Leydig cell function in Klinefelter's syndrome

Leydig Cell Function in Klinefelter’s Syndrome By Monica

Stewart-Bentley

The two major secretion products of the normal testes were measured in peripheral plasma of eight patients with Klinefelter’s syndrome; two agonadal, one castrate, one patient with isolated gonadotropin deficiency, and one patient with unclassified primary hypogonadism. Plasma testosterone and 17-n-hydroxyprogesterone were very low in all but the Klinefelter’s patients. Testosterone values markedly varied, but were generally subnormal in Klinefelter’s, ranging from 25-425 mFg/lOO ml, 187 I? 160. The 17-a-hydroxyprogesterone (17OHP), however, was elevated in three and within the normal range in five [Klinefelter’s 120 X!Z 63 (SD) vs. normal 97 2 21 (SD) m pg/lOO ml, respectively]. The 17-OHP values are inappropriate when considered with the subnormal testosterone values.

and Richard

Horton

One of the youngest patients studied had a subnormal testosterone but a plasma 17OHP that was three times the mean for a normal adult male. The 17-OHP in Klinefelter’s was of testicular origin, since dexamethasone had minimal effect, but testosterone almost completely reduced plasma 17-OHP. The marked circadian variation of 17-OHP in normal males was not present in Klinefelter’s, although noncyclic fluctuations were observed. LH in the form of chorionic gonadotropin produced a variable response. Some of the patients exhibited a rise in plasma 17-OHP and testosterone despite already elevated 17-OHP levels. The studies indicate the presence of a disorder involving Leydig cell steroidogenesis in Klinefelter’s syndrome.

K

SYNDROME is a common type of hypogonadism with LINEFELTER’S small testes, subnormal testosterone production, and varying degrees of sexual underdevelopment and eunuchoidism. 1 The disorder is felt to be typical of primary hypogonadism in that there is hypergonadotropism and reduced responsiveness to LH or HCG stimulation. In addition, there is evidence for testicular damage, such as seminiferous tubular hyalinization and circulating testicular antibodies.” The syndrome now appears to be the result of the presence of excessive X-chromosomal material, usually in the form of Section of Endocrinology, Uni~~ersity of From the Department of Internal Medicine, Southern CaIifornia School of Medicine and the Los Angeles County Hospifal, Los Angeles Calif. Part of this work was presented at the 53rd Meeting of the Endocrine Society Jlrne, 1971 in San Francisco, Calif. Received Supported

for publication December 12, 1972. by USPHS Grant AM-13710 and American

Cancer

Society

Grant

Cl-GQF.

Stewart-Bentley, M.D.: Assistant Professor of Medicine, USC-LAC Medical Center, Los Angeles, Calif. Richard Horton, M.D.: Associate Professor of Medicine, Head Section of Endocrinology, USC-LAC Medical Center, Los Angeles, Calif. Systematic nomenclature for trivial names used in text: FSH, radioimmunoassayable Monica

follicle stimulating hormone; LH, radioimmunoassayable luteinizing hormone or interstitial stimulating hormone; HCG, human chorionic gonadotropin (Maurray Biological Co., Los Angeles); Urine “FSH,” mouse uterine weight bioassay measuring “total urinary gonadotropins”; Testosterone (T), 17&hydroxy-&androsten+one; 17-a-hydroxyprogesterone (17-OHP), 17-a-hydroxy-4-pregnene-3,20-dione; droxy-5-pregnene-20-one; Androstenedione, (lo)-estratriene-3, 17@-diol. 0 1973 by Grune & Stratlon, Inc. Metabolism,

Vol. 22, No. 7 (July),

1973

17-a-hydroxypregnenolone, 4-androstene-3, 17-dione;

38, 17-o-dihyEstradiol, I, 3, s

a75

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STEWART-BENTLEY

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the karyotype XXY, although mosaicism and other variants have been described.3-s No information is known about the actual gene products that result in gonadal deficiency and damage. The pubertal or adult Klinefelter’s patient, however, is different from the castrate or agonadal male. Testicular function, although subnormal,7,8 is present and partial masculinization is typical. The findings of relative and/or absolute Leydig cell hyperplasia. 2*g*10high serum LH despite only subnormal testosterone, and the clinical findings of gynecomastia suggest an altered gonadal secretory pattern. In the study to be reported, measurement of the circulating androgen (testosterone), as well as an early precursor (17-a-hydroxyprogesterone), suggests the presence of a disorder of steroidogenesis in the Klinefelter’s testes. MATERIALS

AND METHODS

Subjects Eight patients, untreated, ranging in age from 17-45 yr, with the clinical findings of Klinefelter’s syndrome, were studied. All had small, firm testes, gynecomastia, increased “FSH,” serum LH, and an abnormal karyotype (6-XXY, bioassayable urinary gonadotropin I-XXXY). One patient met all clinical and endocrine criteria but had a normal peripheral karyotype (46 XY). For comparative purposes, both testosterone and I?‘-a-hydroxyprogesterone were measured in agonadal or castrate males, in a patient with isolated gonadotropin deficiency, and in one patient with unclassified primary hypogonadism. Normal adult values using our methods have been previousIy reported.llJ2 Plasma samples for steroid assay were obtained from 8:00-9:00 a.m. after 1 hr in the sup*i.ne position, for base-line studies. The circadian variation of plasma steroids was observed at 0600, 1200, 1800, and 2400 hr. Human chorionic gonadotropin 4000 IU was given intramuscularly daily for 4 days and plasma samples were obtained at 6, 12, 24, and 96 hr. Two subjects received dexamethasone 1 mg four times a day for 3 days in order to suppress ACTH, and blood for steroid assay was obtained on the third day. Since our previous studies indicated that gonadotropin suppression in chronic hypergonadotropic subjects is quite slow,13 suppression was accomplished by an injection of 400 mg i.m. testosterone propionate, and peripheral plasma samples were obtained at 2 and 6 days postinjection.

Hormone

Assay

Testosterone and 17-a-hydroxyprogesterone were measured by competitive binding methods described previously by us, using the plasma binding proteins TeBG and CBG, respectively. Both methods involve a paper chromatography step for purification. The sensitivity and specificity allow assay of amounts of both steroids in excess of 10 mpg/100 ml. Precision of estimation is 13% for both methods, at values of only 50 and 37 mpg/100 ml, respectively. 12~4 Serum LH was determined by radioimmunoassay, and the specimens were analyzed at two doses in duplicate by a double antibody radioimmunossay described by Ode11 et al.lJJe The intra-assay precision was
Base-line Plasma Values Plasma testosterone variable, ranging from male values previously

values

in the eight Klinefelter’s patients were quite mLcg/lOO ml, means 187 f 160 (SD). Normal reported by us are 660 * 120 (SD) mpg/100 ml.”

25-425

LEYDIG

CELL

FUNCTION

IN KLINEFELTER’S

‘i

300 -

x ti ;

zoo -

SYNDROME

g’ Fig.

1. The

plasma

con-

centration of testosterone and 17-hydroxyprogesterone in a young patient with Klinefelter’s syndrome, during control period, 24 hr post 4000 IU of HCG i.m., at the end of 3 days of 1 mg of dexamethasone q. 6 hr and 6 days after a single injection of 40 mg i.m. testosterone

$ i 100

400

^ A 30’3 i

; P O ‘p 100 r- $

propionate.

Plasma 17-a-hydroxyprogesterone values in the patients with Klinefelter’s syndrome were surprising. The range of values was also very wide, 50-250 mpg/100 ml, mean 120 + 63 (SD) mpg/100 ml (Table I). These values are by themselves striking, since normal adult male values are 97 -t 21 (SD) mpg/loo ml. Therefore, some of the patients had elevated values for 17-ahydroxyprogesterone. One of the youngest subjects (S.R.) actually had a peripheral 17-OHP value, which is three times the normal mean (Fig. 1). The values for plasma l7-OHP are even more surprising when interpreted with testosterone values. Despite subnormal or very low values of testosterone in these patients, 17-OHP is normal or elevated. This relationship is also expressed by comparison of testosterone/l7-OHP ratios, which are 6.7 (N = 12) in normal males and 1.2 (N = 8) in the Klinefelter’s patients. Testosterone and 17-OHP were both very low in agonadal, castrate, isolated hvpogonadotropic, and in unclassified primary hypogonadal subjects Table 1, Fig. 2).

Circadian

Variation

In normal males, there is a marked circadian variation in l7-OHP, with values higher in the morning. l7 We also have confirmed this by observations made throughout the day in two normal males (TabIe 2). In the Klinefelter’s patients, however, there were minimal changes in both plasma testosterone and 17-a-hydroxyprogesterone, but there was no indication of cyclicity, and the testosterone/l7-OHP plasma fluctuations were asynchronous (Table 2). Response

to Gonadotropin

Stimulation

The response to chorionic gonadotropin (HCG 4000 IU/day X 4) was very variable in Klinefelter’s syndrome. Three of eight did not respond, while the remainder showed a moderate increase in both testosterone and l7-OHP. In one subject, there was an increase in 17-OHP without altering plasma testosterone. In the normal subjects the responses were uniform. Normal showed a minimal increase in testosterone, a two- to threefold rise in X7-OHP

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Table 1. Base-line Plasma Testosterone (T) and 17-Hydroxyprogesterone (17-OHP) and LH in Eight Patients With Klinefelter’s Syndrome, as Compared to Other Types of Primary and Secondary Hypogonadism 17-OHP

T Diagnosis

LH

Patient

Age

Kavobw

Klinefalter’s Klinefelter’s Klinefelter’s Klinefelter’s

H.R. W.W. A.C. D.T.

28 29 45 28

47XXY 47XXY 47XXY 46XY

345 45 294 62

223 aa 148 50

120 90 78 -

Klinefelter’s Klinefelter’s

R.P. R.M.

26 46

4axxxY 47XXY

25 43

63 61

63 -

Klinefelter’s Klinefelter’s Mean f SD

S.R. M.R.

25 17

47XXY 47XXY

425 255 187 & 160

249 81 120 ” 63

T.K. J.T.

16 21

46XY 46XY 46XY

660 f 120 20 12

98 k 21 19 28

A.P. R.T.

19 45

46XY 46XY

14 41

G.C.

26

46XY

30 23r 12

Normal males Anarchic Anarchic Isolated gonadotropin deficiency Castrate Unclassified Hypogonadism Mean _+ SD

mCg/lOO

ml

mCLg/lOO ml

mlU/ml

66 70 84k 19

21 30 24 25 ” 4

the first day, and both testosterone and li’-OHP levels were increased two- to threefold and four- to sixfold, respectively, after four daily injections. Therefore, the Klinefelter’s patients have a blunted response, although some subjects appear to further increase their already normal or high 17-OHP levels after gonadotropin (Table 3).

on

600

-

600

-

p;o

400

-

:B I-

200

-

%A 0~ E

I

kg

0 I NORMAL

KLINEFELTER

CASTRATE

Fig. 2. Mean plasma concentration f 1 SD of testosterone (T) and 17+hydroxyprogesterone in normal men, Klinefelter’s. and castrates.

LEYDIG

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FUNCTION

IN KLINEFELTER’S

Table 2. The Mean Plasma Concentration

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SYNDROME

(m/lg/lOO

ml) of Testosterone

(T) and

17 Hydroxyprogesterone (77-OHP) Measured Every 6 hr in Eight Patients With Klinefelter’s Syndrome and in Two Normal Men Patient

6 a.m.

12 noon

6 pm.

12 a.m.

6 a.m.

T 17-OHP T

345 223 45

273 195 36

334 160 56

432 113 44

398 290 57

17-OHP T 17-OHP T 17-OHP

88 294 148 62 50

37 179 80 69 76

48 300 148 46 49

27 214 100 43 97

69 285 86 52 69

R.P.

T 17-OHP

25 63

77 53

56 61

10 74

R.M.

T 17-OHP T 17-OHP T 17-OHP

43 61 425 216 255 81

29 85 261 249 122 78

20 78 354 170 162 48

21 62 390 230 168 63

T 17-OHP

125 f 56 116 rt 25

T 17-OHP T

510 112 630

425 116 580

496 64 620

416 85 520

523 120 -

17-OHP

87

71

32

66

95

HR. W.W. A.C. D.T.

S.R. M.R. Mean 2 SE

130 ?I 34 111 k 30

167 -t 50 159 ” 52

224 -c 62 100 2 24

28 81 425 216 216 75 206&63 135+36

Normals T.K. D.W.

Dexamethasone

Suppression

Dexamethasone I mg four times a day was administered to two patients in order to ascertain the adrenal contribution in the Klinefelter’s patients. Minimal changes in plasma 17-OHP and testosterone were observed in both patients. Values observed are illustrated in Fig. 1 and in Table 4. Suppression

of Gonadotropin

Further evidence that the relatively high 17-OHP is derived from the gonad was provided by a study in which gonadotropin was suppressed. A single injection of 400-mg testosterone propionate markedly reduced l7-OHP levels during the 6 days of study in two patients. A typical study is shown in Fig. I and in Table 4. Plasma LH by immunoassay was correspondingly suppressed to one-fourth of base line in both patients by the sixth day.* DISCUSSION

The two major steroid secretions of the human testes are testosterone (7 mg/day) and 17-a-hydroxyprogesterone (1.8 mglday).“*‘s The secretion *Plasma immunoassayable pital, Torrance, Calif.

LH was performed

by Dr. W. D. Odell, Harbor

General

Hos-

880

STEWART-BENTLEY

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Table 3. Plasma Concentration (mrg/lOO ml) of Testosterone and 17-Hydroxyprogesterone in Two Normal Men and in Eight Patients with Klinefelter’s Syndrome Before and During

the Administration

Post HCG

Base Line 8 a.m.

Patient

H.R.

T 17-OHP

W.W.

T 17-OHP

A.C.

T 17-OHP

D.T.

T 17-OHP

R.P.

T 17-OHP

R.M.

T 17-OHP

S.R.

T 17-OHP

M.R.

T 17-OHP

of 4000 IU of HCG Daily for 4 Days

6 Hr

12 Hr

24 Hr

279 148 146

234 198 80

126 156 174 100 63 11

222 141

89

27 79

272 209 121 270 130 136 76 61 3 75 31 82

249 255 80

334 284 240 116

528 320 204 130

454 400 308 132

640 87 775

-

-

701 250 723

1520 514 1776

109

-

-

262

520

345 223 45 88 294 148 62 50 25 63 43 61 332

156 42 -

77 9

4 Days

224 137 120 97 10 142 700 325 335 158

Normals R.H.

T 17-OHP T 17-OHP

U.I.

Table 4. Effect of Dexamethasone (dex) and Testosterone Propionate Two Patients with Klinefelter’s Syndrome T Propionate Subject

Time

S.R.

Baseline 48 hr post T 6 days post T Base line 48 hr post T 6 days post T

A.C.

Dexamethasone Subject

S.R. A.C.

(T) on

400 mg i.m. LH mllJ/ml

66

4 mg p.o. q.i.d.

Time

Base line 3 Days Postdexamethasone Base line 3 Days Postdexamethasone

17-OHP ng/lOO ml

32 78 52 21

213 116 50 173 90.5 36.8

T ng/loO ml

17-OHP ng/lOO ml

x 3 Days

332 321 167 157

249 206 145 117

LEYDIG

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FUNCTION

IN KLINEFELTER’S

SYNDROME

881

of both steroids is greatly augmented by LH or HCG, although physiologic roIe of 17-a-hydroxyprogesterone is not understood.1g*20 In the adult male, both plasma testosterone and 17-a-hydroxyprogesterone are derived almost exclusively from the testes, and a study of the secretion pattern may reflect Leydig cell efficiency of steroidogenesis. In contrast, the testes secrete almost no androstenedione, and most of the androstenedione in the normal male is derived from the adrenal cortex .21,22 The greater secretion from the normal testes of 17-OHP, in contrast to androstenedione, suggests that the sidechain-splitting enzyme (desmolase) may be an important rate-limiting step in testosterone biosynthesis in the normal male. In the current study, we have simultaneously measured testosterone and I7-a-hydroxyprogesterone in blood under various conditions in patients with Klinefelter’s syndrome. Base-line values for plasma testosterone in our series are quite variable as reported by others,2*7*23 ranging from prepubertal to the lower limit of normal. Even the low normal values are probably pathologic, since a recent report finds very low free testosterone levels in patients with Klinefelter’s syndrome,24 indicating that the Klinefelter’s testes cannot secrete normal amounts of testosterone despite levels of LH equal to, or greater than, observed in male castrates. The plasma levels of 17-a-hydroxyprogesterone are both higher than the mean for normal males12s17 and relatively high as compared to the abnormally low plasma testosterone in Klinefelter’s syndrome. The ratio between testosterone and 17-OHP is markedly altered. In one of the youngest subjects studied, I’?‘-OHP levels were similar to testosterone and the 17-OHP level was 3 times the mean value seen in normal adult males. In contrast to the normal males studied by Strott et a1.17 and ourselves in this article, little or no circadian variation in testosterone or 17-OHP is observed in patients with Klinefelter’s syndrome. A lack of diurnal variation of 17-OHP would be expected in a disorder where gonadotropin stimulation is excessive. In a related study to be published, we have observed marked spontaneous fluctuation in LH (and a lesser degree in FSH) in patients with Klinefelter’s. No circadian pattern was observed. Perhaps the noncyclic fluctuations in 17-OHP are the result of this random variation in gonadotropin release. The established finding of subnormal testosterone production initially suggested the possibility that a late step in testosterone biosynthesis might be involved. However, Paulsen and co-workers found that peripheral androstenedione levels in Klinefelter’s were normal or 10w.~ The finding of variable androstenedione levels in this syndrome by this group is clarified by our present knowledge that androstenedione is primarily an adrenal product in the male.21s22 The adrenal function appears to be normal in Klinefelter’s syndrome. Recently Weinstein et al. have demonstrated that plasma androstenedione in Klinefelter’s syndrome is readily suppressible by dexamethasone and not increased with HCG,25 suggesting that in this syndrome, plasma androstenedione is of adrenal origin; thus an abnormality in the conversion of androstenedione to testosterone in the testes is unlikelv.

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HORTON

Other hypotheses have been proposed to explain the subnormal plasma testosterone in patients with Klinefelter’s syndrome. In 1970 Gabrilove et al. showed that there was an altered conversion of androgens to estrogen in patients with this syndrome. 26 In vitro studies have been inconclusive, although Sharma and Gabrilove recently reported that testicular tissue from Klinefelter’s patients had an increased capacity to convert testosterone to estrogens when 17-a-hydroxyprogesterone (17-OHP) or testosterone (T) was used as a substrate.27 If this exists in vivo, gynecomastia would be explained. The normal or increased l7-OHP levels in Klinefelter’s syndrome are not the result of a reduced rate of peripheral metabolism or an estrogen effect on transcortin (CBG), since plasma cortisol is normal in these patients, and there is no reduction in the metabolic clearance rate of 17-OHP in Klinefelter’s syndrome (MCR l7-OHP = 2500 liter day, 1100 liter/day/sq m, unpublished observations). There are two other possibilities to explain the inappropriate plasma 17-a-hydroxyprogesterone levels. 17-a-hydroxypregnenolone can be converted by peripheral tissue to 17-a-OHP. 28 Plasma 17-OHP in the follicular phase of the female is primarily derived from adrenal 17-OHP-pregnenolone.28 It is possible that the relative increase in plasma 17-OHP is the result of increased conversion of 17-a-hydroxypregnenolone to l7-OHP by peripheral tissues or in the testes. We have shown, however, that l7-OHP in Klinefelter’s is of testicular origin. Studies of the blood production rate and testicular contribution to circulating 17-OHP-pregnenolone are now necessary in Klinefelter’s syndrome. The other possibility is that 17-OHP is derived from a testicular non-Leydig cell source (i.e., Sertoli cells). This cannot be answered at the present time, although suppression of serum LH with parenteral testosterone indicated that both testosterone and 17-OHP are under the control of the leuteinizing hormone. The inappropriate values of 17-OHP in Klinefelter’s are the result of gonadal secretion, since levels of testosterone and l7-OHP are minimally altered by ACTH suppression (dexamethasone) and increased by gonadotropin stimulation (chorionic gonadotropin). The testosterone response to an LH-like substance (HCG) is blunted, suggesting that Leydig cell reserve is minimal. It is important to note that HCG tends to further increase 17-OHP levels in some patients with Klinefelter’s syndrome, despite an already elevated 17-OHP level and subnormal plasma testosterone. LH suppression with testosterone propionate reduced 17-OHP production to levels seen in castrates. We propose, on the basis of the present study, that there is a disorder of steroidogenesis in Klinefelter’s syndrome which prominently involves an intermediate step, such as side-chain removal (lyase). The present evidence implicates the step l7-OHP to androstenedione. Testicular secretion of androstenedione and testosterone is reduced, despite high gonadotropin levels in the presence of increased testicular l7-OHP secretion. The side-chainsplitting enzyme of progesterone-pregnenolone, as well as the C19, 17-P-01dehydrogenase, are located in the microsome.2g A general microsomal disorder would be expected to equally’affect all steps in testosterone biosynthesis after pregnenolone. However, the oxygen-requiring step for side-chain cleavage

LEYDIG

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FUNCTION

IN KLINEFELTER’S

883

SYNDROME

lyase could be affected earlier if the disorder is secondary to microsomal injury.zg We cannot explain the fact that the absolute values of 17-OHP in some of the patients are not elevated, as expected in problems with ciassical enzyme blocks, unless the variability reflects degrees of testicular (Leydig cell) damage. It will be important to study the steroid production of the testes from Klinefelter’s patients during the pubertal period, before any complicating events occur, to further substantiate our hypothesis. It is possible that the gene product in Klinefelter’s syndrome acts by suppression of certain steps in testosterone biosynthesis.

c17-20

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HF Jr,

Reifenstein

EC Jr,

Albright F: Syndrome characterized by gywithout necomastia, aspermatogenesis a-Leydigism, and increased excretion of follicle-stimulating hormone. J Clin Endocrinol Metab 2 :615, 1942 2. Paulsen CA, Gordon DL, Carpenter RW,

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11. Baird D, Horton Steroid prehormones. 11~384, 1968 12. Stewart-Bentley

R, Longcope Perspect M, Horton

C, et al: Biol

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congenital adrenal hyperplasia. J Clin Invest 48 :930, 1969 18. Horton R, Tait JF: In vivo conversion

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of dehvdroisoandrosterone to plasma androstenedione and testosterone in man. J Clin Endocrinol Metab 27~79, 1967 19. Laatikainen T, Laitinen EA. Vihko R:

Electron microscopy of the Leydig cells and hormone assays in Klinefelter’s syndrome. Endokrinologie 55 :129, 1969 10. Ahmad KN, Dykes JRW, FergusonSmith MA, et al: Leydig cell chromatin-positive Klinefelter’s J Clin Endocrinol Metab 33517,

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Secretion of free and sulfate-coniueated neutral steroids by the human testis. Effect of administration of human chorionic gonadotropin. J. Clin Endocrinol Metab 32~59, 1971 20. Eik-Nes KB: The Androgens of the Testis. New York, Marcel Dekker, 21. Gandy HM, Peterson RE:

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ment of testosterone and 17-ketosteroids in plasma by the double isotope dilution derivative technique. J Clin Endocrinol Metab 28 :949, 1968 22. Wieland RG, decourcy C, Levy RI’, et al: C,sOs steroids and some of their precursors in blood from normal human adrenals. J Clin Invest 44:159, 1965 23. Lipsett MB, Wilson H, Kirschner MA: Studies on Leydig cell physiology and pathology: secretion and metabolism of testosterone. Recent Prog Horm Res 22:245, 1966 24. Vermeulen A, Stoica T, Verdonck L: The apparent free plasma testosterone concentration, an index of androgenicity. J Clin Endocrinol Metab 33:759, 1971 25. Weinstein RL, Kaplan SL, Grumbach MM: Gonadal and pituitary insensitivity to HCG and clomiphene stimulation in Klinefelter’s

syndrome.

Prog.

53rd

Meeting

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AND

HORTON

docrine Society, (Abstract), San Francisco, Calif, 1971, p 119 26. Gabrilove JL, Nicolis GL, Havsknecht RV: Urinary testosterone, oestrogen production rate and urinary oestrogen in chromatin positive Klinefelter’s syndrome. Acta Endocrinol (Kbh) 63:499, 1970 27. Sharma DC, Gabrilove JL: Biosynthesis of testosterone and estrogens in vitro bv the testicular tissue from patients with Klinefelter’s syndrome. Acta Endocrinol (Kbh) 66:737, 1971 ~8. Strott CA, Bermudez JA, Lipsett MB: Blood levels and production rate of 17hvdroxypregnenolone in man. J Clin Invest 49 :1999, 1971 29. Machino A, Inano H, Tamaoki B: Studies on enzyme reactions related to steroid biosynthesis. J Steroid Biochem 1 :l, 1969