J. SteroidBiochem. Molec. Biol. Vol. 44, No. 2, pp. 171-177, 1993 0960-0760/93 $6.00+0.00 Printed in Great Britain P¢rgamon Prt~ Ltd

S E C R E T I O N B Y T H E H U M A N T E S T I S O F

E P I T E S T O S T E R O N E , W I T H ITS S U L F O C O N J U G A T E A N D

P R E C U R S O R A N D R O G E N 5 - A N D R O S T E N E - 3 f l , 1 7 o ~ - D I O L

L o u i s D E S E ~ I N

Fondat ion de Recherche en Hormonologie, 67 Boulevard Pasteur, 94260 Fresnes, France

(Received 5 August 1992; accepted 30 September 1992)

Semmary--Epitestosterone (ET) and testosterone (T), free and sulfoconjugated, as well as 5-androstene-3fl,17`'-diol (5AD3flI7`') and its 17fl-epimer have been analyzed, by gas chromatography-mass spectrometry with stable isotope dilution, in peripheral and spermatic venous plasma of patients with left yaricocele. All these androgens are secreted by the testis as evidenced by the significant concentration gradients between peripheral and spermatic venous plasma. Half of the daily ET production is ascribed to the testis, while 95% of T sulfate and roughly 70% of ET sulfate are also of testicular origin. Significant correlations between ET and 5AD3fl17,, are an indication that the 5-ene pathway is also operative for ET biosynthesis. High ratios of spermatic to peripheral venous plasma levels of ET and 5AD3//17,, are also related to the high clearance rates of 17`'-hydroxy-androgens.

I N T R O D U C T I O N

In humans, epitestosterone (ET; 17~-hydroxy- 4-androsten-3-one) is found in various biologi- cal fluids. Firstly excretions of ET glucuronide (ETG) and sulfate (ETS) were reported in urine [1-6]. An intriguing aspect is the strongly increased urinary ETG excretion in hirsute women [7, 8], its origin and biological meaning have still to be clarified. Of up-to-date interest is the use of ETG as a reference for the ex- pression of relative excretions of testosterone giucuronide (TG) in the analytical procedure for detection of testosterone (T) misuse in sports [9].

Male plasma levels of ET are very low (50-250 pg/ml) [10]; they are the consequence of a production rate (200-300 /~g/24 h) [3] which represents only 3% of the daily T production, and of a high metabolic clearance rate (3000-40001/24 h) [11]. Circulating ETS was first evidenced by Dray et al. [12], but they could not detect plasma ET.

Follicular fluid from preovulatory follicles in women with stimulated cycles contains high concentrations of ET (10-50 ng/ml) [13] and this accumulation suggests an ovarian production of ET, although ovarian secretion has not yet been proven, in particular in the case of hirsutism.

Extensive work has been done by the group of Vihko who identified and quantified numer- ous free and sulfoconjugated neutral steroids in testis tissue[14] and in spermatic venous

blood [15]. Conversely, much less endeavor has been devoted to the study of the biological activity of ET, until recent data reported by St~trka and his colleagues[16, 17], indicating that ET has weak antiandrogenic actions evi- denced by the competitive binding to the andro- gen receptor, the moderate suppression of gonadotropins and the competitive inhibition of 5~-reductase.

Our results, demonstrating a drastic decrease of urinary ETG and ETS excretions after long- term T administration to normal men [I 8], have prompted us to apply the analytical procedures, devised therefore and based on gas chromatog- raphy-mass spectrometry (GC-MS) with stable isotope dilution, to investigate the expected secretion of ET by the human testis.

EXPERIMENTAL

The subjects of this study were 16 men (aged 24-36 yr, mean 29 yr), undergoing operations for left varicocele and they gave informed con- sent for the collection of blood samples for this study. Within 30 min after general anesthesia, samples of peripheral blood from the cubital vein and spermatic blood from the spermatic vein of the varicocele side were collected almost simultaneoulsy by needle aspiration into hep- arinized syringes. Samples were centrifuged im- mediately and plasma was stored at -20°C until analysis.

171

172 Louis DEItENNIN

Analysis by GC-MS with Stable Isotope Dilution

Instrumentation

GC was performed on a fused silica capillary column (25 m x 0.32 mm i.d.) coated with OV- 1701 stationary phase (film thickness 0.20 #m), installed in the oven of a gas chromatograph, equipped with a glass solid injection system and heated at an isothermal temperature (between 220 and 240°C). The column was directly coupled to the source (electron impact mode) of a quadrupole mass spectrometer (Model 1010T, Nermag, Argenteuil, France) which was oper- ated under normal ionization and mass filter settings. Data processing was performed with a PDP 11 computer (Digital Equipment France, Rungis, France) and Sidar software (Nermag).

Analytical procedures

Nonconjugated androgens. Samples (1 ml for peripheral plasma and 0.5 ml for spermatic vein plasma) were spiked with known amounts of stable isotope labeled analogs, [3,4-]3C]T (Oris- CEA, Gif-sur-Yvette, France), [I~t,2~t-2H2]ET and [16,16,17 -2 Ha ]5-androstene-3fl,17fl-diol syn- thesized as reported previously[19]. These amounts of isotopic internal standards were close to the amounts of native androgen present in the sample, so that the ratio of labeled over unlabeled species was comprised between 1 and 2. A mixture of n-hexane--diethyl ether 3: 2 (v/v) was used for extraction and residues were fractionated by liquid chromatography on (200 x 5mm) columns of Sephadex LH-20 packed in the eluent which was a mixture of dichloromethane-methanol 95:5 (v/v): the first 4 ml were discarded, the next 1 ml contained ET and T, and the androstenediols were eluted in another 2.5 ml. After evaporation to dryness, ET and T were converted to the bis(hepta- fluorobutyrate) and the androstenediols to the bis(t-butyldimethylsilyl ether). Final residues were taken-up in 30/~1 of iso-octane and 3 or 6/~1 were deposited on the needle of the solid injection system. Selected ion monitoring (SIM) was performed at nominal masses m/z 680 and 682 (ET and T), and m/z 461 and 464 (andro- stenediols). Quantitative results were calculated according to equations outlined previously [20].

Sulfoconjugated androgens. The sulfates of ET (ETS) and T (TS) were analyzed in the sample which had been used for preliminary extraction of the nonconjugated androgens. Concen- trations are expressed in equivalents of noncon- jugated steroid. The internal standard [3,4- ~3 C]T

17 sulfate was synthesized by esterification of [3,4-13C]T with chlorosulfonic acid and purified successively by Amberlite XAD-2 chromatog- raphy and anion exchange chromatography on DEAE-cellulose. Labeled TS was added to the samples (2 ng for 1 ml peripheral plasma and 20ng for 0.5ml spermatic vein plasma) and equilibrated for 1 h at room temperature. After addition of 3 ml methanol and centrifugation, the supernatant was fractionated on small columns (20 x 5 mm) of DEAE-Sephadex (chloride form) packed in methanol. Glucuro- and nonconjugated steroids were washed from the column with 4 ml methanol and sulfates were eluted with 0.3 M LiCI in methanol: 0.5 ml was first discarded and the next 1.5 ml contained sulfates. Solvolysis was carried out by dissolving the dry residue in I ml 0.5 M HC1 in methanol and heating at 60°C for 4 h. Neutralization with 0.5 ml saturated NaHCO3 solution, evaporation of most methanol and extraction with a mixture of n-hexane-diethyl ether 4:1 (v/v) gave upon evaporation a dry residue which was taken-up in 0.2 ml chromatography eluent and purified by Sephadex LH-20 chromatography as described above. Quantification was performed by SIM of the bis(heptofluorobutyrate) derivatives at nom- inal masses m/z 680 and 682.

The internal standard [I~,2ct-EH2]ET 17- sulfate was not suitable for use under solvolysis conditions, since prohibitive exchange for the 2Gt-deuterium for protium occurred. Therefore [3,4-13C]T 17-sulfate was used for both TS and ETS determinations but, while standard ad- ditions of TS were quantitatively recovered, those of ETS had an average yield of 85 + 1% (n = 6). This is due to Wagner-Meerwein re- arrangement catalyzed by the acidic solvolysis medium, and quantitative results had to there- fore be corrected. Enzyme hydrolysis of sulfo- conjugates of ET and T is not feasible by Helix pomatia preparations since these contain to a more or less extent 3fl-hydroxysteroid dehydro- genase/4,5-isomerase which converts 5-andro- stene-3fl,17fl-diol (5AD3fll7fl) and its 17~t- epimer into T and ET, respectively [18].

Accuracy and precision. The accuracy of GC- MS determinations associated with stable iso- tope dilution is principally based on permanent correction of procedural losses, on the specific- ities attained by the high resolution GC column and the selected ion detection and on the purity of the steroids used as primary standards. As expected, precision was better in those cases where the corresponding isotopically labeled

Epitestosterone secretion by the testis

Table 1. Concentrations (in ng of free steroid/ml) in PV and SV 100 plasma and ratios SVfPV of free and sulfoconjugnted androgens

Mean SEM Range

Epitestosterone PV 0.089 0.012 0.030-0.361 50 SV 8.92 1 . 5 8 1.07-23.80 SV/PV 107.9 14.1 35.7-237 co

Epitestosterone sulfate o PV 0.864 0.091 0.320-1.59

e -

SV 8.93 2.27 1.98-36.54 ^o SV/PV 9.87 1 .98 2.28-28.55 ,., 4vx~

5-Androstene-3p, 17= -diol PV 0.17 0.03 0.03-0.46 SV 19.97 3.26 2.52--40.64 _o SV/PV 193.2 54.1 32.4-377 ~ 5O

Testosterone PV 7.29 0.73 2.41-12.74 SV 493.8 71.6 102-961 SVfPV 68.3 10.0 17.4-168

Testosterone sulfate O PV 1.33 0.33 0.380-5.50 SV 12.31 4.04 1.99-64.06 SV/PV 8.67 1 .74 2.86-21.16

5-Androstene-3fl, 17/~ -diol PV 1.45 0.20 0.50-3,52 SV 58.8 1 0 . 2 9.87-1.38 SV/PV 38.9 4.8 10.2-75.9

Mean ratios SV/PV are all significantly different from 1 (one-tailed t-test for normally distributed values, P < 0.05).

173

ET

t % .

I /

0 9 10

MIn

[ IsC]T

11 12

Fig. 2. Window in the selected ion chromatogram of the sulfoconjugated steroid fraction o f 0.5 ml spermatic vein plasma spiked with 13C-labeled TS (2 rig) and analyzed as the bis(heptafluorobutyrate) derivatives at m/z 680 and 682. Endogenous concentrations are: ETS ffi2.89ng/ml; TS-

2.47 ng/rnl.

analogs were available: the coefficient of vari- ation (CV) for inter-assay replicates (n = 10) was then between 3 and 4%. In cases where an isomeric internal standard had to be used, vari- abifty increased significantly (CV between 6 and 10%).

RESULTS

An overview of analytical data is given in Table 1. It can be noticed that for all 17u- hydroxy-androgens the ratio of spermatic (SV)

100

5O @ o t- o

! o ' i

5O

x20

o 8 9

ET

T'dz

40

MIn

T

~K.m/,,,' 680

~lSc]T

mlz 6 8 2

,

11 12 13

Fig. 1. Window in the selected ion chromatogram of the nonconjugated steroid fraction o f 0.5 ml SV plasma, spiked with labeled analogs o f ET (ET-d2= 5ng) and T (I"CTF = 200ng), and analyzed as the bis0teptaltuorobu- tyrate) derivatives at the muss-to-charge ratios (re~z) 680 and 682. Endogenous concentrations are: ET -- 7.04 ng/ml;

T = 448 ng/mL

to peripheral vein (PV) plasma levels (SV/PV) is always higher than for the corresponding 17fl- epimers. The scattering of androgen concen- trations in SV blood is generally higher than in PV blood; this corresponds to what is reported in the literature for normal men and for varico- cele patients [21]. The presence of ET and ETS (together with T and TS) in SV plasma is shown in Figs 1 and 2, which also illustrate the specifi- city of the analytical method.

Significant correlations exist between plasma levels of PV and SV for all investigated par- ameters (middle part of Table 2), except for 5- androstene-3fl,17u-diol (5AD3f117~) (r = 0.40, nonsignificant). Of special interest was also the correlations found in SV plasma (upper part of Table 2), and particularly between ET and its precursor 5AD3f117~.

If it is taken into account that the urinary production rate of ET has an average value of

Table 2. Significant correlations between PV and/or SV plasma androgens

Correlation coefficient

SV (ET vs T) 0.55 SV (ETS vs TS) 0.92 SV (ET vs 5AD3~ 17~,) 0.54 SV (T vs 5AD3pI7~) 0.60 (iV vs SV) ET 0.80 (IV vs SV) ETS 0.54 (IV vs SV) T 0.60 (IV vs SV) TS 0.83 (IV vs SV) 5AD3pI7p 0.68 PV (T vs TS) 0.67 PV (ET vs ETS) 0.51

Spearman's rank correlation co¢flkients with P < 0,05 dS- nificance level, for abbreviations see text.

SBMB 44/2--F

174 Louis DEHENNIN

around 250/~g/24 h (10.5 #g/h) [3, 11], and that the average blood flow through the testis in men is around 10 ml/min [22, 23], then it can be calculated from the mean concentration gradient of ET between PV and SV plasma (=8.8ng/ml) that the testicular ET secretion rate (=8.8 ng/ml x 600ml/h = 5.3/~g/h) rep- resents approx. 50% of the total ET production. For the sake of comparison and as a support to the consistency of our measurements, an analogous calculation with the T concentration gradient found in this study indicates that the testicular secretion rate of T represents about 95% of the total production (7 mg/24 h), and this agrees quite well with current knowledge on T biosynthesis in man. A very similar situation was found for TS: a urinary production rate of around 170#g/24h[24] and a mean concen- tration gradient between PV and SV plasma of l l.0ng/ml indicate that approx. 95% of TS originates from the testis,

The blood production rate of 5AD3f1178 has been determined by Bird et al. [25] who found an average value of 1357 #g/24 h (56.5 #g/h) in normal men; this together with our evaluation of the testicular secretion rate (57.35 ng/ml x 600 ml/h = 34.4 ktg/h) allowed us to estimate at around 60% the gonadal contribution to the total production.

DISCUSSION

When analyzing steroids in SV blood from varicocele patients, a first question arises on the possibility of reduced testicular androgen se- cretion in comparison to normal men. Some controversial data are found in the literature, claiming either normal or impaired testicular endocrine function in these patients, but Scholler et aL [21] have clearly demonstrated that there is no significant difference in the androgen secretory activity between the two testes of patients with unilateral varicocele. Another question concerns the possibility of a retrograde flow of adrenal venous blood through the gonadal vein, consecutive to incom- petent gonadal vein valves and which might cause an elevation of adrenal steroids in SV blood. This does not seem to be the case since peripheral corticosteroid concentrations have been found in SV plasma [26]. Thus, on the basis of these arguments and also of our average T levels which are in agreement with those re- ported by several investigators [27-30], it seems reasonable to admit that the findings of this

study are not particular to varicocele patients, but that they can also be observed in normal men. It has also to be kept in mind that within 30 min of surgery with general anesthesia there is a small but significant decrease of T in SV plasma [31], which is however not seen in PV plasma [32].

The secretion of ET by the human testis is now definitively proven by the concentration gradient between PV and SV blood with a high ratio SV/PV. This secretion was suspected on the basis of previous work demonstrating an increased urinary excretion of ETG upon stimu- lation of Leydig cell function by hCG [3, 33]. The decline of ETG excretion, which we ob- served after chronic T administration, is also directly related to impaired Leydig cell stimu- lation by decreased LH secretion consecutive to the negative feedback exerted by the exogenous T [18]. Such a significant suppression of urinary ETG supposes a substantial contribution of the testis to the total ET production. This is confirmed by our data which indicate that at least half of the daily ET production is of testicular origin. The other half of the ET production is probably due to the adrenal gland, since ACTH increases significantly the urinary ETG excretion in normal men [3, 33]. Some contradictory data have been reported on per- ipheral production of ET in humans [2, 3, 34], but there is no sufficient proof thereof.

Only very scarce information is available on the biochemical pathway(s) leading to produc- tion of ET in general, and in the testis in par- ticular. Up to now, there is no evidence of con- version of androstenedione to ET in the testis since no testicular 17~-hydroxysteroid dehydro- genase activity has been detected. Recently, an interesting proposal was formulated by Weusten et al. [35] on the in vitro synthesis of 5AD3f117ct as a side-product of the 16-ene-synthetase ac- tion, which converts pregnenolone into the cor- responding 5,16-androstadien-3~-ol by a single competitive mechanism. The identification of 5AD3~17~t in human testis tissue[14] and its gonadal secretion [15], as well as the high ratio SV/PV (Table 1) and the significant correlation between ET and 5AD3fl 17~t levels in SV plasma (Table 2) are all pertinent arguments in favor of the statement that this androstenediol is a substrate for 3~-hydroxysteroid dehydrogen- ase/4,5-isomerase and thus a precursor of ET.

The secretion of ETS by the human testis is also clearly evidenced, although the correspond- ing ratio SV/PV is much lower than for ET.

Epitestosterone secretion by the testis 175

A production source of ETS different from sulfoconjugation of circulating ET was hypoth- esized by Dray and Ledru [5] on the basis of the 10 times higher production rate of ET found by measuring ETS as the urinary metabolite in- stead of ETG. This gonadal secretion of ETS is also in keeping with our results indicating a decline of urinary ETS excretions after long- term T administration[18], according to the mechanism outlined above for ET. However, those decreased ETS excretions are not solely due to impaired testicular ETS production; they are also the consequence of suppressed secretion of ET, which is sulfoconjugated peripherally and particularly in the liver, with a 2% overall conversion rate [5]. The biosynthesis of ETS and its subsequent secretion by the human testis supposes sulfotransferase action, either on ET, or on 5AD3fl 17~ with preferential formation of the 17-monosulfate [36] before its conversion to ETS. Both ETS and TS are ring D sulfates which represent a form of inactivation of the unconjugated steroids, since there is little or no evidence of sulfatase action upon them [37]. Although the average concentrations of ET and ETS in SV plasma arc very similar, their PV concentrations are different and this is a conse- quence of the lower metabolic clearance rate of sulfates and also of a higher extragonadal pro- duction of ETS, in particular by sulfoconjuga- tion of circulating ET.

Our TS levels in PV and SV plasma confirm previous data [38, 39]; they are very well corre- lated with ETS concentrations in SV plasma and this may be considered as an argument in favor of a single ring D sulfotransferase enzyme. Moreover there is a remarkable similarity be- tween the mean SV and PV plasma levels of ETS and TS and their concentration ratios; this pleads for a common status of inactive metab- olites. Thus, although the production rate of ETS is still unknown, we have evoked some reasons to assume that it must be rather similar to the TS production rate of 170/zg/24h [24]. Then, on the basis of the mean concentration gradient of ETS between PV and SV plasma (8.0 ng/ml), it may be speculated that the testic- ular secretion rate ( l lS#g/24h) represents roughly 70% of the total production. Yet as for ET, a substantial part of the ETS production may be ascribed to the adrenal, since the conver- sion rate of circulating ET to ETS represents only 2% [5].

Both the PV and SV plasma levels of 5AD3p 17p found in this study agree with those

obtained by radioimmunoassay [21, 25, 40]. The testicular secretion rate determined here is slightly higher than the one reported by Forti et al. [40], and it confirms that a sub- stantial amount of circulating 5AD3~17~ comes from sources outside the testis. Despite the extragonadal production, the correlation between PV and SV plasma levels of 5AD3~ 17~ remains significant (Table 2). It has been shown by Ruokonen [41] that the synthesis of T via the 5-ene pathway is very active in human and boar testis; yet as expected, we have found a significant correlation between T and 5AD3B17p concentrations in SV plasma (Table 2).

Gonadal secretion of 5AD3fll7~ was first shown by Laatikainen et al. [38]; they reported rather high values for SV blood levels of this androstenediol and its 17fl-epimer. The pro- duction rate of 5AD3fll7~ in man is still unknown, but it may be speculated that at least half of the total production can be attributed to the testis, according to what is observed for its metabolite ET and for 5AD3fll7fl. The testicular secretion rate of 5AD3f117~ (19.8 ng/ml × 600 ml/h = 11.9/~g/h) represents approx, one third of the secretion rate of the 17fl-epimer (34.4/zg/h); this proportion is decreased to nearly one tenth for the average circulating levels, thus suggesting a much higher clearance rate of 5AD3p17~, as reported for ET [l 1]. The high SV/PV ratios observed for 5AD3f117~ and ET are a further convincing argument in favor of rapid clearance of 17~-hydroxy-androgens. This is also reflected on the high urinary excretions of the corresponding glucuronides of ET and 5AD3f117~ [18].

Finally, this study is open to criticism because some urinary production rates have been used for the calculation of testicular contribution to the overall production of specific androgens. The accuracy of such calculations might have been better with blood production rates, which were not always available.

In conclusion, this study has demon- strated that ET, its precursor 5AD3fll7~ and its sulfoconjugate ETS are secreted by the human testis, for at least half of the daily production. It remains now to be established if the other half originates mainly in the adrenal gland. The reason why the testes produce and secrete ET is unclear, since no specific physio- logical role can yet be ascribed to this weak androgen.

176 LOUIS DEHENNIN

Acknowledgements--I am very grateful to Dr M. Adjiman, urologist, who provided the blood samples from varicocele patients, by means of Dr C. Blacker. Without the assistance of Messrs J. Dubois and P. Salvador, who are excellent professionals experienced in GC-MS methods, this work could not have been carried out. My sincere gratitude is due therefore.

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Epitestosterone secretion by the testis 177

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