In vitro testosterone synthesis by testicular tissue of old mouse: The metabolism of 3β -hydroxy-5-ene steroids

Exp. Geront. Vol. 12. pp. 43-50. Pergamon Press 1977. Printed in Great Britain.

IN VITRO TESTOSTERONE

SYNTHESIS BY TESTICULAR TISSUE OF OLD MOUSE: THE METABOLISM OF 313 - H Y D R O X Y - 5 - E N E

STEROIDS

Z. CHAP* a n d E. BEDRAK Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel (Received 17 September 1976) Abstract--The conversion of 3B-hydroxysteroid 5-ene C-19 and C-21 steroids into testosterone by testicular homogenate was compared in mature (13-weeks-old) and old (41-weeksold) mouse. With all the substrates used, testosterone production in vitro was higher in the old animal. These data indicate that in testicular homogenate from old animals,tbere isahigher rate of activity of those enzymes involved with the biosynthesis of testosterone via the 5-ene pathway (PREG ~ 17a-OHPREG -+ D H A ---*androstendione ---*T). This pathway, unlike the 4-ene route (P ~ 17aOHP ---*A N D --~ T), was not associated with a rate-limiting reaction for the production of testosterone. The 313-reduction-isomerization of pregnenolone, 17a-hydroxypregnenolone and dehydroepiandrosterone appearing to be unaffecting by ageing. On the basis of the present and previously reported data (Slonim and Bedrak, 1975), it can be concluded that the in vitro production of testosterone is greater in the old mouse, and that this androgen is synthesized primarily via the 5-ene pathway.

RECENT studies (Slonim and Bedrak, 1975) have indicated that, in the mouse, advancing age was accompanied by definite changes in the activity of the enzymes associated with the production of testosterone* by testicular tissue via the 4-ene pathway (P -+ 17a-OHP AND -+ T). These changes included increased activity of 21-hydroxylase, 20~-hydroxysteroid dehydrogenase, 17a-hydroxylase and C17-C20 lyase, and a marked decrease in the activity of 1713-hydroxysteroid oxidoreductase (Fig. I). These data suggested too, that in vivo production of T was increased in the old mouse. This could take place if the synthesis ofT via the 5-ene pathway (PREG -+ 17a-OHPREG -+ DHA ~ androstenediol -+T) is enhanced with advancing age. Since T production in the mouse apparently occurs predominantly along the 5-ene route (Ellis and Berliner, 1965), any changes in the metabolization of steroids via this route would be, therefore, more effective in modifying the production of T. We present here, for the first time, a systematic survey of the metabolization of 313-hydroxy-5-ene C-21 and C-19 steroids by testicular tissue of mature and old mice. Furthermore, the data demonstrate that, in most cases when these steroids are used as substrates, the in vitro production rate of T is markedly increased in the old mouse. MATERIALS AND METHODS Animals and preparation of homogenate Newly weaned male mice (C57/BL/6) were obtained from a colony raised at the Weizmann Institute, *In partial fulfilment of requirements for the degree of MSc. *Abbreviation and Nomenclature. PREG, pregnenolone = 3ct-hydroxypregn-5-en-20-one; P, progesterone = pregn-4-en-3, 20-dione; DOC = 21-hydroxypregn-4-en-3, 20-dione; 20a-OHP = 20ahydroxyprogesterone, 20a-hydroxypregn-4-en-3, 20-dione; 2013-OHP = 20B-hydroxyprogesterone, 2013hydroxypregn-4-en-3,20-dione; 17a,20a-diOHP, 17a,20a-dihydroxy-progesterone = 17a,20a-dihydroxypregn-4-en-3-one; 17a, 2013-dihydroxy-progesterone = 17a, 20B-dihydroxypregn-4-en-3-one; 17a-OHPREG, 17a-hydroxypregnenolone = 313, 17a-dihydroxypregn-5-en-20-one; 17a-OHP, 17a-hydroxyprogesterone = l Ta-hydroxypregn-4-en-3,20-dione; D H A = 313-hydroxyandrost-5-en-17-one; AND, androstenedione = androst-4-en-3, 17-dione; T, testosterone = 17B-hydroxyandrost-4-en-3-one; androstenediol = androst-5en-3B, 1713-diol; CHOLES, cholesterol. 43

44

Z. ('HAP AND E. BEDRAK

Cholesterol I 1 i

k

Pregnenolone

Progesterone

°1

17a- Hydro×ypregnenelone - -

De hyd roeplandrosterone

A ndrostenedlol

-

17a- Hydroxyprogesterone - -

L

~

Deoxycor tlcosterone

~

1 7 a , 2 0 a - D~hydroxyorogesterone

1'

_~ A n d r o s t e n e d l o n e

Testosterone

Fl~. 1. Generalized scheme for androgen biosynthesis. Enzymes operating at the various steps are: a - 5ene 17ct-hydroxylase; b = 5-ene 17-20 lyase; c = 5-ene 17B-hydroxysteroid oxidoreductase; d, e, f and g = 3B-hydroxysteroid dehydrogenase and 5-ene isomerase; h = 4-ene 17ct-hydroxylase; i = 4-ene 17-20 lyase; j : 4-ene 17B-hydroxysteroid oxidoreductase; k : 21-hydroxylase; 1 = 20a-hydroxysteroid, oxidoreductase Dashed arrow indicates that more than one step is involved. Rehovoth. The animals were kept in a temperate environment (20-22°C, 30-50 % relative humidity) with a lighting regimen of 14 hr daylight and 10 hr darkness. Tap water and rat-chow were freely available throughout the experiment. Two age-groups were investigated: 13 weeks old (mature) and 41 weeks old (old). At the appropriate age, the animals were killed by severing the spinal cord, and testicular homogenate was prepared as previously described (Chap et al., 1976).

Radioactive steroids, preparation of substrate and incubation The radioactive steroids (obtained from the Radiochemical Centre, Amersham, England) were checked for purity by thin-layer chromatography (TLC) developed in a benzene : acetone (4 : 1, v/v) system. The radioactive androstenediol was prepared by reduction of (14C)DHA with sodium borohydride in darkness for 4 hr. The reduction was stopped by addition of a few drops of acetic acid. The product was dissolved in H~O, extracted four times in dichloromethane and purified by chromatography in hexane : benzene (I : 1 v/v)/formamide. The (14C)-labelled compounds used as substrates were diluted with corresponding nonradioactive steroids (Ikapharm, R a m a t Gan, Israel) to a specific activity (SA) of 5"0 laCi/lamol and the (3H)-labelled compounds to a SA of 25 laCi/lamol. An N A D P H generating system was employed throughout the incubation experiments. Details of the procedure and conditions of the incubation experiments were reported previously (Bedrak and Samuels, 1969). For quantitative assessment of the formation of a specific steroid produced in a one-step reaction, out of a sequence of reactions, it was necessary to trap the radioactive steroid product. This was achieved by introducing into each tube, prior to incubation, 100 lag of non-radioactive trapping agent. The radioactive metabolite formed was thus trapped in the non-radioactive material and so facilitated the quantitative estimation of the conversion of the substrate to the steroid in question. When (~4C) pregnenolone was used as substrate, P and 17ct-OHPREG served as trappers, whereas 17ct-OHP and D H A were employed as trappers when (SH) 17ct-hydroxy-pregnenolone was the substrate. Finally, when (1~C) dehydroepiandrosterone was used as substrate, A N D and androstenediol were employed as trappers. The incubation experiments were carried out as described above.

Extraction, isolation and identification of products After the incubation experiments, the mixtures were acidified with 0.05 ml I N HCL, the tubes were placed in an ice-bath and non-radioactive steroids (reference standards) at the level of 50-100 lag were added. The

In vitro TESTOSTERONESYNTHESISBY TESTICULARTISSUEOF OLD MOUSE

45

tubes were extracted four times with 5 ml ethyl-ether : chloroform (4 : 1, v/v). Recovery at this stage was more than 90~o. The steroids were isolated as previously described (Chap et al., 1976). The isolated steroids were finally identified by recrystallization to constant SA with 20-25 mg of authentic non-radioactive steroids from different solvents (Axelrod et al., 1965). Representative data are shown in Table 1. The yield of each individual compounds was calculated as described previously (Bcdrak and Sarnuels, 1969). Enzymatic activity was estimated by the amount and nature of the product formed upon incubation of individual substrates with the appropriate cofactors (Samuels et al., 1964). Protein was determined by the method of Lowry et al. (1951). TABLE 1. RECRYSTALLIZATIONOF STEROmS TO CONSTANTSPECIFIC ACTIVITY( D P M / M G ) Steroids Original No. of recrystallization solution 1 2 3 Pregnenolone-3-acetate 1181 958 950 942 957 17a-hydroxypregnenolone-3-acetate 624 595 604 615 619 Progesterone 1662 1221 1297 1259 1256 17a-hydroxyprogesterone 720 698 682 700 708 Androstenediol-3B,1713-diacetate 400 376 389 362 361 Androstenedione 1206 1218 1188 1176 1178 DHA-acetate 1534 1438 1426 1405 1400 Testosterone-acetate 915 894 895 874 887

RESULTS

Effects of age on body weight, weight of testes and seminal vesicles Under the prevailing experimental conditions the old mice were heavier and possessed a larger testicular mass than the mature animals. Mean body weights were 23.6 g (mean of 40 animals) and 28.7 g (mean of 30 animals) for the mature and old animals, respectively. Mean testes weight was 0.204 g and 0.227 g for the mature and old animals, respectively. The weight of the seminal vesicles, emptied, was much the same in both groups, i.e. 71 and 69 mg for the mature and old mice, respectively. Metabolism of (14C)pregnenolone by testicular homogenate of mature and old mouse in the presence of NADPH A typical distribution of (Iac) pregnenolone into 5-ene and 4-ene steroidal metabolites, upon incubation of testicular homogenate in the presence of an NADPH generating system, is presented in Table 2. A higher percentage of the substrate was converted to T by testicular tissue of the old animals, i.e. 8.62 and 6.37 % for the old and mature animals, TABLE 2. DISTRIBUTION OF STEROIDS FOLLOWING 20 min INCUBATION OF 20 nmoles (t'C)PREGNENOLONE WITH N A D P H GE~RATV~G SYSTEMAND TESTICULARHOMOGENATEFROM MATUREAND OLD MICE Steroid PREG 17a-OHPREG DHA

Mature ~o Product'i" Distrib.* (66.89) 4.34 0.68 0"56

0'09

Old ~ Distrib. (63.02) 2"44

Product

0'45

0"08

0.44

Androstenediol 0.44 0.07 0"43 0.08 P 8.72 1.36 15.64 2.80 17a-OHP 2.79 0.44 4.27 0.77 AND 1-20 0-19 0-48 0.09 T 6.37 0.99 8.62 1.54 Unidentified 1 3"80 0"59 2-27 0-41 Unidentified 2 4-89 0.76 2.38 0.43 *Per cent distribution of substrate into steroid metabolites. tnmole product formed/mg protein/20 min. Figures in parenthesis represent unmetabolized substrate.

46

z . CHAP AND E. BEDRAK

respectively. The difference between the two age-groups was also maintained when the data was corrected for protein content of the homogenate used in the incubating media, i.e. 1.54 and 0.99 nmol testosterone produced/mg protein/20 rain incubation by testicular homogenate of old and mature animals, respectively. These data also reveal that the conversion of P R E G to its 4-ene homologue, P, by testicular homogenate of the old mouse was greater than that of the mature animal, i.e. 15.64 and 8.72 ~ for the old and mature mouse, respectively. This observation does not necessarily suggest that the activity of the enzyme system 313-hydroxysteroid dehydrogenase, 5-ene isomerase metabolizing pregnenolone is greater in the old animal, but rather that the activity could be a direct result of the rate limiting step in the 4-ene pathway, i.e. the conversion of A N D to T, known to be in the testicular tissue of old mouse. Inhibition of the activity of 1713-hydroxysteroid oxidoreductase would explain the accumulation of 4-ene intermediates such as P and 17a-OHP. The data also indicate that the conversion rate of P R E G to 5-ene metabolites (17~-OHPREG. D H A and androstenediol) was lower in the old animals. This implies that in the old animal there is at least one rate-limiting step in the 5-ene pathway or that the reaction rate at the various steps in the 5-ene route is faster in the old mouse. The latter possibility could account for the lower levels of the 3[3-hydroxy-5-ene metabolites and higher levels of T present at the end of the incubation of testicular homogenate prepared from old mouse. Addition of 100 lag P or 17ct-OHPREG as trapping agents inhibited the 5-ene hydroxylation and 313-reduction of P R E G (Table 3). This can be observed by comparison of the percentages of unmetabolized substrate remaining after the incubation experiments (Tables 2 and 3). It is not wholly certain, therefore, that the 17a-hydroxylation of P R E G is elevated TABLE 3. EFFECT OF TRAPPING AGENTS; PROGESTERONE A N D 17o.-HYDROXYPREGNENOLONE ; ON TFt~ MI-.TAMETABOLISM OF (z4C) PREGNENOLONE BY TESTICULAR TISSUE I N THE PRESENCE OF NADPH

Progesterone 17ct-hydroxypregnenolone Mature Old Mature Old PREG (94'98) (92"92) (88' 11) (95-53) 170t-OHPREG -0'02 0"49 0"02 DHA -0"04 0'03 Androstenediol .... 0"53 -P 0.44 0.93 1.10 0"11 17ct-OHP -0'13 -0"19 AND 0"32 0"05 0"36 0'04 T 0"23 0"07 0'37 0'40 Unidentified 1 -0"02 0.02 Unidentified 2 -0.02 -0-02 Figures in parenthesis represent unmetabolized substrate at the end of 20 rain incubation. Other values represent nmol product formed/rag protein/20 rain incubation. Mean of two experiments. Steroid

in the mature animals, although we found that 0-49 and 0-02 nmol 17ct-OHPREG were produced by mature and old animals, respectively. Similarly, despite the values found for the 313-reduction isomerization reaction of P R E G in the old and the mature mice namely, 0.93 and 0.44 nmol of P, respectively (Table 3), it cannot be categorically stated that this reaction is enhanced in old animals. Additional and different techniques should be employed to confirm these findings.

Metabolism of (3H)17ct-hydroxypregnenolone by testicular homogenate of mature and old mouse in the presence of NADPH Testicular preparation from old animals metabolized (14C)17ct-OHPREG faster than did

In vitro TESTOSTERONESYNTHESISBYTF-.STICULARTISSUEOF OLDMOUSE

47

that of mature mice. After 40 min of incubation 45.52 ~ of the substrate remained unmetabolized when incubated with testicular homogenate of old animals as compared with 60.07 ~o for mature mice (Table 4). The production rate of 5-ene steroids by testicular homogenate of old animals is several fold greater than that of mature mice, i.e. the per cent of the substrate converted to D H A and androstenediol combined, was 7.37 ~o for the old and 1.52 for the mature animals. The same trend was observed when the data were corrected for protein content of the incubating media. On the other hand, the conversion of the substrate to 17u-OHP was lower (2.87 ~ ) in the old mice than in the mature animals (4.75 ~o). The levels of A N D and T present at the end of the incubation experiments were relatively equal in both groups. These data imply that the activity of the 313-hydroxysteroid dehydrogenase5-ene isomerase system converting 17a-OHPREG to 17a-OHP is lower in the old animals. In contrast, the activity of 5-ene 17-20 lyase appeared to be higher in the old animals. The results obtained in the experiments in which 17a-OHP and D H A were used as trapping agents (Table 5) supports the conclusion drawn after incubating the substrate without trapping agents (Table 4). In the presence of 17a-OHP as a trapping agent, the conversion rate of (3H)17a-OHPREG to 17a-OHP is markedly lower in the old than in the TABLE4. METABOLISMOF( ' H ) 17~-HYDROXYPREGNENOLONE WITHNADPH GENERATINGSYSTEMAND TESTICULARHOMOGENATEFROMMATUREANDOLDMICE* Mature Old Steriod ~ Product ~o Product Distrib. Distrib. 17a-OHPREG (60.07) (45.52) 17a-OHP 4-75 0.74 2.87 0"51 DHA 1.52 0.24 6.70 1'20 Androstenediol --0"67 0"12 AND 3.58 0.56 3.72 0"67 T 30.08 4.70 26.92 4"82 Unidentified --13.60 2"44 *Incubation experiment carried out for 40 min, for legends see Table 2. TABLE5. EFFECTOFTRAPPINGAGENTS,17~t-HYDROXYPROGESTERONEANDDEHYDROEPIANDROSTERONE,ONTHE METABOLISMOF 17~-HYDROXYPREGNENOLONEBYTESTICULARTISSUEIN THEPRESENCEOF NADPH 17a-hydroxyprogesterone Dehydroepiandrostrone Steroid Mature Old Mature Old 17a-OHPREG (73.36) (51"09) (77.54) (60.80) DHA 0"13 1"00 1'98 3"04 Androstenediol -0'04 -0"25 17a-OHP 2"56 0'92 0"57 0"19 AND 0"43 0"47 0-19 0"11 T 1"04 2"59 0"77 1'65 Unidentified -1'94 -1"95 Figures in parenthesis represent unmetabolized substrate at the end of 40 min incubation. Other values represent nmole product formed/rag protein/40 min incubation. Mean of two experiments. mature animal, being 0.92 and 2.56 nmol/mg protein/20 min incubation, respectively. This is a clear indication that the 313-reduction-isomerization reaction of 1713-OHPREG in the old mouse is lower than in the mature animal. The increased activity of 5-ene-17-20 lyase in the old animal can be deduced from experiments in which D H A was used as a trapping agent. Here the conversion o f the substrate to the combined 5-ene intermediates D H A and androstenediol was 1.98 and 3.29 nmol/mg protein/40 min incubation, for the mature and old mice, respectively (Table 5).

48

z, CHAP AND E. BEDRAK

Metabolism of 3~-hydroxy-5-ene C-19 steroids by testicular homogenate in the presence o]" NADPH When DHA is used as a substrate, T can be produced by a two-step reaction through either the 5-ene (DHA ~ androstenediol ~ T) or the 4-ene route (DHA -> AND -+ T). In this experiment, however, supplementing the reaction media with cofactor NADPH, favours the production of T via the 5-ene pathway. This two-step reaction involves several enzymes, i.e. the 5-ene 1713-hydroxysteroid oxidoreductase and 313-hydroxysteroid dehydrogenase-5-ene isomerase system. The production rate of T from DHA by testicular homogenate prepared from old animals is higher by 32.8 ~ than that obtained from mature mice (Table 6) (0.89 nmol/mg protein/l 5 min incubation for old animals as against 0-67 nmol/mg protein/15 min incubation for mature ones). TABLE 6. METABOLISMO F (14C) DEHYDROEPIANDROSTERONEWITH NADPH GENERATINGSYSTEMAND TESTI('ULAR HOMOGENATE FROM MATURE AND OLD MICE* Mature Steroids DHA Androstenediol AND T Unidentified

% Distrib. (69"96) 23" 72 2-04 4'28 --

Old Product

3.70 0.32 0.67 --

~ Distrib. (69'26) 20' 54 3'22 4"90 2"08

Product

3' 68 0.58 0'89 0'38

*Incubation experiment carried out for 15 min, for legends see Table 2.

To differentiate between the activity of the 17fl-hydroxysteroid oxidoreductase and that of the 313-hydroxysteroid dehydrogenase-5-ene isomerase systems, trapping experiments were introduced. The use of androstenediol as a trapping agent indicated that advancing age is associated with enhanced activity of 5-ene 1713-hydroxysteroid oxidoreductase. In these experiments, the conversion of DHA and androstenediol by testicular homogenate of mature and old animals was respectively, 2.95 and 3.13 nmol/mg protein/15 min incubation (Table 7). Similarly, the activity of the enzyme system participating in the conversion of androstenediol to T, i.e. the 313-hydroxysteroid dehydrogenase-5-ene isomerase system, was TABLE 7. THE EFFECT OF TRAPPING AGENTS; ANDROSTENEDIONEAND ANDROSTENED1OL; ON THE METABOLISM OF DEHYDROEPIANDROSTERONEBY TESTICULAR HOMOGENATEIN THE PRESENCE OF N A D P H Steriods DHA Androstenediol AND T Unidentified 1 Unidentified 2

Androstenedione Mature Old

Androstenediol Mature Old

(95'41) 0.21 0"26 0'25 ---

(77'56) 2'95 0"28 0'27 ---

(90.81) 0-52 0'41 0'11 0.61 --

(66'71) 3"13 0' 16 1" 11 0.37 1"20

Figures in parenthesis represent per cent unmetabolized substrate at the end of 15 min incubation. Other values represent nmole p r o d u c t formed/rag protein/15 min incubation. Mean of two experiments.

increased in the old mouse (Table 8). It is clearly demonstrated (Table 8) that the activity of this enzyme system in the old animal surpassed by about 30.45 ~ that observed in the mature mice, being, respectively, 5.74 and 4.40 nmol T produced/mg protein 30 min incubation.

In vitro TESTOSTERONE SYNTHESIS BY TESTICULAR TISSUE OF OLD MOUSE TABLE 8. METABOLISM o r

(a4C) ANDRO~TENEDIOL W I T H NADPH GENERATING

49

SYSTEM AND TESTICULAR HOMO °

GENATE FROM MATURE A N D OLD MICE*

Mature Old ~ Product ~ Product Distrib. Distrib. Androstenediol (71.80) (58.38) Testosterone 28.20 4"40 41.62 5-74 *Incubationexperimentcarried out for 30 min, for legendssee Table 2. Steroid

The incorporation of AND as trapping agent with (14C) dehydroepiandosterone as substrate induced, irrespective of the animal's age, a marked inhibitory effect on the activity of the 313-hydroxysteroid dehydrogenase-5-ene isomerase system metabolizing DHA. This can be observed by comparing both age groups for the amount of unmetabolized substrate remaining after incubation (Tables 6 and 7). This fact makes it impossible to estimate exactly the activity of the enzyme system involved in the conversion of DHA to AND. DISCUSSION The data presented here indicate differences between mature and old mouse with respect to the activity of several NADPH-dependent enzymes which are associated with the production of T via the 5-ene pathway. The activity of these enzymes operating in steps a,b,c and g (Fig. 1) changes with advancing age in a different manner from the corresponding steps (h, i and j) operating in the 4-ene route (Slonim and Bedrak, 1975). The conversion from the 5-ene to the 4-ene route can occur at various stages along the pathway of synthesis, and are marked here as steps d, e and f (Fig. 1). In mature mouse the synthesis of T is primarily carried out via the 5-ene route (Ellis and Bediner, 1965). It can be assumed that the in vivo output of androgens is dependent upon the activity of the enzymes operating in the gonads. Hence, a deviation in the action of one or more enzymes participating in androgen synthesis via the preferred route will evoke a reduction in T concentration in peripheral blood. That this is the case in the rat, where T is primarily produced via the 4-ene pathway (Slaunwhite and BurgeR, 1965), was shown previously (Amatayakul et al., 1971 ; Bedrak e t a / . , 1973). Likewise, the lower activity of 4-ene 17-20 lyase (step i, Fig. 1) observed in testicular tissue of a 61-year-old man (Axelrod, 1965) would offer a partial explanation for the lower level of plasma T which occurs in old man (Vermeulen et al., 1971). As already mentioned, the synthesis of androgens in the mouse is primarily carried out via the 5-ene route (Ellis and Bediner, 1965) involving reaction steps a, b, c and g (Fig. 1). Hence the reduction in enzyme activity in the 4-ene pathway which accompanies ageing (Slonim and Bedrak, 1975), would not imply a concomitant reduction in T concentration in the peripheral blood. Significant changes in the in vivo production of T would rather stem from alteration in the activity of the enzymes participating in the synthesis of the androgen via the 5-ene route. This would be true if the conversion of the 5-ene to the 4-ene steroids was not affected by advancing age. Indeed, our data do not indicate that ageing is coupled with an increased conversion rate of 313-hydroxy 5-ene intermediates into their 4-ene homologues. Consequently, any alteration in the production rate of T should be associated with changes in the activity of enzymes involved in the biosynthesis of the androgen via the 5-ene route. The results of the work presented here indicate that, in the old mouse, the activity of the enzymes participating in T production via the 5-ene pathway is higher than in the mature animal (steps a, b, c and g, Fig. 1). Our results confirm those of Ellis and Berliner (1965), namely, that in the presence of NADPH, androstenediol is an

50

Z. CHAP AND E. BEDRAK

obligatory intermediate between D H A and T. Moreover, they demonstrate that, unhke the 4-ene route, ageing is not associated with a rate limiting reaction for T production via the 5-ene pathway. The present data together with previously reported information (Slonim and Bedrak, 1975), indicates that the synthesis of T in the old mouse is greater than in the mature animal, and that more androgen is produced via the 5-ene route. The validity of using in vitro experiments for interpreting in vivo conditions has been questioned (Matsumoto and Samuels, 1969). However, in addition to being essential to fundamental knowledge of the subject, these data also have tangible advantages in that those factors which cannot be controlled in vivo, are controlled in incubation experiments. The data presented here demonstrate the capacity of the endocrine gland to produce, but can only suggest what actually takes place in the intact animal. Supplementary data obtained from in vivo trials, in mice of varying ages up to the end of senescence, i.e. death, coupled with double labelling experiments, are essential for comprehensive understanding of the relative roles of the 5-ene and 4-ene routes in the production of T during advancing age. REFERENCES AMATAYAKUL, K . , RYAN, R., UOZUMI,T. and ALBERT, A. AXELROD, L. R. (1965) Biochim. biophys. Acta 97, 551.

(1971) Endocrinology 88, 872.

AXELROD,L. R., MATTHIJSSEN,C., GOLDZICHER,J. W. and PULLIAM,J. E. (1965) Acta Endocr. 49, Supp. 99, 1. BEDRAK,E. and SAMUELS,L. T. (1969) Endocrinology 85, 1186. BEDRAK,E., SAMOILOFF,V. and SoD-MORIAH,U. A. (1973) J. Endocr. 53, 207. CHAP, Z., BEDRAK,E. and SoD-MORIAH,U. A. (1976) Israel J. rned. Sci. In press. ELLIS,L. C. and BERLtNER,D. L. (1965) Endocrinology 76, 591. LOWRY,O., ROSEBROUGIq,N. J., FARR,A. I. and RANDALL,R. J. (1951) J. Biol. Chem. 193, 265. MATSUMOTO,K. and SAMUELS,T. (1969) Endocrinology 85, 402. SAMUELS,L. T., SNORT,G. J. and HUSEBY,R. A. (1964) Acta Endocrinol. 45, 487. SLAUNWHtTE,W. R. JR. and BURGETT,M. J. (1965), Steroid 6, 721. SLONIM,A. and BEDRAK,E. (1975) 5th Int. Conference of Endocrinology, London, p. 72. VERMEULEN,A., RUBENS,B. and VERDONCH,L. (1971) ,4cta Endocr. 67, Supp. 155, 63.