 1972; Saez, Morera and Bertrand, 1972; Winter and Faiman, 1971 ; Attanasio et aI, 1973; Bidlingmaier et al, 1973; Faiman and Winter, 1974; Knorr et ai, 1974; Gupta, Attanasio and Raaf, in press) in children during their pubertal development. Urinary excretion In the current literature there are only a few reports on the excretion levels of testosterone by pre-school, pre-adolescent and adolescent children. Rosner et al (1965) in a study of five boys aged from seven to 12 years found a mean excretion of 6 3 Jlg/24 hours. Vermeulen (1966) on the other hand, found a much higher level in eight children (mean 12.2 Jlg/24 hours). Knorr's (1967) results in nine ten-year-old children were also higher (mean 11 Ilg/ 24 hours). In our own studies (Gupta, 1967; Gupta and Butler, 1969) we found that, from the ages of three and 11 years, the boys and the girls tend to have similar excretion patterns. After the age of 11.5 years the boys had a much steeper rise. There was also greater variation between individual boys, when examined on the basis of chronological age. This difference seemed to disappear when the data were examined in relation to skeletal age. The girls, on the other hand, were more or less low excretors and those with higher skeletal age did not differ substantially from the subjects with low skeletal ages. The ratio of testosterone excretion values for adult males to pre-school boys is 50; for adult males to pre-adolescent boys it is 9.4, and for adult males to adolescent boys 1.8. This ratio for adult females to pre-school, pre-adolescent and adolescent girls is 10, 2.9 and I.l respect ively. ln the excretion of epite stosterone there was no obvious difference between boys and girls, nor was there any obvious relationship to skeletal age. Wilson and Lipsett (1966) have demonstrated that this steroid does not come from testosterone and, although its production rate is only three per cent of that of testosterone, its excretion is about 33 per cent that of testosterone in the adult male. Martin (1966), however, showed that the interconversion of testosterone and epitestosterone was not a common metabolic pathway for these steroids in the human, although it might come into play if high amounts of testosterone and epitestosterone had been administered. Although no sex difference could be observed in the excretion of testosterone and epitestosterone in younger children (when the excretions were related to skeletal maturity score) it became apparent that boys had a higher mean rate of excretion than the girls (Figure 4). This figure also illustrates that the difference between the two sexes in the excretion of epitestosterone is not as pronounced as that found for testosterone. Figure 5 demonstrates a comparable pattern of excretion for three representative steroids : testosterone, a steroid primarily of gonadal origin;

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androsterone, of mixed origin; and cortisol, mainly of adrenal origin. The values are adjusted to the children's body weight and arranged according to their sexual maturation ratings. The excretions of testosterone and androsterone, although rising sharply in the boys from pubertal developmental stage 3 to 5, virtually did not change in girls throughout their maturational stages from I to 5. For the cortisol metabolites, both sexes had similar patterns of excretion. The urinary excretion of oestrone, oestradiol and oestriol increases with age and sexual maturation in the female (Pennington and Dewhurst, 1969). Winter and Faiman (1972) recorded low levels (2.2 Ilg/24 hours) at pubertal stage I in male children, that more than doubled (5.2 Ilg/24 hours) at stage 2, and increased to a new level of 16.2 Ilg/24 hours at stage 3, reflecting either a direct secretion by the adrenal or the gonads, or the peripheral conversion of the androgens to oestrogens.

Circulating levels Plasma concentrations of testosterone, dihydrotestosterone, androstenedione, androstenediol and androstanediol increase progressively from pre-pubertal to adult levels during sexual maturation in the male children. Faiman and Winter (1974) observed no significant change in plasma testosterone concentration occurring during childhood, with all values being below 40 ng/IOO rnl, At the onset of puberty (stage 2) there was a more rapid rate of testicular enlargement which usually preceded any signs of an androgen effect. Between 10 and 17 years of age, plasma testosterone concentration increased twentyfold, accompanied by increased phallic growth and an appearance of pubic hair and axillary hair. Faiman and Winter (1974) did not detect plasma oestradiol in a large majority of the pre-pubertal girls. Levels from 1.0 to 1.7 ng/IOO ml were seen in a few of the older girls before the onset of breast development. The transition towards pubertal stages 2 and 3 was accompanied by a rise in the mean plasma concentration of oestradiol and testosterone in the girls. During pubertal stages 4 and 5 and the attainment of menarche, oestradiol concentration reached the adult range. Bidlingmaier et al (1973) found values for oestrone and oestradiol below 15 Ilg/ml in normal boys up to eight years of age. During the next five years, the values gradually increased to those of the adult males. In most cases, oestrone exceeded oestradiol. Up to the age of seven years these investigators did not observe any difference between the sexes. However, during the next four to five years, there was a sharp increase exceeding that for the boys and reaching the values of mature women. Saez, Morera and Bertrand (1972) also made similar observations regarding the plasma concentrations of the oestrogens in developing children. Boon et al (1972) observed a marked rise in all plasma androgens, especially that of testosterone in normal boys aged 9 to 18 years. Only a moderate increase in plasma androgen concentration was observed in the girls of the corresponding age grouping. Jenner et al (1972) did not detect any oestradiol in four pre-pubertal boys of stage I rating. Sixty per cent of stage 2 girls had detectable levels of plasma oestradiol that steadily rose during pubertal

39

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

development. The difference in the levels between stage I and stages 2 and 3, and between stages 2 and 3 and stages 4 and 5 were significant. These investigators also observed that plasma concentrations of oestradiol had a significant correlation to chronological and skeletal ages, but the best correlation was with the pubertal developmental stages between 2 and 5. In our own series of investigations (Attanasio et ai, 1973; Gupta, Attanasio and Raaf, in press) variations between individuals in steroid hormone concentrations at certain chronological ages were found to be considerably reduced when the same data were related to the pubertal developmental stages. In the majority of cases, the concentration of oestradiol and oestrone had a close association with the girls' sexual maturation. Figure 6a shows the individual values for the plasma concentrations of oestrone for successive chronological ages of boys and girls. There is great variation among individuals, girls tending to show higher plasma concentrations even from the age of 10 years. Boys start showing higher concentrations after the age of 14 years. When the same data were plotted against sexual maturation ratings (Figure 6b) the variations between individuals were greatly reduced. At pubertal stages I and 2 the results were homogeneous, with boys and girls

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barely showing any difference. At stage 5 the girls showed more scatter than the boys. This increase in the s.d. of the oestrogen values with sexual maturation in girls was not previously stressed b y other investigators. During pubertal stages 3 and 4 the s.d. for oestrone and oestradiol was 0.26 and 0.80 respectively, increasing to 0.82 and 1.49 in stage 5. Th is increase perhaps reflects the onset of cyclic acti vity. Figure 7a and b demonstrates the plasma concentration of oestradiol in children, according to chronological age, and related to sexual maturation ratings. Boys remain uniformly low throughout the increment of chronological

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Figure 7. Plasma oestradiol concentrations in children (a) when plotted against chronological age; and (b) related to pubertal developmental stages.

age, but girls show wide scatt er in the ir plasma concentrations at different ages. When th ese data are related to sexual maturation ratings (F igure 6b), the boys did not show any appreciable change, but the early developing girls had the points shifted towards the ir higher maturational stages.

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

41

Figure 8a and b demonstrates the plasma concentration of testosterone in relation to chronological age and pubertal stages. Whether as a function of age or of pubertal development, the girls' plasma testosterone concentrations tended to be low and steady. From these figures it appears that boys and girls have the same amount of testosterone during stages I and 2. The sex difference as noted in plasma concentrations begins to be apparent at stage 3 with boys showing a steep increase during later stages.

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Figure 8. Plasma testosterone concentrations in children (a) when plotted against chronological age; and (b) related to pubertal developmental stages.

Individual values for dihydrotestosterone, plotted against chronological ages and pubertal stages, are illustrated in Figure 9a and b. Dihydrotestosterone concentration in girls surprisingly shows a higher increment than that seen for testosterone. When related to pubertal stages (Figure 9b) boys show a clear upward trend in concentration and the sex difference then becomes discernible at stage 4 rather than at stage 3, as was seen with testosterone.

42

DEREK GUPTA

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Figure 9. Plasma dihydrotestosterone concentrations in children (a) when plotted against chronological age; and (b) related to pubertal developmental stages.

The majority of the fully sexually mature boys (genitalia stage 5) showed plasma levelsof testosterone and dihydrotestosterone below the normal adult male range. Similar observations were made earlier (Gupta, MacCafferty and Rager, 1972; Gupta, 1970; Gupta and Butler, 1969) with the urinary and plasma androgens, estimated by different techniques. The current results indicate that the levels of androgen in boys rise even after full sexual maturation. When the relationship of the oestrogens to testosterone is examined, no sex difference emerges during pubertal stages I and 2 (Figure 10). However, at stage 3 the figure clearly indicates a decrease in the ratios for the boys, whereas there is a steep increment for both ratios in the girls. The ratios oestrone/testosterone and oestradiolJtestosterone increase from about 0.05 to 0.15 and 0.25 respectively during stage I to the stage of full maturation. For the same period in the boys steep decrements are observed in both the ratios. While the bulk of oestradiol in the girls is secreted by the ovary, a significant portion of oestrone is perhaps derived from the circulating androgens.

43

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

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Figure 10. Mean trends in oestrogen/testosterone ratios (EdT and Ez/T) during pubertal development in children.

The ratio of oestrone/oestradiol (Figure 11) does not show any marked deviation nor any sex difference, in children between the pubertal stages 1 and 2. Later, however, a sex difference becomes discernible and the ratio progressively decreases from 1.2, during pubertal stage 2, to 0.6 in stage 5. Among boys, on the other hand, the ratio rises from 1.2 during stage 2, to 1.6 with a full sexual maturation. Figure 12 demonstrates the relationship between testosterone and the two oestrogens in the peripheral plasma of boys. The correlation coefficient is 0.784 with an equation for the line of regression being y = 0.008x- + 2.511, indicating that perhaps a significant portion of the oestrogens is derived from the circulating androgens and not from the gonadal sources. For the girls, no such relation was observed, inferring that here oestrogens have a more independent origin.

44

DEREK GUPTA

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Figure II. Mean trends in oestrone/oestradiol ratios during pubertal development in children. In recent years another steroid metabolite , Sn-androstane-Ja-I'jfj-diol (androstanediol) has gained some importance with relation to the question of androgenic function. The Sa-reduction of testosterone to dihydrotestosterone has been recognised as an important event in androgen action . Dihydrotestosterone after being reduced from testosterone is further metabolised in the target cells by a conversion to androstanediol, the rate of which has been noted to be higher in men than in women (Mahoudeau, Bardin and Lipsett, 1971). Mauvais-Jarvis, Charransol and Bobas-Masson (1973) have measured this substance in urine, and recently Kinouchi and Horton (1974) have described a method for the estimation of androstanediol in the peripheral plasma. Our research (Klemm and Gupta, 1974) has produced a radioimmunoassay method for the estimation of the circulating level of this substance in pre-pubertal and pubertal boys during their sexual maturation. From a level of 0.56 ng/IOO mI in stage I, the peripheral concentration of androstanediol leaps to a I3-fold higher concentration in stage 5. When the relationship between androstanediol and testosterone is examined in the form of a ratio, a sharp rise is recorded from stage I to stage 2 (Figure 13). This rise, however, declines sharply during the transition from stage 2 to

45

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

4 and reaches a plateau thereafter. The change in the relationship between dihydrotestosterone and testosterone parallels the androstanediol/testosterone curve, indicating that at the beginning of puberty there is an increment of 5a-reductase activity. y

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46

DEREK GUPTA

Sex steroid binding protein It has been currently established that a large fraction of the non-conjugated steroids is bound to plasma proteins and only a very small fraction remains unbound. This unbound fraction is considered to be the 'biologically active' form of the hormone. The percentage of hormone bound to proteins is influenced by several factors. These are: (1) concentration of the steroid binding protein; (2) num ber of binding sites on that protein; (3) concentration of the steroid hormone; and (4) existing association constant between the protein and that particular steroid. Pearlman, Crepy and Murphy (1967), Mercier-Bodard, Alfsen and Baulieu (1970) and Rosner and Deakins (1968) demonstrated that in fact the concentration of the binding protein is the main factor responsible for the increased binding of testosterone observed in pregnant females, and in oestrogen-treated males when compared to non-pregnant females and normal adult males respectively. Although Forest and Migeon (1970) did not observe any influence of age or sex on the percentage binding of testosterone or androstenedione in a group of children (four months to 12 years), August, Tkachuk and Grumbach (1969) correlated testosterone and testosterone binding affinity levels in the plasma of boys from the ages of one to 12 years, and found that the testosterone binding affinity decreased with an increase in age. In our own investigations (Gupta, Huenges and Rager, 1970, 1971; Gupta and Bundschu, 1972) we observed that as early as stage 3 of pubertal development the difference regarding the percentage binding of testosterone between sexes was significant. When the values of adult females were compared to those of adult males and pre-adolescent children, it was found that adult males had minimum binding while the adult female had the maximum. The pre-adolescent children had values between the adult male and the adult female. It seems that in the boys the time interval between the development of secondary sexual characteristics and the initial testosterone increase, together with the decrease in testosterone binding affinity, may be related to the amount of unbound testosterone available. Mean trends in hormonal increments The following two figures (Figures 14 and 15) demonstrate a composite picture of the chronological age and the stages of sexual maturation in boys and girls respectively, where the plasma concentrations of testosterone, dihydrotestosterone, oestradiol, oestrone and percentage binding of testosterone first exceed pre-pubertal levels. Along with these, the data for penis size, testis length and prostate weight are given in Figure 14. Figure 15 illustrates ovarian weight and uterine weight with additional hormonal values. It can be seen that the mean chronological age at which testosterone, dihydrotestosterone and androstanediol begin to rise in boys is 13 years (stage 3), with the prostate weight, testis length and penis size also demonstrating simultaneous increments. The levels of plasma oestrone and oestradiol in boys do not show such comparable increments. Percentage binding of testosterone decreases at the same time towards the adult level. The corresponding age when oestradiol shows signs of a marked increment in girls is

47

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY CHRONOLOGICAL AGE 1,.lllofS I

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before 12 years (stage 2), whereas oestrone starts to rise at a mean age of 12.5 years (stage 3). Marked increments in the growth of the ovary and the uterus also synchronise at that stage of life. Thus the rise toward the adult levels of the androgens and oestrogens begins one to two years earlier in the girls. Appreciable sexual maturation in both sexes can be associated with the period when the plasma concentrations of the androgens in boys and the oestrogens in girls have begun to rise rapidly towards adult level. The subsequent time course of the plasma concentrations of the gonadal hormones parallels the growth of the reproductive organs in both sexes. It can be seen, from the above figures, that the androgen concentrations in boys and oestrogens concentration in girls tend to rise progressively over a period of approximately four years, but stabilisation in increment does not come with the attainment of adult-type secondary sexual characteristics in the boys (G5). Post-pubertal concentrations of the gonadal hormones in the boys tend to increase further in the young adults and reach a plateau thereafter. The changes in all the gonadal steroid hormone levels accompanying the transition from late puberty (B5jG5) to adulthood are highly variable, and partly reflect the limitations of cross-sectional investigations. These limitations

48

DEREK GUPTA 1

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are magnified in the case of the females where it is almost impossible to obtain samples at a definite period of the cycle. It is, therefore, most important to appreciate the fact that the real magnitude of a hormonal increment in a given individual during his sexual maturation can only be known through a longitudinal study. Longitudinal investigation

Figure 16 depicts two longitudinal investigations at present carried out independently in two different parts of the world. The first is on the excretion of urinary testosterone and epitestosterone (Gupta and Butler, 1969) and the second is on the serum concentration of circulating testosterone (Faiman and Winter, 1974)in sexually maturing boys. In spite of the enormous procedural differences, when the two sets of data are compared, a striking parallel between the mean trends in the hormone excretion and hormone concentration emerges. Utilising the same method of calculation for mean trends in longitudinal investigation (Tanner and Gupta, 1968), these results illustrate that during pubertal stage 1, testosterone levels in blood and urine are low and the increments are gradual. The most marked increment in plasma concentration and urinary excretion occurs between ages 12 and 14 years.

49

GONADAL AND ADRENAL STEROJl) CHANG ES IN PUBERTY

When the data are exam ined in relation to pubertal stages, the increments a re significant between stages 2 and 3, 3 and 4, 4 and 5. The total increment, whether for the urinary excretion or for the pla sma concentration, of the stage 5 bo ys is about ten-fold higher than the stage 2 boys.

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Figure 16. Mean trend in the increment of urina ry excretion and plasma concent ration of testosterone in boys stud ied longitudinally as a function of chronological age and sexual maturation stages. Redrawn from the data of Gupta and Butler (1969) and Faiman and Winter (1974).

The excretion of epitesto sterone, on the other hand , shows gradual Illcrement which is favourably associated with the increments in body size rather than with the pubertal developmental stage s. In bo th st udies the population was not large, and the biological importance is limited due to the mixed nature of the series. The potenti aliti es of a purely longitudinal design a re much more than that ofa mixed series but its execution requires a strenuo us effort. In fact , such a project on pure longitudinal in vestigation is und er way (a joint study between London a nd Tiibingen), where a simu lta neo us estimation of urinary gonadal steroids and gonadotro pins, together with morphological changes during adolescent growth, are being carried ou t.

50

DEREK GUPTA

GONADAL STEROIDS IN EXPERIMENTAL ANIMALS DURING PUBERTAL GROWTH: A DYNAMIC RELA nON WITH GONADOTROPINS The production of the gonadal steroid hormones during sexual maturation is entirely under the control of the pituitary gland. Before puberty, when no mature Leydig cells are identifiable in the testis, levels of plasma LH are low. Rising levels of testosterone and gonadotropins correlate well with the pubertal developmental stages and Leydig cell maturation. Fairnan and Winter (1970) suggested the possible existence of a critical threshold level in plasma gonadotropins, the attainment of which initiates the pubertal spurt in testicular growth and surge in testosterone production. Since the changes in gonadotropin levels in children during adolescent growth have been dealt with in detail in a subsequent chapter, a very brief description is given in this section on the relationship of gonadal steroids with gonadotropins in experimental male rats during pubertal growth. A number of investigators have already measured testosterone (Resko, Feder and Goy, 1968; Knorr, Vanha-Perttula and Lipsett, 1970; Grota, 1971; Miyachi, Nieschlag and Lipsett, 1973; Gupta et aI, in press) or LH and FSH (Swerdloff, Walsh and Odell, 1971; Goldman et ai, 1971; Amatayakul et aI, 1971; Yamamoto, Diebel and Bogdanove, 1970; Negro-Vilar, Krulich and McCann, 1973), but none had simultaneously studied the circulating levels of the gonadal steroids and gonadotropins in laboratory animals. In a pilot study, we have looked into the levels of circulating testosterone, dihydrotestosterone, LH and FSH in sexually maturing male rats at daily intervals (Gupta et aI, in press). These investigations reveal that the surge in testosterone level is dramatic in the male rat after the 25th day of life, and in three days, a level is reached that is five-fold higher than the level seen earlier. After this critical period, there are some fluctuations in testosterone concentrations, but the peak value seen in the 70-day-old animal is only II I per cent of the plasma testosterone concentration already reached at the 26th day of life. For dihydrotestosterone the surge is noted to occur on the 26th day of life, but the peak is reached later (day 33), than that seen in the profile of testosterone. On the 38th day dihydrotestosterone reaches a new concentration level and remains constant throughout adulthood. Our observations on the plasma profiles of LH and FSH for intact male rats corresponded well with those recently reported by Negro-Vilar, Krulich and McCann (I973). Plasma LH has a clear peak between the ages of 25 to 30 days after which it declines only to rise again at age 70 days. The rate of increase in plasma LH concentration also indicates that this is greatest during the 25- to 30-day-old period when simultaneously the rate of increment for the other two hormones, FSH and testosterone, is also the greatest. Plasma FSH in the male rat has a clearly discernible peak, thereafter declining. Between 25 and 35 days of age, FSH values almost double, reaching a peak level at age 33 days. Thereafter, the levels fall at 40 days and decline further to a minimum value at age 70 days. Examination of the velocity of increment in the plasma FSH concentration reveals that it is highest between

GONADAL AND ADRENAL STEROID CHANGES IN PUBERTY

51

16 and 35 days and the sharp decline begins at age 35 days, i.e., just before mature sperm appears in the tubules, and then remains steadily constant throughout the later phase of sexual development. These cumulative experimental data illustrate that between 16 and 20 days of age the rate of increment in the plasma concentration of FSH is greater than at any other time and is followed by a spurt in the LH concentration after the 20th day. The abrupt increase in plasma testosterone concentration seen between 25 and 30 days is perhaps mediated by the sudden rise in the rate of increments in the two gonadotropins. The higher gain in dihydrotestosterone is, however, little delayed. These events of higher hormonal activities in the male rat are perhaps related to the initiation of the Leydig cell differentiation seen at about the same time (Resko, Feder and Goy, 1968; Knorr, Vanha-Perttula and Lipsett, 1970). It is, however, surprising to note that at age 40 days when the growth rates of sex accessories such as seminal vesicle and ventral prostate weights are the greatest (Bloch et ai, 1974), the circulating levels of these hormones demonstrate a sharp decline in the rate of gain in concentration. Following bilateral gonadectomy, plasma levels of LH and FSH increased within four days in both juvenile and adult male rats, indicating the existence of an intact feedback relation between the gonadal sex steroids and the pituitary gonadotropins, long before the onset of puberty. In contrast to the results seen in the orchidectomised animals, the experimentally cryptorchid groups present such evidence which demonstrates that the gonadal steroidgonadotropin feedback cannot be the only factor in initiating puberty. The plasma FSH concentrations are not different from those of the age-matched controls at earlier stages of development. At 60 days of age, FSH concentration in the cryptorchid animals continue to rise, while falling in the intact rats. In contrast, LH levels in the cryptorchid animals are higher even in the earliest stage of sex.ual maturation, while this difference gradually increases in the later stages. The fact that the increments in the LH and FSH concentrations in the ex.perimentally cryptorchid animals are not as great as those seen in the gonadectomised animals suggests the involvement of other factors in the regulation of the feedback process. In contrast to the changes in the testosterone levels seen in the intact, pre- and post-pubertal animals, we observed that FSH secretion correlates more uniformly with the dihydrotestosterone levels. It has also been recently noted (Swerdloff, Walsh and Odell, 1972; Zanisi, Motta and Martini, 1973; Rager et ai, 1974), that dihydrotestosterone and androstanediol effectively inhibit the plasma FSH release in the intact and in the orchidectomised rat. Besides the androgenic inhibition of FSH secretion, it has been additionally postulated that the germinal epithelium exerts a negative feedback on FSH production (Swerdloff, Walsh and Odell, 1971). When these postulations are taken together with the early observations on the apparent difference in the regulatory mechanism of FSH secretion, one can infer (on the basis of the current data), that the steady levels of plasma FSH in the cryptorchid immature rats are a result of the absence of the FSH-inhibiting factor(s) in the immature gonad and no reflected change in dihydrotestosterone concentration. The higher magnitude of increment

52

DEREK GUPTA

in the FSH levels seen in the later stages of maturation in the cryptorchid animals over the control animals is perhaps due to the destruction of the germinal epithelium in the cryptorchid group. The feedback inhibition provided by the still existing low levels of the circulating dihydrotestosterone prevents plasma FSH reaching the high levels of the castrated animals. These observations indicate that in the mechanism of the onset of puberty, the role of the gonadal steroids is of prime importance, because of their inhibitory action on the release of the pituitary gonadotropins, and the rapid metabolic degradation in the pituitary-hypothalamic apparatus. In trying to understand the mechanism of the onset of puberty, however, we should always look beyond the apparent endocrinological events to the species difference between the human and the rodent, especially in terms of pubertal development. The rat enters puberty shortly after weaning, while man takes a considerable length of time between weaning and the attainment of puberty.

ACKNOWLEDGEMENTS

It IS a pleasure to thank the Deutsche Forschungsgerneinschaft for generous grants, Professor Klyne of the Steroid Reference Collection, London, for authentic steroids and Drs Rager, Attanasio and Klemm for many fruitful critical discussions.

REFERENCES Amatayakul, K., Ryan, R. T., Uozumi, T. & Albert, A. (1971)A reinvestigation of testicular anterior pituitary relationship in the rat: I. Effect of castration and cryptorchidism. Endocrinology, 88, 872-880. Attanasio, A., McCafferty, E., Reaf, S., Rager, K. & Gupta, D. (1973) The interrelationship of the plasma oestrogens with the androgens in sexually maturing children. Acta Endocrinologica, Supplement 177, 307. August, G. P., Tkachuk, M. & Grumbach, M. M. (1969) Plasma testosterone-binding affinity and testosterone in umbilical cord plasma, late pregnancy, prepubertal children and adults. Journal of Clinical Endocrinology, 29, 891-899. August, G. P., Grumbach, M. M. & Kaplan, S. (1972) Hormonal changes in puberty: III. Correlation of plasma testosterone, LH, FSH, testicular size, and bone age with male pubertal development. Journal of Clinical Endocrinology, 34, 319-329. Bidlingmaier, F., Wagner-Barnack, M., Butenandt, O. & Knorr, D. (1973) Plasma oestrogens in childhood and puberty under physiologic and pathologic conditions. Pediatric Research, 7, 901-907. 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, 947-951. Blunck, W. (1968) Die n-Ketotischen Cortisol- und Corticosteron-metaboliten sowie die l l-oxy- und II-Deoxy-17-Ketosteroide im Urin von Kindem. Acta Endocrinologica, Supplement 134, 1-1l2. Boon, D. A., Keenan, R. E., Slaunwhite, W. R. Jr & Aceto, T. Jr (1972) Conjugated and unconjugated plasma androgens in normal children. Pediatric Research, 6, 111-118. Buus, 0., Bro-Rasmussen, F., Trolle, D. & Schacht, S. (1962) Adrenal steroids in mother and infant at birth. Excerpta Medica, 52, 278-283. Camacho, A. M. & Migeon, C. J. (1964) Studies on the origin of testosterone in the urine of normal adult subjects and patients with various endocrine disorders. Journal of Clinical Investigation, 43, 1083-1089. Camacho, A. M. & Migeon, C. J. (1966) Testosterone excretion and production rate in normal adults and in patients with congenital adrenal hyperplasia. Journal of Clinical Endocrinology, 6, 893-896.

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