Steroid metabolism in testicular tissue of genetic mutant mice

.lournol

of Sterord

Bmchemwr~.

1978. Vol. 9, pp. 41-45.

STEROID

Pergamon

Press.

Printed

in Great

Britain

METABOLISM IN TESTICULAR OF GENETIC MUTANT MICE

TISSUE

J. D. DALEY and E. V. YQUNGLAI Department of Obstetrics and Gynecology, McMaster University Medical Centre, Hamilton, Ontario, Canada L8S 459 (Received 14 March 1977) SUMMARY The conversion of testosterone and progesterone to other products was studied in testes of normal BALBjC mice, testicular. feminized (TJ%I o+) and testicular feminized mice carrying the protective (oh”)gene. The formation of testosterone from progesterone was 11 +_ 1.3% in normal mice, 2.20 If: 0.8% in (Tfm oh”) and 0.8 + 0.2% in (Tfm o+) mice. With androstenedione as substrate, both (7”m o+) and (Tfm oh”) synthesized testosterone equally well. Testicular progesterone metabolism was also compared between normal and sex reversed (sxr) mice. The data suggested that the 178 hydroxysteroid dehydrogenase was more active in normal mice whereas the testes from the sex reversed (Tfm oh”) gave higher conversions to androstenedione. Some conversion to dihydrotestosterone and androstanediol was found in all the testicular incubations.

were normal XY carrying the Tfm gene, i.e., (Tfm d+), Tfm with the modifying (oh”) gene [17], testicular Decreased production of testicular androgens was the feminized, sex reversed (7’fmC(oh’)Blo/++ ‘,sxr/‘~) first suspected abnormality of steroid metabolism in [18] and normal sex reversed (+Ta++/+++,sxr/+J). testicular feminization [l]. Increased conjugation or Normal BALBjC mice were obtained from Health peripheral aromatization has also been proposed [2]. Research Inc. In the studies to date, aberrant activities have been Radiochemicak. All radioactive steroids were purfound in the following enzymes: decreased 3/%hydroxchased from New England Nuclear except for ysteroid dehydrogenase [2,3], decreased 5a-reductase [7c(-3H]-androstenedione (30 Ci/mmol) from Amer[4], decreased 17c+hydroxylase, 17P-hydroxysteroid sham/Searle. The specific activities of the steroids dehydrogenase and increased 20-keto reductase [S]. used were-[4-14C]-androstenedione (58.8 mCi/ There are a number of reports on steroid metabommol), [7a-3H]-testosterone (25 Ci/mmol), [4-14C]lism in testes obtained from patients with this syntestosterone (52.8 mCi/mmol) and [4-14C]-progesterdrome [2-lo], pseudohermaphroditic rat [l l-141 and one (52.8 mCi/mmol). testicular feminized mice 115,161. Testicular steroid Chromatography. Solvent systems (by volume) used metabolism in Tfm mice has not been extensively in chromatography were: Paper Chromatography studied. It was the aim of this investigation to deter(Whatman No. 1): PC-l heptane-methanol-water mine the extent of metabolism of progesterone, (5:4: 1, by vol.); PC-2 heptane-benzene-methanolandrostenedione and testosterone in these mice and water (33:17:40:10, by vol.); PC-3 toluene-light to compare them with other variants and normal petroleum-methanol-water (5 : 5 : 8 : 2, by vol.). Thin male mice. layer chromatography (Silica gel F-254, 0.25 mm thickness, Merck): t.l.c.-1 chloroform-methanol (1: 1, MATERIALSAND METHODS v/v); t.l.c.-2 chloroform-methanol (49:1, v/v); t.l.c.-3 Animals. Mice, 50-190 days old, were maintained benzene-ethyl acetate (2: 1, v/v); t.l.c.-4 benzene-ethyl acetate (13:1, v/v). at the City of Hope National Medical Centre. The Procedures. At the time of sacrifice by decapitation, genotypes were ascertained at this point. Those used blood was collected. Testes were decapsulated and The following abbreviations and trivial names have been minced with scissors. The minced tissue (l&28 mg used: androstenedione = 4-androstene-3,17-dione; testoswet wt) was incubated in 5 ml medium 199 (Gibco). terone = 17p-hydroxy-4-androsten-3-one; dihydrotestosCo-factors were not added. The radioactive steroids terone = 17/J-hydroxy-5a-androstan-3-one; androstaneglycol diol = 5a-androstane-3a,l7b-diol; dehydroepiandroster - were dissolved in two drops of propylene before the addition of medium and tissue. Incubations one = 3b-hydroxy-S-androsten-17-one; estrone = 3-hydroxy-1,3,5(10)-estratriene-17-one; estradiol = 1,3,5(10)-es- were carried out in a Dubnoff metabolic shaker for tratriene-3,17fl-diol: estriol = 1,3,5(10)-estratriene- 3,16cr, 3 h at 37°C with air as the gas phase. At the end of 17gtriol; progesterone = 4-pregnen-3,20-dione; 17x-hythe incubation the medium and tissue were frozen. droxyprogesterone = 17cc-hydroxy-4-pregnen-3-one; pregTissue and medium were extracted with ether and nenolone = 3b-hydroxy-5-pregnen-20-one; NADPH = rethe dried ether extract partitioned between toluene duced nicotinamide adenine dinucleotide phosphate. INTRODUCTION

41

42

.I. D. DAL.I:Y and E. V.

and IN sodium hydroxide to give neutral and phenolit fractions. Following purification by several chromatography steps, the labelled metabolites were recrystallized to constant S.A. with the appropriate carrier steroid before and after derivative formation wherever possible. Corrections for recoveries throughout all the procedures were not carried out. hence conversions must be regarded as minimal. Radioi~nmunos.sa~.s. Serum samples were analysed for testosterone and androstcnedione using established radioimmunoassay procedures on ether extracts of plasma. Testosterone antiserum was purchased from Diagnostics Biochem (Canada) Ltd. and used as previously described 1191. Androstenedione antiserum, Sl557 No. 2, which had a 25”,, cross-reaction with dehydroepiandrosterone, was a gift from Dr. G. E. Abraham, and used as directed. The characteristics of this antiserum has been described [20]. The limits of sensitivity for the assay of testosterone and androstenedione were 20 pg and 5 pg respectively and the inter-assay coefficients of variation for replicate analyses were 9.7”,, and I I”,, respectively. RESULTS

Incuhutions terone

with [3H]-testosferone

cd

[ “C]-proge.v

A total of ten incubations were done with testes from normal male BALB/C, Tfnl o+, and T/in 0”‘. Tissue minces, 10-28 mg wet wt were incubated, per animal, with 0.2 $Ji [ “C]-progesterone and 0.5 &i [3H]-testosterone. Following extraction and partitioning the phenolic fraction was chromatographed on PC-3. Elution of the areas corresponding to estrone and estradiol and recrystallization failed to confirm any conversion to estrogens. The neutral extract was chromatographed on PC-l. Two radioactive peaks with R, values of 0.33 and 0.85 could not be identified. Three other labelled zones had R, values of 0.12, 0.44 and 0.64. The zone with RF 0.12 was chromatographed on t.l.c.-3 which separated testosterone from I7a-hydroxyprogesterone. The material with R, of 0.44 was chromatographed on t.l.c.-2 to separate androstenedione from dihydrotestosterone. Progesterone with R,; of 0.64 Table acetate

YOUN(;L,AI

was further purified on PC-2 After chromatography. each labelled metabolite was recrystallized to constant S.A. before and after derivative formation as far as possible. Recoveries of starting material ranged from 70-KY’, after extraction of the incubates. No evidence was obtained for the presence of conjugates after ether extraction. In Table I are shown representative recrystallization data for testosterone acetate from one incubation for each genotype of mice. Since approximately the same amount of carrier was used it appears that more radioactivity was associated with testosterone in the BALB,IC incubation. Table 2 gives the combined results from the testes incubations with double labels. No significant dillerence was found in the manner by which different genotypes metabolized testosterone. However. progesterone was metabolized differently. The normal mice formed more testosterone than the two types of testicular feminized mice which produced more androstenedione than the normal mice.

Tissue minces (28-46 mg wet wt) were incubated with 0.2 &i [i4C]-progesterone as previously described. The neutral extracts were chromatographed on thin layer plates previously washed in chloroformmethanol (I : 1. v/v), in t.l.c.-2 at 4-C and again on t.l.c.-4 at room temperature. After autoradiography a number of radioactive zones were observed (Fig. 1). The material from zone III was chromatographed on t.l.c.-3 to separate testosterone from 17x-hydroxyprogesterone. All metabolites were recrystallized to constant S.A. with carrier steroids. Unhindered hydroxyl groups were acetylated and the products recrystallized again. The results obtained are shown in Table 3. The recoveries of progesterone were similar in all incubations. Sex reversed mice produced more androstenedione than normal which produced more testosterone. Incubations with [3H]-undroster~etiione The main purpose of this experiment was to determine whether the testes of 7’fl-rimice could aromatize androstenedione. One T/in (o+) and 2 Tfm (0”‘) were used. Incubations were carried out with 1 LtCi

I. Representative recrystallization data for following chromatography and acetylation [‘VI-progesterone

[“YJ-testosterone of extracts with

Steroid metabolism Table 2. Metabolism of [3H]-testosterone

43

and [t4C]-progesterone

‘/*Conversion Genotype

n

“/; Recovery of teStosteTOne

of testosterone to androstenedione

% Recovery of progesterone*

BALB/c Tfm (Of) Tfm (ohb)

6 4 4

25.4 k 2.8 262 * 1.9 19.3 i 2.3*

1.5 f 0.8’ 0.8 + 0.1 0.8 f 0.2

23 3 k 2.8 34 3 k 2.3 13.3 i 1.6

by mouse testes

9<, Conversion progesterone testosterone* 11.0 * 1.3 0.8 * 0.2 2.2 + 0.8

of to

:/, Conversion of progesterone to androstenedione* 0.7 + 0.1 7.6 f 0.8 1.3 *02

Results, mean + S.E.M., are expressed as percent per 1Omg tissue per 3 h. n = number of incubations. *All differences between genotypes are significant at P < 0.05 level using the Student’s “t” test. All steroids were purified by various chromatography steps prior to recrystallization to constant S.A. with carrier steroid before and after derivative formation wherever possible. The percentages were computed from the final specific activities of crystals and mother liquor and the amount-of carrier added. -

[3H]androstenedione. The phenolic fractions were chromatographed on paper in system PC-3. The scans from both genotypes showed a radioactive peak at RF 0.17. This material contained about lo4 c.p.m. and was not identical to estriol. Further characterization was not attempted. Elution of the areas corresponding to estrone and estradiol yielded insufficient material to allow for further processing. With the neutral fractions, Tfm (o+) testes produced mainly androstenedione but Tfm (oh”) had several more metabolites with R,s of 0.09, 0.24, 0.55 and 0.81 which were not identified. The conversions to testosterone were similar by both genotypes (Table 4). Peripheral

that the 17jGhydroxysteroid dehydrogenase enzyme is most active in the BALBjC testes and least in the Tfm (o+). This conclusion is supported by the concen-

androgen levels

Serum from BALBjC mice contained 22.1 + 1.5 ng/ml testosterone while levels were not detectable in Tfm (oh”), or Tfm+(oh’)B/o/+++,sxr. Levels of 1.32 + 0.37 ng/ml were found in Tfm o+ and of 2.29 f 2.0 ng/ml in + Ta + + ‘1’ + +sxr mice. Androstenedione was not detectable in the plasma of any of the different mice. DISCUSSION

The results of the present study confirm and extend the data of previous investigations. New data was derived from incubations with Tfm (oh”) and sex reversed mice and for the metabolism of androstenedione by testes of testicular feminized mice. Endogenous levels of substrates and intermediates were not measured in any of these incubations. Product inhibition and protein binding may have some effect on conversion rates and thus the results of the present incubations can best be interpreted qualitatively because of possible inherent systematic errors. In the double label experiments the most striking differences were found in the relative amounts of testosterone formed from progesterone. Normal BALBjC testes converted progesterone to testosterone in 11% yield while the corresponding conversions by Tfm (o+) and Tfm (oh”) were OX’? and 2.2% respectively. The conversions of progesterone to androstenedione were 7.5% in the Tfm (o+), 1.3% in Tfm (0”“) and 0.7% in BALB/C. These data suggest

Fig. 1. Autoradiogram after thin layer chromatography in t.l.c.-2 at 4°C and t.l.c.-4 at 22”C, of organic extract from testes incubations with [4-‘4C]-progesterone. (a) Tfm + (o”‘)Blo/+++, sxrle6. (b) +Ta++/+++, sxr/+$. (c) BALB/ ‘6. i = origin, ii = androstanediols, iii = testosterone, iv = androstenedione, v = dihydrotestosterone, vi = progesterone, vii and viii-unidentified metabolites.

.I. D.

DALEY

and E. I’ YOUY~LAI

Table 3. Metabolism of [r4CJ-progesterone

by testes of sex reversed

Results are mean & S.E.M. and are expressed as percent per 10 mg tissue per 3 h. cation and percentages were carried out as for Table 2. Table 4. Conversion of androstenedione to testosterone by testes of testicular feminized mice

) T/m(0”‘) r/m (0.

I 3

x25 517

63 5x

Results are expressed as percent per 10 mg tissue. Identification and percentages were carried out as for the data of Table 2.

trations of testosterone found in the sera of these animals and the results of other investigators with Tfm (0’) mice [15], man [lo], and rat [21]. Leydig cells in the tTut+t/+tt,~~r mice in spite of their original XX constitution, must produce sufficient testosterone to maintain development of the wolffian duct and urogenital sinus derivatives [22]. This is supported by the results of the present study. Testes from + Tu+ +/’ + +,SXY mice produced about 60:; as much testosterone as those from normal male mice. The sex reversed mice testes are about one-tenth the size of normal testes and devoid of germ cells. Comparison of the amounts of androstenedione and testosterone produced from progesterone suggest that the 17/%hydroxysteroid dehydrogenase is most active in normal mice and least in the sex reversed carrying the Tfm (oh”) alleles. Thus it would appear that the Tfm (oh”) has some effect on steroid metabolism although the exact mechanism could not be determined. Further evidence that the Tfm (oh‘) influences steroid metabolism is seen in the data of Table 4. In this study the conversion of androstenedione to testosterone was similar but androstenedione was metabolized to more unidentified products by testes from Tfm (oh”) mice. A similar pattern of increased metabolism by the testes of Tfm (oh”) mice was observed when the substrates were testosterone and progesterone (Tables 2 and 3). However, the livers of these animals were less capable of metabolizing testosterone [23]. The lower production of testosterone by the Tfin (ohV) mice therefore appears to be a reflection of increased metabolism at the site of production rather than increased peripheral metabolism. The significance of the larger number of metabolites in the incubations of the Tfm (oh”) testes remains to be determined. Although it was not possible to identify these metabolites at the present time their

17 =

mice

number of incubations. Identifi-

characterization may be of interest in view of the differences which have been described between the ?“jn (o+) and rjin (o”~) strains [22]. Thus it is possible that the metabolites of androstenedione and testosterone may be involved in the altered gonadotrophin feedback response observed in these mice 1241. The purpose of incubating testicular tissue of genetic mutant mice with androstenedione was to determine whether the aromatizing enzyme system in the testes were active. This stemmed from the observation that rat Sertoli cells have the ability to aromatize androgens [25]. The failure to isolate labelled estrogens in the present study could be due to the small amount of radioactivity used, the presence of other cell types and the lack of suitable gonadotrophin stimulation. Isolation of pure Sertoli cells from these genetic mutant mice would be needed to answer this question. Acknowledgemenrs~This work was supported by the Medical Research Council of Canada, MT 4192. The authors are grateful to Dr. A. Clark for gifts of labelled androstanediols, mice.

and to Dr. S. Ohno

for the genetic

mutant

REFERENCES

1. Hauser G. A.: In Irrrersr.~ucllitr (Edited by C. Overzier). Academic Press, New York (1963) p. 255. 2. Morris J. M. and Mahesh V. B.: Am. J. Ohster. Gynrc. 87 (1963) 731-748. 3. Bell J. B. G.: C/in. Endow. 4 (1975) 343-356. 4. Mauvais-Jarvis P., Bercovici J. P., Crepy 0. and Gauthier F.: J. c/in. Incest. 49 (1970) 3140. 5. Bardin C. W., Bullock C. P., Sherins R. J. and Mowszowicz I.: Recent hog. Harm. Res. 29 (1973) 65-109. 6. Griffiths K., Grant J. K. and Whyte W. G.: J. (,/in. Endocr. Metab. 23 (1963) 10441055. 7. Pion R. J., Dignam W. J.. Lamb E. J., Moore J. G., Frankland M. V. and Simmer H. H.: Am. J. Ohsrrr. Gynec. 93 (1965) 1067-1075. E. and Villee C. A.: J. c&n. Endocr. Metuh. 8. Charreau 28 (1968) 1741~1746. 9. Wade A. P., Wilkinson G. S., Davis J. C. and Jeffcoate T. N. A.: J. Endocr. 42 (1968) 391-403. 10. Schindler A. E.: Endokrinologie 67 (1976) 1419. 1 Schneider G. and Bardin C. W.: Endocrirrolog~ 87 (1970) 864873. Coffey J. C., Aronin P. A., French F. S. and Nayfeh S. N.: Steroids 19 (1972) 4333454. Bardin C. W., Bullock L., Schneider G., Allison J. E. and Stanley A. J.: Science 167 (1970) 11361137. 1 Bullock L. and Bardin C. W.: J. steroid Biochem. 4 (1973) 139-151.

Steroid metabolism 15. Blackburn W. R., Chung K. W., Bullock L. and Bardin C. W.: Biol. Reprod. 9 (1973) 9-23. 16. Goldstein J. L. and Wilson J. I).: d. clin. Invest. 51 (1972) 1647-1658. 17. Ohno S., Christian L., Attardi B. J. and Kan J.: Nature New Biol. 245 (1973) 92-93. 18. Cattanach B. M., Pollard C. E. and Hawkes S. G.: Cytogenetics 10 (1971) 318-337. 19. Moor B. C. and YoungLai E. V.: J. reprod. Fert. 42 (1975) 259-266. 20. Abraham G. E. and Chakmakjian Z. H.: J. clin. Endocr. Met&. 37 (1973) 581-587.

45

21. Aronin P. A., Coffey J. C., French F. S. and Nayfeh S. N.: Steroids 24 (1974) 139-150. 22. Drews U., Blecher S. R., Owen D. A. and Ohno S.: Cell 1 (1974) 3-8. 23. Daley J. D., Ohno S. and YoungLai E. V.: Experientia 32 (1976) 1607-1608. 24. Kan J., MacKinnon P. C. B., Ohno S. and YoungLai E. V.: J. Physiol. 242 (1974) 103. 25. Dorrington J. H. and Armstrong D. T.: Proc. Natn. Acad. Sci. U.S.A. 72 (1975) 2677-2681.