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Progesterone metabolism in vitro by testes from germ cell-free mice of different ages
127
Bioehimica et Biophysics @ ~~~r~No~h-Holl~d
Acta, 486 (1977) BiomedicaI
127-135
Press
BBA 56903
PROGES~RO~E MET~OL~~ CELL-FREE MICE OF DIFFERS
MASAHIRO KITAMURA
IN VITRO BY TESTES FROM GERM AGES
KUWATA *, MORIO YAMADA b, MASAMITSU c and KEISHI MATSUMOTO d
TAMAI c, YUKIHIKO
a Research ~~tit~te for ~~c~b~~ ~~~, &aka Uni~~i~, b ~~~rnent of Patho~o~, Hyogo College of Medicine, and c Department of Pathology and d Institute for Cancer Research, Osaka University Medical School, Osaka (Japan) (Received March 2nd, 1976) (Revised manuscript received September
8th, 1976)
Androgen biosynthesis in vitro by testes from (C57BL X WN)Fi-WV/W” (congeni~y germ cell-free) and +/+ (normal) mice of different ages was investigated using [ 3H) progesterone as substrate. At all ages examined, the seminiferous tubules of the W/W” genotype were largely devoid of germ cells. However, the age dependent pattern of progesterone metabolism by germ cell-free testes was similar to that by normal testes. Conversion of progesterone to bareduced 17-hydroxy-C?i and C&-steroids such as 3a,l?a_dihydroxy-fia-pregnanQOsne,
ages [ 2 J . However, androgen biosyn~es~ in the germ cell-free testes has not been clarified. Although the activity of 5a-reductase for A’-3-ketosteroids began to fall after 40 days of age in testes of normal rats and mice [3-81, the activity remained persistently elevated in testes of Stanley-Gumbreck pseudohermaphrodite rats [9,10]. The seminiferous tubules of the pseudohermaphrodites had a few cells in the diplotene stage at 51 and 60 days, but at 140 days most of the sperm cell elements were gone [lo]. These findings seem to indicate that testicular Fiar-reductase activity changes with age in such a fashion as to suggest an association with the differentiation of germ cells present in the testes. This possible association was examined in (C5’7BL X WN~-F~-W~~Wn (congeni~ly germ cell-free) mice of different ages. However, this hypoth~~ is not supported by the data presented, since the formation of 5rr-reduced metabolites from progesterone in testes of different ages of mice was not influenced by the presence of germ cells in the seminiferous tubules. Materials and Methods Animals. The BWNF, hybrid male mice of various ages, 20,28,35, 52,120 and 150 days, were raised in our laboratory. Parent mice were C57BL-WY/+ and WN-W”/+. C57BL/6J-WI+ mice were purchased from the Jackson Laboratory (Bar Harbar, Maine, U.S.A.) and then raised in our laboratory by repeated backcrossing with our C57BL mice. The WN strain was es~blished as an inbred strain at Nagoya University [ll], and obtained through Dr. Nishimura, Wakayama Medical College. A cross between these two inbred strains results in congenic animals, designated BWNF1, with four possible genotypes at the W locus, +/+, WV/+, Wn/+, W/W*. These W series genotypes can be recognized on the basis of coat color: +/+ mice are all black; W/+ are grey with a white belly spot; W”/+ are black with numerous white spots over the whole body; and WV/ W” are black-eyed white-coated. W/W* male mice were used as germ cell-free mice and +/+ male mice as control animals. In one experiment, 4 normal and 4 germ cell-free mice were given human chorionic gonadotropin plus pregnaut mare’s serum gonadotropin subcutaneously (10 I.U. of each hormone) each day, for 7 or 12 days. The gonado~opin treated and control mice were killed 28 days after birth. histological technique. The testes were examined as previously reported 1121. Chemicals. [ 1,2-3H] progesterone (45 Ci/mmol), obtained from Daiichi Pure Chemical Co Ltd. (Japan) was purified by paper chromatography using the hexane/formamide system [13] just before use. The radiochemical purity of the purified [ 3H] progesterone was 99% by repeated crystallizations. Furthermore, the purified [3H] progesterone behaved as a single compound in paper Cl33 and column [14] chromatographies and there was no or very small amounts of other radioactive steroids (less than 0.2% of each) in 13 steroid fractions (Tables 11). Non~dioactive steroids were obtained from Steraloids; Inc. and Ikapharm. Pregnant mare’s serum go~ado~op~ and human chorionic gonadotropin were obtained from Teikokuzoki Co. Ltd. Japan. Other reagents were of analytical grade.
129
Incubation procedure. The testes obtained from 2-8 mice of different ages (Table I) were weighed and were homogenized in 0.25 M sucrose containing 1 mM EDTA. The purified [‘HI progesterone (1 nmol : 1 j&i per vessel) was introduced into 2 X 10 cm tubes and dissolved in 0.02 ml ethanol. To each tube, 0.5 ml of buffer co-factor solution was added. The buffer co-factor solution consisted of 0.3 M potassium phosphate buffer (pH 7.4), 0.06 M nicotinamide, 2 mM MgClz and 2.5 mM NADPH. 0.5 ml of the tissue homogenate containing 0.4-30 mg of tissue was then introduced to make the total volume of the incubation mixture 1.0 ml. The samples were incubated in a shaking water bath in air at 34°C for 30 min. At the end of incubation, the mixtures were immediately acidified with 0.1 ml 1 M HCl and mixed with 3 ml of ether to stop the reaction. In all incubations, the metabolism of progesterone was examined in duplicate for each condition tested. Mean values of the two tubes are used throughout this report except in isolated instances where a sample was lost during the procedure. Analysis and identification of steroids. To the incubation mixtures, 5-50 pg quantities of the 14 steroids shown in Table II together with 2Ocu-hydroxypregn4-en-3-one and 17a-hydroxy-5e-pregnane-3,2Odione were added as nonradioactive carriers. The extraction of steroids from the incubation mixtures and the analysis of these 16 steroids by paper [ 131 and elution [ 141 chromatographies with acetylation of steroids were the same as previously described [6,15] . The percentage of the total radioactivity on the first chromatogram present in each zone was calculated. This percentage was distributed among th.ose compounds which showed significant radioactivity, according to the proportion of each compound present, as indicated by succeeding chromatograms. Finally, all the purified radioactive steroids or their acetates except for 3-hydroxy&pregnan-20-one, 17e-hydroxy-5a-pregnane-3,20-dione and 3cr,l7cy-dihydroxy5ar-pregnan-20-one were repeatedly crystallized with lo-15 mg nonradioactive TABLE I MEAN WEIGHT FREE MICE
OF BODY,
TESTES
AND SEMINAL
VESICLES
Normal
Age
No. (days) 20 28 35 62 120 159 28 (G X 7) * 28(GX 12):
3 8 5 2 3 2 2 2
OF NORMAL
AND GERM CELL-
Germ cell-free Body
Testes
Seminal vesicles
(B)
(me)
7 12 19 23 23 27 11 13
38 69 92 206 140 I83 42 63
No.
Body
Testes
Seminal vesicles
(ms)
(9)
(mr)
(mr)
2 6 28 104 103 118 30 58
6 9 9 14 28 30 6 11
10 15 12 17 31 36 15 24
2 4 6 14 130 119 16 60
* Mice were given humau chorionic gonadotropin PIUS premmnt mare’s serum gonadotropin subcutaneously (10 1.U.. of each hormone) each day for 7 or 12 days. The mice were killed on the day of the last injection (aged 28 days).
II
FORMATION
OF [3H]STEROIDS
FROM
[3H]PROGESTERONE
BY TESTICULAR
HOMOGENATES
8.1 38.6 1.8 5.4 1.5 2.6 4.5 4.1 20.5 67.7
9.1 31.0 3.1 6.2 2.6 6.5 2.1 6.1 26.6 66.7
2.1 2.1 <0.2 0.0 0.0
Androstenedione Testosterone 5a-Androstane-3.17-dione ll&OH-5a-Androstan-3-one Androsterone 3&OHda-Androstan-ll-one Sa-Aadrostane-3 a.1 1 @dial ScY-Androstane-3 p.11 &diol Total 5&-H-C* 9 steroids TotaI C 19 steroids -
2.4 4.8 0.1
2.4 1.3 1.3
1.9 <0.4 0.0
11~Hydroxyprogesterone 3a.17a-OH-5a-Pregnan-2O-one 38.1Ta-OH-5a-Pregnan-20-one
6.5 <0.3 <0.5
35
8.1 0.7 1.8
28
79.0 2.8 4.8
20
Progesterone (unchanged) 5cu-Pregnane-3.2O-dione 3-Hydroxy-5a-pregnan-20-one
Days after birth:
Normal mice
10.0 <0.2
0.1 <0.2 0.0 6.5 71.7 <0.2 1.3 0.2 0.5 1.3 1.4 4.1 88.9
120
4.3 <0.2 <0.4
52
6.5 5.5 0.0 0.0
6.4 0.7 0.0
53.4 3.2 12.8
20
mice
10.3 36.5 2.2 3.7 2.0 5.6 2.2 6.0 21.1 68.5
1.1 4.5 0.6
8.9 0.3 1.1
28
Germ cePfree
9.1 49.0 0.8 2.5 1.9 2.4 5.3 3.2 16.1 14.2
2.6 4.7 0.0
4.3 <0.4 2.7
35
Testicular homogenates prepared from 30 mg of testes of mice of aII ages employed were incubated with [3H]progesterone at 34Oc for 30 min in 1 ml. Values were obtained after 4 repeated crystaIIizations to constant specific activity.
FREE MICE
PERCENTAGE
TABLE
6.2 66.1 <0.4 2.1 0.1 <0.6 4.7 1.9 9.4 81.7
0.3 1.1 0.0
2.6 <0.2 co.4
52
(1 nmol
FROM
:
AND
GERM
CELL-
69.6
10.0
0.2 <0.5
6.3 <0.2
120
1 /Xi per vessel) and NADPH
NORMAL
131
standard steroids to constant specific activity in order to identify the steroids formed. 3-Hydroxy-Be-pregnar&O-one, i7cu-hy~~~~Ekr-pi;egnane-3,2Odione and 3a ,17adihydroxy-5a-pregnan+ZO-one were $&&vely. identified by derivative formation (acetylation and oxidation with &hronWn :t&&ii) ad rttpbd crystallizations of the derivatives formed with chromium~tkioxide to, cerktant specific activity, as previously described [15] ; lhal evidence of identification or tentative identification required that the specific activities of three consecutive crystallizations were within +5% of the mean. The amount of each steroid on the final chromatogram was corrected by the decrease of specific radioactivity on 4 repeated crystallizations to constant specific activity, which was mostly less than 20%. The above procedure for calculating the rate of formation of metabolites can permit an approximate estimation of the percent conversion of [3H] progesterone, which seems to be of use for the present purpose. Results Testes and seminal vesicles of normal and germ cell-free mice of different ages
The mean weight of body, testes and seminal vesicles of normal and germ cell-free mice used in the present incubation studies are shown in Table I. At all ages examined, the weight of testes of germ cell-free mice was much lower than that of normal mice. On the other hand, the maximum seminal vesicle weights at 120 and 150 days were similar in the two groups of mice. However, between 28 and 52 days, seminal vesicle weights were greater in normal than in germ cell-free mice. The increase in the weights of seminal vesicles was observed in 28day-old mice receiving gonadotropin. The results of the microscopic examination in normal mouse testes were as follows. At 52 days, full spermatogenesis was noted (Fig. 1). Earlier stages (not shown) revealed the following: at 20 days, primary spermatocytes, including pachytene spermatocytes, occupied most parts of the tubules with a few
Fig. 1. Photomicrograph of a section from 62-dau-old normal mouee testis (X600). posed of seminiferous tubules and.interstitial cells.
The section is com-
132
Fig. 2. (X 500, aged 62 days) is representative of the histological appearance of germ cell-free mice of all ages examined. It is seen that no germ cells are present in the seminiferous tubules which contain a single layer of Sertoli cells.
immature spermatids; at 28 days, the immature spermatids had increased with a few mature spermatids; at 35 days, the mature spermatids were increased. The results in the germ cell-free mice were as follows. At all ages examined, each tubule consisted of a single layer of Sertoli cells (Fig. 2). The testes were largely devoid of germ cells’and very few germ cells in a very few tubules were always spermatogonia. No differences were noted among both groups of mice in morphology of interstitial tissue (Figs. 1 and 2). The microscopic findings of testes from the present W/W” mice were similar to those reported previously in testes from W/W mice [2]. Progesterone metabolism different ages
by testes from germ cell-free and normal mice of
The percentage formation of [3H] steroids from [3H] progesterone by testicular homogenates from germ cell-free and normal mice at different stages of development is shown in Table II. 30 mg tissue were incubated with 1 nmol of [3H] progesterone and NADPH for 30 min in 1 ml. Conversion of progesterone to 5cu-reduced 17-hydroxy-Czl and C19-steroids such as 3ar,l7adihydroxy5ol-pregnan-20-one, androsterone, 3/3-hydroxy-5c-androstan-17-one, Eia-androstane-3a,l7P-dial and 5a-androstane-3fiJ’Ipdiol was signific&t in testes of 2% and 35-day-old mice of both groups, germ cell-free and normal mice. However, these 5a-reduced metabolites were found to be formed in very small amounts or not formed at all in testes of 20- and 120-day-oM mjce of both groups, in which conversion of progesterone to l’lar-hy&ox terone, androstenedione and testosterone was observed. Conversion of proge&terone to 5cy-reduced steroids was low at 52 days of age in both groups. Since substrate progesterone was almost completely consumed in most of the incubations shown in Table II, the following incubations by testes from 35- and 15Oday old mice of both groups were attempted.
133 TABLE EFFECT
III OF GONADOTROPIN
ONE BY 39 MG TESTICULAR
ADMINISTRATION HOMOGENATES
ON IN VITRO FROM
PI-DAY-OLD
METABOLISM NORMAL
OF PROGBSTERAND
GERM
CELL-
FREE MICE TestIcuIar homogenste wss Incubsted with [3H]progcste?Onc (1 run01 : 1 ~ICI per VWseI) snd NADPH Pt 34OC for 30 mIn in 1 ml. V&es were obtaIned after 4 repeated crystsIBsatIons to constant sppsciiicsctkity. Results represent amounts formed or remshdng In pmol. Mice were given human chodonic gonsdotropin plus pregnant mare’s serum gonadotropin subcutaneous& (10 I.U. of each hormone) each day for 7 or 12 days, starting from 22 or 17 days after birth. The mice were kIIIed on the day of the Isst injection. aged 28 days. -
---
Days after treatment:
-Germ cell-free
NormsI
-
0
7
12
0
7
Progesterone (unchanged) 6a-Reduced P * 17a-Hydroxyprogesterone 6a-Reduced 17.OH-P * * Androstenedione Testosterone 6a-Reduced CI c steroids
33 99 26 201 28 147 337
26 10 1 16 8 71 767
26 13 1 33 11 117 663
69 22 11 61 103 366 222
63 18 1 12 41 326 461
(6a-H-steroids/total)
(0.66)
(0.91)
(0.84)
(0.32)
(0.66)
l **
-
12 33 11 1 21 16 106 700 (0.86)
* 6a-Reduced metsboIItes of progesterone. ** ba-Reduced metsholites of 17a-hydroryprogesteroone. *** Ratio of 6a-reduced Cl9 steroids to totsI CI9 steroids.
When homogenate8 from 1.5-6.0 mg of normal testes or those from 0.43.0 mg of germ cell-free testes were incubated with [ 3H] progesterone (1 nmol : 1 @i per vesel) and NADPH at 34°C for 30 min in 1 ml, more than half of the substrate progesterone remained unchanged and the rates of production of total of C19-steroids or total of 17.hydroxy-Cz,-steroids plus C1&eroid8 were roughly proportional to the weights of each tissue used. Conversion of progesterone to 5a-reduced l’ldeoxy-C,,-, 17.hydroxy-C2, and C19-steroid8 wa8 low but significant in testes of 35day-old mice of both group8, but these 5areduced metabolites were not formed at all in testes of 150day-old mice of both groups (data are not presented). However, conversion of progesterone to 17a-hydroxyprogesterone, androstenedione and testosterone was similar in testes of prepubertal and adult mice. There were no significant differences in the ratios of the sum of 5a-reduced C1&eroid8 to total C.&&eroids (approx. 0.1) and those of the sum of Sa-reduced 17.hydroxy-CzI-steroids to total 17. hydroxy-C,l-steroids (approx. 0.1) between prepubertal testes from germ cellfree and normal mice. These findings indicate that the age dependent pattern of progesterone metabolism by congenitally germ cell-free testes was similar to that by normal testes. The formation of products from [3H] progesterone by 30 mg of testicular homogenates from control and gonadotropin injected 28day-old mice is shown in Table III. Control mice were given saline each day for 12 days, starting from 17 days after birth. Gonadotropin injected mice were given gonadotropins each day for 7 or 12 days, starting from 22 or 17 day8 after birth. The mice were killed at 28 days of age, on the day of the last injection. Conversion of progesterone to 5o-reduced C,&eroids wa8 found to be high in testes of 23day-old mice of both groups receiving saline (control) and those receiving gonado-
134
tropins. Treatment of immature mice with large amounts of gonadotropins for 7 and 12 days did not reduce but increased the conversion of progesterone to 5a-reduced steroids relative to A4-3-ketosteroids in testes of 28day-old normal and germ cell-free mice (Table III). As shown in Table II, the formation of 5areduced steroids in testes of adult mice (52 and 120 days of age) of both groups was much lower than that in testes of prepubertal mice (28 and 35 days of age) indicating that there was a reduction of 5e-reductase activity in normal and germ eel&free testes during maturation of the tests. These findings seem to suggest that there is an increase of FicY-reductaseactivity in prepuce testes stimulated by gonadotropins and that the influence of age upon the reduction of 5e-reductase activity in testes of both groups does not results from the action of gonadotropins. However, further studies on the effects of hypophysectomy and administration of gonadotropins in normal and germ cell-free mice of different ages upon the activity of testicular 5a-reductase are necessary in order’ to clarify the mechanism of age dependent change of testicular 5areductase . Discussion The present results shown in Table II derno~~~ that prog~~rone is actively metabolized to C,&eroids in vitro in testes of (C57BL X ~)F,-WV/ W” (germ cell-free) mice, regardless of the age of mice and that the characteristic age-dependent pattern of progesterone conversion to &-reduced- and A4-3ketoG&eroids in testes of W/W” (congenitally germ cell-free) mice is similar to that in testes of +/+ (normal) mice. This age dependent pattern of progesterone metabolism shown by testes of (C57BL X WN)F, mice is in agreement with a previous report on testes of albino mice of the d.d. strain [ 7 ] . To the best of our knowledge, the present results demonstrate for the first time the agedependent formation of ficu-reduced C,&eroids in congenitally germ cell-free testes. These results seem to indicate that this age-dependent change in progesterone metabolism is unrelated to germ cell development in the ~rn~~erous tubules and that the persistently high activity of 5e-reductase in testes of adult Stanley-Gumbreck pseudohermaphrodite rats [9,10] may not be due to lack of germ cells in the seminiferous tubules. . Folman et al. [16] and Matsumoto and Yamada [17] reported that in prepubertal rat testes, tja-reduction of testosterone in vitro in the presence of NADPH was much higher in the in~~titi~ tissue than in the tubules. However, Rivarola et al. [18] reported an opposite result by incubation of isolated tubules and interstitial tissue from prepubertal rats in the absence of NADPH. This discrepancy may be due to a deficiency of co-factors in the interstitial tissue isolated by Rivarola et al. [18] . Since the age-dependent formation of Sa-reduced &#eroids from progesterone was found to be unrelated to the presence of germ cells (Table II) and the 5c~reductase was much higher in the interstitial tissue than in the seminiferous tubules in prepubertal rat t&m [ 16,171, this pattern of metabolism in testes seems to represent agedependent change in androgen biosynthesis by the interstitial cells. However, the contribution of Sertoli cells present in testes of both groups of mice cannot be ruled out in the present study. Although 5cr-reductase in the seminiferous tubules of
136
prepubertal rats was shown to be distributed mainly in germ cells [12,19],
a
recent study demonstrated the presence of Ew-reductase in Sertoli cells isolated from prepubertal rat testes [20]. Contrary to the re8ulte ahown in Table II, the results shown in Table III demonstrate that in prepubertal mice, the germ cellfree t~&.es produce more androstenedione and kto8terone and less k-reduced C,+&eroids than normal testes. It is possible that this may be due to the presence of !5a-reductase in germ cell8 [12,19,20]. Although the activity of 5areducta8e wa8 shown to be much higher in the interstitial tissue than in germ cell8 in prepubertal rats [ 12,16,17] , this activity in germ cell8 wa8 still found to be similar to that in the ventral prostate [12]. The data in Table II appear to support the present statement that the pattern of progesterone metabolism is approximately the 8ame in testicular preparations from germ cell-free and normal mice of similar ages. However, the percentage of conversion per unit weight a8 shown in Table II could be misleading. The population of cell8 in homogenate8 prepared from immature or adult germ cell-free testes consist primarily of Leydig cells, Sertoli cell8 and peritubular myoid cells. In contrast, the germinal cell8 would contribute a8 much a8 60-70’S of the weight of testes from mature normal mice. Consequently, if germ cell8 have a low capacity to metabolize progesterone, it i8 not possible to compare rate8 between preparation8 from germ cell-free and normal t&es on a weight basis alone. These considerations influence investigations of the quantitative aspects of progesterone metabolism, and may alter interpretation of Borne of the qualitative observations. In the present study, however, the major fact remain8 that in the absence of germinal cells, there is a normal increase and reduction of !kreducta8e activity in one or more classes of remaining cell8 during maturation of the testis. Reference8 E.8.(1967) J.L.MIXRUSMU.
1 2 3 4 6 6 7 8 9 10
Mint& B. and Russell. J. EXP. 2001.134,207-237 Coulombre. E.8. (19644) J. Bxp. 2001.126, 277395 Filcher. M. and Steinbergor. IL (1971) Acta Badoainol. 68.286-292 Coffey, J.C., Fmach. F.S. and Nayfob. 8.N. (1971) Rudocdnology 89.865-872 Inaao. H.. Hori, Y. and Tamaoki. B. (1967) Ciba Fomxl. Colloa. Bndocrinol. 18.106-119 Yamada. M. and hfatsumoto, K. (1974) Endocrinology 94.777-784 Tmkiimura, T. and bfatsumoto. K. (1974) Endocdnology 94.288-290 Tm&mura. T.. Mizutani. 9. and Mataunoto. K. (1976) Bndocrinology 96. Sib-618 Coffey, J.C.. Aronin, P.A.. French. F.S. and Nayfeh. S.N. (1972) steroids 19.4334b4 Goldmao. AS. and KlIagele. D.A. (1974) Endocrinology 94.1-16
11 12 13 14 16 18 17 18 19 20
Ntiimura. ht. (1972) EXR. AnIm. 21.188-141 (in Japa~ec) Yamada, M.. Yuue. 9. and Mataumoto. K. (1972) Aeta Endocxinol. 71.893408 Zaffuoni, A. and Burton. R.B. (19Sl) J. Blol. Chem. 198.749-787 SCM. T. and Matsumoto, K. (1967) J. Chromatogr. 27.423-490 Y-da, M.. Yasue. 9. and Matuumoto. K. (1973) Endocrlaology 93.81-87 Folmm. Y., Ahmad. N.. Sowell. 1.0. and JUk-Ner, K.B. (1973) Endocrinology 92.4147 Mat8umoto. K. and Yamada, M. (1973) Radocrlnology 98.255-266 Rivuola. M.A.. Podesta. E.J. aad Chemer, H.E. (1972) Endocrinology 91. S37-642 Doningtoo. J.H. and Ritz. I.B. (1978) Biocbem. Blophys. Rar. Commun. M.142bl431 Dotigton. J.H. md Mtz. LB. (1976) Endocrinology S&879-889
Bioehimica et Biophysics @ ~~~r~No~h-Holl~d
Acta, 486 (1977) BiomedicaI
127-135
Press
BBA 56903
PROGES~RO~E MET~OL~~ CELL-FREE MICE OF DIFFERS
MASAHIRO KITAMURA
IN VITRO BY TESTES FROM GERM AGES
KUWATA *, MORIO YAMADA b, MASAMITSU c and KEISHI MATSUMOTO d
TAMAI c, YUKIHIKO
a Research ~~tit~te for ~~c~b~~ ~~~, &aka Uni~~i~, b ~~~rnent of Patho~o~, Hyogo College of Medicine, and c Department of Pathology and d Institute for Cancer Research, Osaka University Medical School, Osaka (Japan) (Received March 2nd, 1976) (Revised manuscript received September
8th, 1976)
Androgen biosynthesis in vitro by testes from (C57BL X WN)Fi-WV/W” (congeni~y germ cell-free) and +/+ (normal) mice of different ages was investigated using [ 3H) progesterone as substrate. At all ages examined, the seminiferous tubules of the W/W” genotype were largely devoid of germ cells. However, the age dependent pattern of progesterone metabolism by germ cell-free testes was similar to that by normal testes. Conversion of progesterone to bareduced 17-hydroxy-C?i and C&-steroids such as 3a,l?a_dihydroxy-fia-pregnanQOsne,
ages [ 2 J . However, androgen biosyn~es~ in the germ cell-free testes has not been clarified. Although the activity of 5a-reductase for A’-3-ketosteroids began to fall after 40 days of age in testes of normal rats and mice [3-81, the activity remained persistently elevated in testes of Stanley-Gumbreck pseudohermaphrodite rats [9,10]. The seminiferous tubules of the pseudohermaphrodites had a few cells in the diplotene stage at 51 and 60 days, but at 140 days most of the sperm cell elements were gone [lo]. These findings seem to indicate that testicular Fiar-reductase activity changes with age in such a fashion as to suggest an association with the differentiation of germ cells present in the testes. This possible association was examined in (C5’7BL X WN~-F~-W~~Wn (congeni~ly germ cell-free) mice of different ages. However, this hypoth~~ is not supported by the data presented, since the formation of 5rr-reduced metabolites from progesterone in testes of different ages of mice was not influenced by the presence of germ cells in the seminiferous tubules. Materials and Methods Animals. The BWNF, hybrid male mice of various ages, 20,28,35, 52,120 and 150 days, were raised in our laboratory. Parent mice were C57BL-WY/+ and WN-W”/+. C57BL/6J-WI+ mice were purchased from the Jackson Laboratory (Bar Harbar, Maine, U.S.A.) and then raised in our laboratory by repeated backcrossing with our C57BL mice. The WN strain was es~blished as an inbred strain at Nagoya University [ll], and obtained through Dr. Nishimura, Wakayama Medical College. A cross between these two inbred strains results in congenic animals, designated BWNF1, with four possible genotypes at the W locus, +/+, WV/+, Wn/+, W/W*. These W series genotypes can be recognized on the basis of coat color: +/+ mice are all black; W/+ are grey with a white belly spot; W”/+ are black with numerous white spots over the whole body; and WV/ W” are black-eyed white-coated. W/W* male mice were used as germ cell-free mice and +/+ male mice as control animals. In one experiment, 4 normal and 4 germ cell-free mice were given human chorionic gonadotropin plus pregnaut mare’s serum gonadotropin subcutaneously (10 I.U. of each hormone) each day, for 7 or 12 days. The gonado~opin treated and control mice were killed 28 days after birth. histological technique. The testes were examined as previously reported 1121. Chemicals. [ 1,2-3H] progesterone (45 Ci/mmol), obtained from Daiichi Pure Chemical Co Ltd. (Japan) was purified by paper chromatography using the hexane/formamide system [13] just before use. The radiochemical purity of the purified [ 3H] progesterone was 99% by repeated crystallizations. Furthermore, the purified [3H] progesterone behaved as a single compound in paper Cl33 and column [14] chromatographies and there was no or very small amounts of other radioactive steroids (less than 0.2% of each) in 13 steroid fractions (Tables 11). Non~dioactive steroids were obtained from Steraloids; Inc. and Ikapharm. Pregnant mare’s serum go~ado~op~ and human chorionic gonadotropin were obtained from Teikokuzoki Co. Ltd. Japan. Other reagents were of analytical grade.
129
Incubation procedure. The testes obtained from 2-8 mice of different ages (Table I) were weighed and were homogenized in 0.25 M sucrose containing 1 mM EDTA. The purified [‘HI progesterone (1 nmol : 1 j&i per vessel) was introduced into 2 X 10 cm tubes and dissolved in 0.02 ml ethanol. To each tube, 0.5 ml of buffer co-factor solution was added. The buffer co-factor solution consisted of 0.3 M potassium phosphate buffer (pH 7.4), 0.06 M nicotinamide, 2 mM MgClz and 2.5 mM NADPH. 0.5 ml of the tissue homogenate containing 0.4-30 mg of tissue was then introduced to make the total volume of the incubation mixture 1.0 ml. The samples were incubated in a shaking water bath in air at 34°C for 30 min. At the end of incubation, the mixtures were immediately acidified with 0.1 ml 1 M HCl and mixed with 3 ml of ether to stop the reaction. In all incubations, the metabolism of progesterone was examined in duplicate for each condition tested. Mean values of the two tubes are used throughout this report except in isolated instances where a sample was lost during the procedure. Analysis and identification of steroids. To the incubation mixtures, 5-50 pg quantities of the 14 steroids shown in Table II together with 2Ocu-hydroxypregn4-en-3-one and 17a-hydroxy-5e-pregnane-3,2Odione were added as nonradioactive carriers. The extraction of steroids from the incubation mixtures and the analysis of these 16 steroids by paper [ 131 and elution [ 141 chromatographies with acetylation of steroids were the same as previously described [6,15] . The percentage of the total radioactivity on the first chromatogram present in each zone was calculated. This percentage was distributed among th.ose compounds which showed significant radioactivity, according to the proportion of each compound present, as indicated by succeeding chromatograms. Finally, all the purified radioactive steroids or their acetates except for 3-hydroxy&pregnan-20-one, 17e-hydroxy-5a-pregnane-3,20-dione and 3cr,l7cy-dihydroxy5ar-pregnan-20-one were repeatedly crystallized with lo-15 mg nonradioactive TABLE I MEAN WEIGHT FREE MICE
OF BODY,
TESTES
AND SEMINAL
VESICLES
Normal
Age
No. (days) 20 28 35 62 120 159 28 (G X 7) * 28(GX 12):
3 8 5 2 3 2 2 2
OF NORMAL
AND GERM CELL-
Germ cell-free Body
Testes
Seminal vesicles
(B)
(me)
7 12 19 23 23 27 11 13
38 69 92 206 140 I83 42 63
No.
Body
Testes
Seminal vesicles
(ms)
(9)
(mr)
(mr)
2 6 28 104 103 118 30 58
6 9 9 14 28 30 6 11
10 15 12 17 31 36 15 24
2 4 6 14 130 119 16 60
* Mice were given humau chorionic gonadotropin PIUS premmnt mare’s serum gonadotropin subcutaneously (10 1.U.. of each hormone) each day for 7 or 12 days. The mice were killed on the day of the last injection (aged 28 days).
II
FORMATION
OF [3H]STEROIDS
FROM
[3H]PROGESTERONE
BY TESTICULAR
HOMOGENATES
8.1 38.6 1.8 5.4 1.5 2.6 4.5 4.1 20.5 67.7
9.1 31.0 3.1 6.2 2.6 6.5 2.1 6.1 26.6 66.7
2.1 2.1 <0.2 0.0 0.0
Androstenedione Testosterone 5a-Androstane-3.17-dione ll&OH-5a-Androstan-3-one Androsterone 3&OHda-Androstan-ll-one Sa-Aadrostane-3 a.1 1 @dial ScY-Androstane-3 p.11 &diol Total 5&-H-C* 9 steroids TotaI C 19 steroids -
2.4 4.8 0.1
2.4 1.3 1.3
1.9 <0.4 0.0
11~Hydroxyprogesterone 3a.17a-OH-5a-Pregnan-2O-one 38.1Ta-OH-5a-Pregnan-20-one
6.5 <0.3 <0.5
35
8.1 0.7 1.8
28
79.0 2.8 4.8
20
Progesterone (unchanged) 5cu-Pregnane-3.2O-dione 3-Hydroxy-5a-pregnan-20-one
Days after birth:
Normal mice
10.0 <0.2
0.1 <0.2 0.0 6.5 71.7 <0.2 1.3 0.2 0.5 1.3 1.4 4.1 88.9
120
4.3 <0.2 <0.4
52
6.5 5.5 0.0 0.0
6.4 0.7 0.0
53.4 3.2 12.8
20
mice
10.3 36.5 2.2 3.7 2.0 5.6 2.2 6.0 21.1 68.5
1.1 4.5 0.6
8.9 0.3 1.1
28
Germ cePfree
9.1 49.0 0.8 2.5 1.9 2.4 5.3 3.2 16.1 14.2
2.6 4.7 0.0
4.3 <0.4 2.7
35
Testicular homogenates prepared from 30 mg of testes of mice of aII ages employed were incubated with [3H]progesterone at 34Oc for 30 min in 1 ml. Values were obtained after 4 repeated crystaIIizations to constant specific activity.
FREE MICE
PERCENTAGE
TABLE
6.2 66.1 <0.4 2.1 0.1 <0.6 4.7 1.9 9.4 81.7
0.3 1.1 0.0
2.6 <0.2 co.4
52
(1 nmol
FROM
:
AND
GERM
CELL-
69.6
10.0
0.2 <0.5
6.3 <0.2
120
1 /Xi per vessel) and NADPH
NORMAL
131
standard steroids to constant specific activity in order to identify the steroids formed. 3-Hydroxy-Be-pregnar&O-one, i7cu-hy~~~~Ekr-pi;egnane-3,2Odione and 3a ,17adihydroxy-5a-pregnan+ZO-one were $&&vely. identified by derivative formation (acetylation and oxidation with &hronWn :t&&ii) ad rttpbd crystallizations of the derivatives formed with chromium~tkioxide to, cerktant specific activity, as previously described [15] ; lhal evidence of identification or tentative identification required that the specific activities of three consecutive crystallizations were within +5% of the mean. The amount of each steroid on the final chromatogram was corrected by the decrease of specific radioactivity on 4 repeated crystallizations to constant specific activity, which was mostly less than 20%. The above procedure for calculating the rate of formation of metabolites can permit an approximate estimation of the percent conversion of [3H] progesterone, which seems to be of use for the present purpose. Results Testes and seminal vesicles of normal and germ cell-free mice of different ages
The mean weight of body, testes and seminal vesicles of normal and germ cell-free mice used in the present incubation studies are shown in Table I. At all ages examined, the weight of testes of germ cell-free mice was much lower than that of normal mice. On the other hand, the maximum seminal vesicle weights at 120 and 150 days were similar in the two groups of mice. However, between 28 and 52 days, seminal vesicle weights were greater in normal than in germ cell-free mice. The increase in the weights of seminal vesicles was observed in 28day-old mice receiving gonadotropin. The results of the microscopic examination in normal mouse testes were as follows. At 52 days, full spermatogenesis was noted (Fig. 1). Earlier stages (not shown) revealed the following: at 20 days, primary spermatocytes, including pachytene spermatocytes, occupied most parts of the tubules with a few
Fig. 1. Photomicrograph of a section from 62-dau-old normal mouee testis (X600). posed of seminiferous tubules and.interstitial cells.
The section is com-
132
Fig. 2. (X 500, aged 62 days) is representative of the histological appearance of germ cell-free mice of all ages examined. It is seen that no germ cells are present in the seminiferous tubules which contain a single layer of Sertoli cells.
immature spermatids; at 28 days, the immature spermatids had increased with a few mature spermatids; at 35 days, the mature spermatids were increased. The results in the germ cell-free mice were as follows. At all ages examined, each tubule consisted of a single layer of Sertoli cells (Fig. 2). The testes were largely devoid of germ cells’and very few germ cells in a very few tubules were always spermatogonia. No differences were noted among both groups of mice in morphology of interstitial tissue (Figs. 1 and 2). The microscopic findings of testes from the present W/W” mice were similar to those reported previously in testes from W/W mice [2]. Progesterone metabolism different ages
by testes from germ cell-free and normal mice of
The percentage formation of [3H] steroids from [3H] progesterone by testicular homogenates from germ cell-free and normal mice at different stages of development is shown in Table II. 30 mg tissue were incubated with 1 nmol of [3H] progesterone and NADPH for 30 min in 1 ml. Conversion of progesterone to 5cu-reduced 17-hydroxy-Czl and C19-steroids such as 3ar,l7adihydroxy5ol-pregnan-20-one, androsterone, 3/3-hydroxy-5c-androstan-17-one, Eia-androstane-3a,l7P-dial and 5a-androstane-3fiJ’Ipdiol was signific&t in testes of 2% and 35-day-old mice of both groups, germ cell-free and normal mice. However, these 5a-reduced metabolites were found to be formed in very small amounts or not formed at all in testes of 20- and 120-day-oM mjce of both groups, in which conversion of progesterone to l’lar-hy&ox terone, androstenedione and testosterone was observed. Conversion of proge&terone to 5cy-reduced steroids was low at 52 days of age in both groups. Since substrate progesterone was almost completely consumed in most of the incubations shown in Table II, the following incubations by testes from 35- and 15Oday old mice of both groups were attempted.
133 TABLE EFFECT
III OF GONADOTROPIN
ONE BY 39 MG TESTICULAR
ADMINISTRATION HOMOGENATES
ON IN VITRO FROM
PI-DAY-OLD
METABOLISM NORMAL
OF PROGBSTERAND
GERM
CELL-
FREE MICE TestIcuIar homogenste wss Incubsted with [3H]progcste?Onc (1 run01 : 1 ~ICI per VWseI) snd NADPH Pt 34OC for 30 mIn in 1 ml. V&es were obtaIned after 4 repeated crystsIBsatIons to constant sppsciiicsctkity. Results represent amounts formed or remshdng In pmol. Mice were given human chodonic gonsdotropin plus pregnant mare’s serum gonadotropin subcutaneous& (10 I.U. of each hormone) each day for 7 or 12 days, starting from 22 or 17 days after birth. The mice were kIIIed on the day of the Isst injection. aged 28 days. -
---
Days after treatment:
-Germ cell-free
NormsI
-
0
7
12
0
7
Progesterone (unchanged) 6a-Reduced P * 17a-Hydroxyprogesterone 6a-Reduced 17.OH-P * * Androstenedione Testosterone 6a-Reduced CI c steroids
33 99 26 201 28 147 337
26 10 1 16 8 71 767
26 13 1 33 11 117 663
69 22 11 61 103 366 222
63 18 1 12 41 326 461
(6a-H-steroids/total)
(0.66)
(0.91)
(0.84)
(0.32)
(0.66)
l **
-
12 33 11 1 21 16 106 700 (0.86)
* 6a-Reduced metsboIItes of progesterone. ** ba-Reduced metsholites of 17a-hydroryprogesteroone. *** Ratio of 6a-reduced Cl9 steroids to totsI CI9 steroids.
When homogenate8 from 1.5-6.0 mg of normal testes or those from 0.43.0 mg of germ cell-free testes were incubated with [ 3H] progesterone (1 nmol : 1 @i per vesel) and NADPH at 34°C for 30 min in 1 ml, more than half of the substrate progesterone remained unchanged and the rates of production of total of C19-steroids or total of 17.hydroxy-Cz,-steroids plus C1&eroid8 were roughly proportional to the weights of each tissue used. Conversion of progesterone to 5a-reduced l’ldeoxy-C,,-, 17.hydroxy-C2, and C19-steroid8 wa8 low but significant in testes of 35day-old mice of both group8, but these 5areduced metabolites were not formed at all in testes of 150day-old mice of both groups (data are not presented). However, conversion of progesterone to 17a-hydroxyprogesterone, androstenedione and testosterone was similar in testes of prepubertal and adult mice. There were no significant differences in the ratios of the sum of 5a-reduced C1&eroid8 to total C.&&eroids (approx. 0.1) and those of the sum of Sa-reduced 17.hydroxy-CzI-steroids to total 17. hydroxy-C,l-steroids (approx. 0.1) between prepubertal testes from germ cellfree and normal mice. These findings indicate that the age dependent pattern of progesterone metabolism by congenitally germ cell-free testes was similar to that by normal testes. The formation of products from [3H] progesterone by 30 mg of testicular homogenates from control and gonadotropin injected 28day-old mice is shown in Table III. Control mice were given saline each day for 12 days, starting from 17 days after birth. Gonadotropin injected mice were given gonadotropins each day for 7 or 12 days, starting from 22 or 17 day8 after birth. The mice were killed at 28 days of age, on the day of the last injection. Conversion of progesterone to 5o-reduced C,&eroids wa8 found to be high in testes of 23day-old mice of both groups receiving saline (control) and those receiving gonado-
134
tropins. Treatment of immature mice with large amounts of gonadotropins for 7 and 12 days did not reduce but increased the conversion of progesterone to 5a-reduced steroids relative to A4-3-ketosteroids in testes of 28day-old normal and germ cell-free mice (Table III). As shown in Table II, the formation of 5areduced steroids in testes of adult mice (52 and 120 days of age) of both groups was much lower than that in testes of prepubertal mice (28 and 35 days of age) indicating that there was a reduction of 5e-reductase activity in normal and germ eel&free testes during maturation of the tests. These findings seem to suggest that there is an increase of FicY-reductaseactivity in prepuce testes stimulated by gonadotropins and that the influence of age upon the reduction of 5e-reductase activity in testes of both groups does not results from the action of gonadotropins. However, further studies on the effects of hypophysectomy and administration of gonadotropins in normal and germ cell-free mice of different ages upon the activity of testicular 5a-reductase are necessary in order’ to clarify the mechanism of age dependent change of testicular 5areductase . Discussion The present results shown in Table II derno~~~ that prog~~rone is actively metabolized to C,&eroids in vitro in testes of (C57BL X ~)F,-WV/ W” (germ cell-free) mice, regardless of the age of mice and that the characteristic age-dependent pattern of progesterone conversion to &-reduced- and A4-3ketoG&eroids in testes of W/W” (congenitally germ cell-free) mice is similar to that in testes of +/+ (normal) mice. This age dependent pattern of progesterone metabolism shown by testes of (C57BL X WN)F, mice is in agreement with a previous report on testes of albino mice of the d.d. strain [ 7 ] . To the best of our knowledge, the present results demonstrate for the first time the agedependent formation of ficu-reduced C,&eroids in congenitally germ cell-free testes. These results seem to indicate that this age-dependent change in progesterone metabolism is unrelated to germ cell development in the ~rn~~erous tubules and that the persistently high activity of 5e-reductase in testes of adult Stanley-Gumbreck pseudohermaphrodite rats [9,10] may not be due to lack of germ cells in the seminiferous tubules. . Folman et al. [16] and Matsumoto and Yamada [17] reported that in prepubertal rat testes, tja-reduction of testosterone in vitro in the presence of NADPH was much higher in the in~~titi~ tissue than in the tubules. However, Rivarola et al. [18] reported an opposite result by incubation of isolated tubules and interstitial tissue from prepubertal rats in the absence of NADPH. This discrepancy may be due to a deficiency of co-factors in the interstitial tissue isolated by Rivarola et al. [18] . Since the age-dependent formation of Sa-reduced &#eroids from progesterone was found to be unrelated to the presence of germ cells (Table II) and the 5c~reductase was much higher in the interstitial tissue than in the seminiferous tubules in prepubertal rat t&m [ 16,171, this pattern of metabolism in testes seems to represent agedependent change in androgen biosynthesis by the interstitial cells. However, the contribution of Sertoli cells present in testes of both groups of mice cannot be ruled out in the present study. Although 5cr-reductase in the seminiferous tubules of
136
prepubertal rats was shown to be distributed mainly in germ cells [12,19],
a
recent study demonstrated the presence of Ew-reductase in Sertoli cells isolated from prepubertal rat testes [20]. Contrary to the re8ulte ahown in Table II, the results shown in Table III demonstrate that in prepubertal mice, the germ cellfree t~&.es produce more androstenedione and kto8terone and less k-reduced C,+&eroids than normal testes. It is possible that this may be due to the presence of !5a-reductase in germ cell8 [12,19,20]. Although the activity of 5areducta8e wa8 shown to be much higher in the interstitial tissue than in germ cell8 in prepubertal rats [ 12,16,17] , this activity in germ cell8 wa8 still found to be similar to that in the ventral prostate [12]. The data in Table II appear to support the present statement that the pattern of progesterone metabolism is approximately the 8ame in testicular preparations from germ cell-free and normal mice of similar ages. However, the percentage of conversion per unit weight a8 shown in Table II could be misleading. The population of cell8 in homogenate8 prepared from immature or adult germ cell-free testes consist primarily of Leydig cells, Sertoli cell8 and peritubular myoid cells. In contrast, the germinal cell8 would contribute a8 much a8 60-70’S of the weight of testes from mature normal mice. Consequently, if germ cell8 have a low capacity to metabolize progesterone, it i8 not possible to compare rate8 between preparation8 from germ cell-free and normal t&es on a weight basis alone. These considerations influence investigations of the quantitative aspects of progesterone metabolism, and may alter interpretation of Borne of the qualitative observations. In the present study, however, the major fact remain8 that in the absence of germinal cells, there is a normal increase and reduction of !kreducta8e activity in one or more classes of remaining cell8 during maturation of the testis. Reference8 E.8.(1967) J.L.MIXRUSMU.
1 2 3 4 6 6 7 8 9 10
Mint& B. and Russell. J. EXP. 2001.134,207-237 Coulombre. E.8. (19644) J. Bxp. 2001.126, 277395 Filcher. M. and Steinbergor. IL (1971) Acta Badoainol. 68.286-292 Coffey, J.C., Fmach. F.S. and Nayfob. 8.N. (1971) Rudocdnology 89.865-872 Inaao. H.. Hori, Y. and Tamaoki. B. (1967) Ciba Fomxl. Colloa. Bndocrinol. 18.106-119 Yamada. M. and hfatsumoto, K. (1974) Endocrinology 94.777-784 Tmkiimura, T. and bfatsumoto. K. (1974) Endocdnology 94.288-290 Tm&mura. T.. Mizutani. 9. and Mataunoto. K. (1976) Bndocrinology 96. Sib-618 Coffey, J.C.. Aronin, P.A.. French. F.S. and Nayfeh. S.N. (1972) steroids 19.4334b4 Goldmao. AS. and KlIagele. D.A. (1974) Endocrinology 94.1-16
11 12 13 14 16 18 17 18 19 20
Ntiimura. ht. (1972) EXR. AnIm. 21.188-141 (in Japa~ec) Yamada, M.. Yuue. 9. and Mataumoto. K. (1972) Aeta Endocxinol. 71.893408 Zaffuoni, A. and Burton. R.B. (19Sl) J. Blol. Chem. 198.749-787 SCM. T. and Matsumoto, K. (1967) J. Chromatogr. 27.423-490 Y-da, M.. Yasue. 9. and Matuumoto. K. (1973) Endocrlaology 93.81-87 Folmm. Y., Ahmad. N.. Sowell. 1.0. and JUk-Ner, K.B. (1973) Endocrinology 92.4147 Mat8umoto. K. and Yamada, M. (1973) Radocrlnology 98.255-266 Rivuola. M.A.. Podesta. E.J. aad Chemer, H.E. (1972) Endocrinology 91. S37-642 Doningtoo. J.H. and Ritz. I.B. (1978) Biocbem. Blophys. Rar. Commun. M.142bl431 Dotigton. J.H. md Mtz. LB. (1976) Endocrinology S&879-889