2359

495

IN VITRO METABOLISM OF TESTOSTERONE BY CULTURED SERTOLI CELLS AND THE _EFFECT OF FSH. Robert K. Tcholakian and Anna Steinberger Department of Reproductive Medicine and Biology The Duiversity of Texas Medical School at Houston Houston, Texas 77030 Received 8-7-78 ABSTRACT Cultures of Sertoli cells isolated from testes of 18-and 36-day-old Long Evans rats were used to investigate their capacity to metabolize testosterone and the effect of FSH on such metabolism. Three different approaches were used: 1) investigation of the metabolism of radiolabeled testosterone under saturating substrate conditions; 2) study of the metabolism of radiolabeled testosterone utilizing trace amounts of high specific activity substrates; 3) the utilization of radioimmunoassay for measurement of estradiol-178. The following steroids were isolated and identified by recrystallization to constant specific acitvity from the control and FSH-treated cultures: testosterone (unconverted substrate), androstenedione, dihyand Sa-androstanedrotestosterone, 3o-hydroxy-5a-androstan-17-one 3a,17S-diol. Radioimmunoassay data suggests that the Sertoli cells produce an estradiol-178-like compound from unlabeled testosterone and that this production is stimulated by FSH. However, the radioactive metabolite from all our studies that behaved chromatographically like estradiol-17S failed to crystallize to constant specific activity, while in each experiment, authentic radiolabeled estradiol-176 added as recovery tracer did. The data demonstrate that: 1) cultures of Sertoli cells from iamature rats have 5o-reductase, 3a- and 17S-hydroxysteroid oxidoreductase activities; 2) these enzymes may be affected by FSH; 3) based on radiolabeled metabolic techniques, Sertoli cells were unable to biotransform testosterone to estradiol-178 even in the presence of FSH. INTRODUCTION It

is well

accepted

that

vitro are enzyme mediated. to

convert

metabolize

cholesterol

(2-3)

(3) and metabolize

5

s

and

in vivo --

tubule preparations to

(4-pregnene-3,20-dione)

(4-androstene-3,17-dione)

Vo[IL?Zwne 33, Number

Seminiferous

bioconversions

f5-cholesten-38-01)

progesterone

androsten-3-one)

steroid

various to

testosterone

and -in

were shown

steroids

androstenedione (17S-hydroxy-4-

testosterone to androstenedione

II?IJEOXDE)I

(11,

May,

and

1979

So-reduced tubules

androgens

contain

these

out

in --

the steroidogenic

Sertoli

cells.

structural

data

reticulum,

an

This

organelle

usually

of

investigators

the

(2,3)

the seminiferous

was made

the

that

enzymes capable of carrying

Some

activity

conclusion

indicate

observations

vitro.

demonstrating

steroidogenesis, Recently,

These

an array of steroidogenic

bioconversions

attributed the

(4-9).

on

abundance associated

tubules

the basis

of

smooth

with

a

have

of

to

ultra-

endoplasmic

capacity

for

in the Sertoli cells (10).

we

have

demonstrated

that

cultures

of

Sertoli

cells

isolated from adult rat testes are capable of metabolizing progesterone to

3-one)

(11).

reported

Sertoli basis

This

for

biochemical

have

and

testosterone

dihydrotestosterone

metabolic

tubule

as

demonstrated

testes

are

the

postulated

of morphological that

capable

dihydrotestosterone

of

is

preparations

evidence' for

cells

pattern

similar

Christensen

of

and

Sertoli

other

Sertoli

metabolizing

and

that

capacity

Fawcett

cells

testosterone

from to

that

cells

enzymes:

17c+hydroxylase,

reductase

(17S-OR),

%-20

Sa-reductase

possess

the

lyase, and

the

(10) on

the

(12,131

immature

rat

androstenedione,

following

These

studies

steroidogenic

17S-hydroxysteroid

3a-hydroxysteriod

direct of

investigators

5a-androstane-3a,l7b-diol.

indicate

previously

represents

biosynthetic

data. Furthermore, cultures

to

and

(2,3)

steroid

by

fl7B-hydroxy-5u.-androstan-

oxido-

oxidoreductase

(3o-OR). FSH was

also reported

to stimulate

17&OR,

5a-reductase and 3a-OR

in Settoli cells from lo-day-old rats, and 17S-OR in Sertoli cells from 16 and ZO-day-old rat testes (12). On the other hand, FSH significantly suppressed 5o-reductase and 3a-OR activity in Sertoli cells from 16 and

20-day-old rats, and 178-OR, .W-reductase

and 3a-OR

in Sertoli

cells

on the basis of radioimmunoassay

(RIA)

from 24-day rat testes (12). It has also been suggested data,

that

capable

cultures

of

of

converting

Sertoli

testosterone

activity

is significantly

presence

of

These

FSH

stimulated

responsive

observations

cells

have

not

C-19 been

from

to

imnature

estradiol-176

by FSH

and

testes that

(14-161, alluding

aromatizing verified

rat

enzymes

by

are this

to the

in these cells.

radiolabeled

metabolic

studies, or by authentication of the estradiol-17B by recrystallization to constant specific metabolism

activity. Thus, FSH appears

from

immature

rat

androgen

immunoassayable estrogen (14-16). The

(12) and to stimulate

current study was designed to further explore cells

to suppress

testes

to

the capacity of Sertoli

metabolize

testosterone

and

the

effect of FSH on such metabolism. Three different approaches were used: 1)

Investigation

Sertoli

of

the

metabolism

cells under saturating

metabolism

of

radiolabeled

of

substrate

testosterone

radiolabeled conditions; by

Sertoli

testosterone 2)

Study of

cells

by the

utilizing

trace amounts of high specific activity substrates; 3) The utilization of radioimmunoassay techniques for measurement of estradiol-178. ME~ODS

ADD MATERIALS

Sertoli cell cultures: Sertoli cells were isolated from testes of 18-day or 36-day-old Long Evans rats raised in our colony under controlled temperature and light conditions. The cells were cultured at 34OC in Eagle's minimum essential medium without, serum, as previously described (17,181. Using morphological (light and electron microscopic), biochemical (FSH binding and stimulation of endogenous cyclic AMP) and cytochemical (localization of 3B-hydroxysteroid oxidoreductase in Sertoli cells) parameters it was determined that the cultures contained only Sertoli cells ( > 90%) and germ cells ( ~10%) and that they were free of Leydig cells and peritubular cells (11). For each experiment 3 or 4 individual cultures each containing approximately 1~10~ Sertoli cells were used. The cultures were incubated for 24 h at which time the media was changed and incubation was continued for an additional 24 h with or without FSH. At this time,

the cultures were rinsed twice with 2 ml Hank's balanced salt solution and utilized for experiments as outlined below. Experimental Design: I. Metabolism of 14C-testosterone by Sertoli cell cultures derived from 18-day-old rats utilizing saturating (low specific activity) substrate conditions. A. Cultures grown for 48 hours in the absence of FSH were incubated at 34OC in 2 ml Eagle's minimum essential medium containing 0.25 uCi [4-14C]-testosterone (57 mCi/mnol) for an additional 6 h. B. Cultures grown for 24 h in the presence of FSH were incubated at 34OC in 2 ml Eagle's minimum essential medium containing 0.25 uCi [4-14C]-testosterone (57 mCi/mnol) and FSH (5 ng/ml; NIH-FSH-Sll) for an additional 6 h. II. Metabolism of 3H-testosterone by Sertoli cell cultures derived specific activity substrate from 18-day-old high rats utilizing conditions. A. Cultures grown for 48 h in the absence of FSH were incubated at 34OC in 2ml Eagle's minimum essential medium containing 1.25 PCi [la,2a-3H]-testosterone (1 mCi/4.9 ug) for an additional 6 h. B. Cultures grown for 24 h in the presence of FSH were incubated at 34OC in 2ml Eagle's minimum essential medium containing 1.25 (1 mCi/4.9 up) and FSH (5 pg/ml; uCi [la,2a-3H]-testosterone NIH-FSH-Sll) for an additional 6 h. III. Metabolism of testosterone by Sertoli cell cultures derived from 18-day-old rats utilizing saturating substrate (unlabeled) conditions. A. Cultures grown for 48 h in the absence of FSH were incubated at 340C in 4 ml Eagle's minimum essential medium containing unlabeled testosterone (5 X 10-7M) for an additional 6 h. B. Cultures grown for 24 h in the presence of FSH were incubated at 340C in 4 ml Eagle's minimum essential medium containing unlabeled testosterone (5 x 10m7M) and FSH (5 ugg/ml; NIHFSH-Sll) for an additional 6 h. IV. Metabolism of 14C-testosterone by Sertoli cell cultures derived from 36-day-old rats utilizing saturating (low specific activity) substrate conditions. A. Cultures grown for 48 h in the absence of FSH were incubated at 340C in 2 ml Eagle's minimum essential medium containing 1 pCi [4-14C]-testosterone (57 mCi/nsnol) for an additional 6 h. B. Cultures grown for 24 h in the presence of FSH were incubated at 34OC in 2 ml Eagle's minimum essential medium containing 1.0 and FSH (5 ug/ml); uCi [4-14C]-testosterone (57 mCi/mnol) NIH-FSH-Sll) for an additional 6 h. Extraction, separation and purification of radiolabeled steroids: At the end of the 6 h incubation, the reactions were terminated by the addition of 0.1 ml of 1N HC1. Ten ug of each of the following carrier steroids: testosterone, androstenedione, dihydrotestosterone, (3a-hydroxy- 5a - androstanandrosterone 5a-androstane-3a,l7$-diol, and estrone 17-one), estradiol-178 (1,3,5(10)-estratriene-3,17S-dial)

3f3-hydroxy-1,3,5(10)-estratriene-17-one) were added at this time to the incubates. Correction for methodological losses: 1) In experiments utilizing saturating substrate 14C-testosterone conditions the following tracer steroids were used: [7(n)-3H]-testosterone (12.4 Ci/rmzol), [1,2,-3H(N)]-androstenedione (40 Ci/mmol), [1,2,-31i -dihydrotestosterone, 40 Ci/mmol), (35 Ci/mmol), [1,2(n)- 3HI-5o-androstane-3a,17&diol [ 2,4,6,7(n)-3H]-estradiol-176(85 Ci/xmnol) and [2,4,6,7(n)-3H]-estrone (80 Ci/nzsol). 2)In experiments utilizing high specific activity 3H-testosterone the following 14C-labeled tracer steroids were used: testosterone (59 mCi/mmol), androstenedione (60 mCi/mmol), dihydrotestosterone (59 mCi/nmol), estradiol-17B (55 qCi/zsaol). utilizing 14C-testosterone subThe samples from experiments strates were processed at a different time than those utilizing 3H-testosterone to avoid any possibility of sample contamination. Extraction: Samples (cells plus media) were extracted to background with 1.5 volumes of ether:chloroform (4:1, v/v>. The extracts were dried and concentrated under nitrogen. Separation and purification: che extracts were chromatographed on 50 cm paper strips (Whatmann #l)in hexane:formamide to the front, and rechromatographed- in hexane:benzene (1:l):formamide to the front: The chromatograsm were dried at 40°C overnight and scanned for radio(Model No. 7210). The activity using a Packard paper scanner chromatograms were then divided into specific zones using carrier steroids as markers, with each zone representing a group of steroids with similar polarity. The zones were eluted with methanol (5 x 10 ml) and the eluates were evaporated to dryness. Radioactive material in the unknown zones with chromatographic behavior of authentic androstene5o-androstane-3a,l7&diol, estrone, dione, dihydrotestosterone, estradiol-178 and testosterone (unconverted substrate) were processed through various TLC systems and where applicable, derivatized and then recrystallized to constant specific activity from several different solvent combinations as indicated in Tables 6-17 (Appendix). Testoswere recrystallized as acetates, terone and dihydrotestosterone 5o-androstane-3o,178-dial as diacetate, while androstenedione, estrone and estradiol-176 were recrystallized in their natural forms. The techniques and procedures used for handling each steroid were the same as previously published (11). Substrate bioconversions were expressed as: a) Percent conversion (The amount of biosynthesized steroid as percentage of the substrate) derived as follows: r3H] or [14C l(dpm/mg) mean of 3 crystals [14~]Converted (dpm)-total steroids crystallized % Recovery % substrate: converted =Total

[%l

or [14C]converted (dpm)

Substrate [-'HIor [14C ](dpm)

Xl00

X 100

S

500

'EmPEOXDI

(The amount of biosynthesized steroid as conversion. b) ng nanograms of substrate) is based on percent conversion of the substrate concentration: % substrate converted X

ng substrate converted =

substrate concentration (ng) 100

Protein determinations: Two cultures of 18-day-old rats without added substrate containing about 1 x 106 cells were used for protein determination using a method similar to that described by Layne (19). Extraction and purification of estradiol-173 for radioimmunoassay: Samples consisting of 0.3 ml of culture media from experiment III A and B were pipetted into 35 ml glass-stoppered conical centrifuge tubes. Distilled water (0.5 ml) and 1800 dpm 2,4,6,7-3H-estradiol-17B (equivalent to 2.5 pg-Amersham/Searle) were for added recovery estimations. The samples were adjusted to pH 9-10 with O.lN NaOH before extraction with (2 x 5 ml) ether:chloroform (4:1, v/v), and the resultant extracts were washed with distilled water (2 x 2 ml> and evaporated to dryness under nitrogen. The extracts were chromatographed on silica gel (TLC) (20 x 20 cm plates coated with Silica-TLC 7GF-Mallinckrodt-0.25 mm thickness) in chloroform:methanol (98:2, v/v) Rf:estrone=0.34, estradiol-1%=0.14, estriol (1,3,5(10)-estratriene3,16@,178-triol)=origin. The TLC areas chromatographing like authentic estradiol-178 were scraped and extracted with ethylacetate (2 x 5 ml). The extracts were dried and reconstituted in 0.8 ml methanol, a 60% aliquot was transferred into 10 x 75 mm glass assay tube, dried and radioiumunoassayed. An additional 30% aliquot of each sample was pipetted into a scintillation vial for recovery estimation. antiserum ~dioi~noassay of estradiol-17B: The estradiol-178 f029-14-received courtesy of Dr. Ian Thornevcroft) was used at a final dilution of 1:4,000,000 The antiserum crossreacts 100% with estrone and estradiol-178. Radiolabeled estradiol-17B (2,4,6,7-3H-estradio1-17B, 18,000 dpm/sample equivalent to 19.5 pg) in 0.5 ml diluent (0.1% bovine gamma globulin-Fraction II-Sigma in 0.85% saline) and 0.5 ml estradiol-17Bantiserum in diluent (1:2,000,000) were added to each tube, agitated and incubated overnight (18h) at 4OC. Subsequently, 0.2 ml of dextran-coated charcoal in diluent was added, incubated for 20 min and centrifuged for 20 min at 2000 XG at 4OC. The supernatant (bound fraction) was decanted into 3 ml of p-dioxane, 10 ml of scintillation fluid added and counted in a Packard Tricarb Liquid Scintillation Spectrometer, Model 2450. Intra-assay and inter-assay coefficients of variation were 4% and 7% respectively. The computations and statistical analysis of the data were performed in our calculator laboratory using a Hewlett Packard 9830A programmable

according Statistical on ratios

to

the Fortran Computer Program of Rodbard and Lewald (20). evaluation of the data was done using analysis of variance calculated for individual samples. RESULTS

I.

Metabolism

14

of

18-day-old

C-testosterone

rats

utilizing

activityjsubstrate

The low specific

metabolized 5o-andros

of

in

saturating this

testosterone

were

activity

with

observed

by

steroids

authentic

by

for

to

substrate

concentration

of

.

Cultured

steroids,

of

recovery

the

(Appendix,

constant

specific

to

activity

like

cells

All

constant

to

authentic Tables

1.3

dihydrotestosterone,

and

chromatographically

Serto li

tan-17-one.

crystallization

unlabeled

behaving

crystallize

specific

andros tened ione,

crystallization

metabolite(s)

from

(low

and 3~hydroxy-5~andros

ident if ied

added

derived

saturating

experiment

to

tane-3u,l7f3-diol

compounds

cells

FSH:

activity

used

was

Sertoli

conditions:

A. In the absence

pgfsample

by

specific

constant

ratio

‘H-labeled 6-9).

C-labeled

estradiol-17S Table

as

tracer

14

The

(Appendix,

these

did 10,

not

samples

l-4). The total 22

ng/mg

amount of

protein.

lized

compounds that activity Tables

6h or

remained

representing (Table

other at

eliminated 6-91.

was 37.5

1).

79 ng of Unidentified

compounds the

origin

during

_t 1.9% or 447 +

dihydrotestosterone,

and Sa-androstane-3a,178-diol

metabolism in

metabolized

Androstenedione,

5u-androstan-17-one total

substrate

in

Table

of

the

purification

comprised

testosterone radioactivity 1 include paper

3 a-hydroxy6.7% of

substrate shown various

chromatograsm

and crystallization

metaboas

14

the

polar

C-steroids

and

radio-

(Appendix,

502

s

TmICOXDN

TABLE 1 MEAN TESTOSTERONE CONVERSIONS (+ S.E.M.) BY SERTOLI CELL CULTURES FROM IS-DAY-OLD RATS. SUBSTRATE = [4-14CI-TESTOSTERONE (57 mCi/mmol), 0.25 pCi

No Treatment

FSH

(N=4)

(N=4)

wlw % Converted Testosterone (5)

n&w

Protein

% Converted

Protein

63

+2 -

745

Androstenedione

4

+l

45

+

9

4.6 + 0.6

52+-

6

Dihydrotestosterone

1

_+o

12

+

2

0.4 -+ 0.1

4+-

1

0.2 -+ 0.0

2

-+l

0.3 + 0.2

4+-

2

178-diol

1.7 -+ 0.2

21

2.3 -+ 0.5

26+-

5

Estradiol-178

0.0

Polar Compounds

6.8 + 0.6

81

Other Compounds

24.1 + 1.6

Total metabolism

37.5 + 1.9

+ 27

52.5 + 3.8

592 f. 51

3a-hydroxy-5aandrostan-17-one 5a-androstane-3a,

5

-+

2

0

0

0.0 7

5.9 -+ 0.4

288

+ 19

34.0 -+ 3.8

382 + 39

447

+ 22 -

47.6 + 3.8

534 + 36

-+

66+-

5

See Key to Appendix

B. Xn the Presence of FSH: Testosterone

was

metabolized

terone, 5a-androstane-3a,l7S-diol (Appendix,

Tables

6-9).

Similar

to and to

androstenedione,

dihydrotestos-

3x-hydroxy-5x-androstan-17-one the

samples

without

FSH

the

14

C-labeled

178 did IO).

not

The

metabolite

behaving

crystallize

to

14C-label

significantly deviation

from

14

the

activity, 10,

presented

C-metabolite 3

22

to

recrystallize

substrate

8.6%

of

metabolism.

Thus,

86 ng of

(Table

4.6%

a proportionately

with

(2.3%)

and

1) .

lesser

dihydrotestosterone

II.

If]-day-old

of

rats

amounts

In

this

Table FSH are

was 47.6

per

-+ 3.8%

mg or

dihydrotestosterone,

or

18.1%

of

of

to

the

total

the

above

to

androstenedione

was

5u-androstane-3o,l76-diol

3a-hydroxy-5a-androstan-17-one

and

formed.

under high of

experiment

specific

of

was metabolized

amount

s- testosterone

A. In the absence

95%.

5u-androstane-3o,i7S-diol

metabolism

of

to

(Appendix,

presence

metabolized

testosterone

large

consecutive

and as ng substrate

converted

Testosterone

being

Metabolism

substrate

percent

constant

did

the

and

represented

androgens

in

decreased

92% and 21 to

tracer

Table

the

three

Androstenedione,

3o-hydroxy-Sa-androstan-17-one the

as

conversion

protein.

ngfmg

85%,

formed

percent

with

35 to

to

added

amount of

(Appendix,

of

30

failed

1 as

The total

-+

from

estradiol-

compound

activity

specific

The metabolites

Table

activity

recrystallizations

II-estradiol-178

S-8).

in

protein. 534

ranging

the

Samples

mean

specific

like

estradiol-17S-like

the

consecutive

the

recrystallizations While

constant

in

during

chromatographically

by

specific

Sertoli

activity

cells

substrate

derived

from

conditions.

FSH

a high

specific

activity

substrate

of

6.13

ng

n

(1.25 to

n Ci)

per

sample

androstenedione,

was used.

The cells

dihydrotestosterone,

3a-hydroxy-5a-androstan-17-one

(Appendix,

metabolized

‘H-testosterone

Sa-androstane-3a,l7&diol Tables

11-14).

and The

3H-

S

504

labeled failed

metabolites to

-l?BEOXD-

behaving

crystallize

to

chromatographically

constant

specific

like

activity

estradiol-176

while

the

14C_

estradiol-178 added as recovery tracer did (Appendix, Table 15, Samples 9-11). Since the mass of the substrate utilized

in these experiments was

small (6.13 ng per sample) the metabolites produced by the cultures are presented as percent conversion of the substrate (Table 2). TABLE 2 MEAN TESTOSTERONE CONVERSIONS (+ S.E.M.) BY SERTOLI CELL CULTURES FROM 18-DAY-OLD RATS. SUBSTRATE = [lu,2u-3Hl-TESTOSTERONE (1 mCi/4.9 ug), 1.25 PCi % Substrate Converted No Treatment

FSH

(N=3)

(N=4)

Testosterone(')

30.2 + 6.0

38.2 + 5.5

Androstenedione

2.7 + 0.7

3.5 + 0.3

Dihydrotestosterone

1.1 + 0.3

+ 0.1 0.8 -

3c-hydroxy-5u-androstan-17-one

0.8 -+ 0.2

0.3 -+ 0.1

5c-androstane-3c,l7b-diol

1.1 -+ 0.0

4.9 + 0.6

Estradiol-178

0.0

0.0

Polar Compounds

12.6 + 2.9

9.3 -+ 0.6

Other Compounds

42.9 + 5.7

42.9 + 4.0

Total Metabolism

69.8 + 6.0

61.7 -+ 5.1

5See Key to Appendix

B. In the presence of FSH: Testosterone

was

metabolized

terone, Sa-ahdrostane-3x,178-dial

to and

androstenedione,

dihydrotestos-

3a-hydroxy-5a-androstan -17-one

S Tables

(Appendix, 3

11-14).

TDEOXDI Similar

to

the

samples

metabolite

chromatographing

like

crystallize

to

specific

(Appendix,

consecutive

recrystallizations

H-labeled

metabolite mean from the

constant

decreased

specific 21 to

activity

of

recrystallize

to

added

as

tracer

lites

formed

first,

constant did

with

2 to

3

Fig.

1:Radioiuznunoassay from 18-day-old

of rats.

ions

second

the

12-15).

T+FSH

T

Effect

in Sertoli FSH.

and

the

ranged 132% in

failed

to

The metaboconverted.

T+FSH

96

of

During

l4 C-estradiol-17b

substrate

estradiol-17B

not

from

and 60 to

metabolite

samples

hr

did 15).

deviation

recrystallizat

15,

the

estradiol-178-like

2 as percent

1

0

the

activity,

Table

Table

percent

H-labeled

specific

in Table

in

the

FSH,

estradiol-178

23% in the

the

(Appendix,

are presented

H-label

consecutive

While

sample.

3

the

significantly

101% in the

third

activity

without

hr

cell

cultures

III.

The effect of FSH on the metabolism of testosterone by Sertoli cells

derived

from

l&day-old

rats

utilizing

saturating

substrate (unlabeled) - conditions. Radioimmunoassay:

RIA results

indicate

that

an estradiol-17S-like

compound was present in Sertoli cell cultures from 18-day-old rats. It persisted

throughout

the

and cross reacted with estradiol-17b-like

purification

procedures

the estadiol-176

used

antiserum.

prior

to assay

FSH increased this

compound by 333% during 96 hours of incubation (Fig.

1). IV.

14 C-testosterone

Metabolism

of

36-day-old

rats

utilizing

by

Sertoli

saturating

cells

(low

derived

specific

from

activity)

substrate conditions: A. In the absence of FSH: The cultured dione,

Sertoli cells metabolized

dihydrotestosterone

compounds

were

and

identified

by

crystallization

authentic unlabeled

observed

crystallization

steroids

added

for

recovery

crystallize 14 C-label

to in

from

during

and

178.

The

l4C-metabolite

and

constant

specific

to constant

authentic Table

activity

3

ratio

H-labeled

16).

(Appendix,

estradiol-17B-like

specific

lizations reached 0 for estrone

to

The

these

as

tracer

14C-labeled

like estrone and estradiol-17B did not

consecutive

the mean

the

(Appendix,

specific

estrone-like

significantly deviation

constant

steroids,

of

metabolites that chromatographed

to androstene-

5cr-androstane-3cr,178-diol. All

activity with by

testosterone

and ranged

of

The

decreased

The

consecutive

from 5-62X

failed to recrystallize

16).

compounds

recrystallizations. activity

Table

percent

recrystal-

for estradiol-

to constant

specific

S activity

while

3 I-I-estradiol-178 added as tracer did. The metabo-

the

lites formed are presented substrate

converted

metabolized

substrate terone

and

or 41.6%

metabolized

most

protein.

The

total

of

represented

the total metabolism.

to the above androgens

predominant

Thus

of

substrate

(3%)

with (2.3%)

a

6.4%

275 ng

of

of

lesser

large

amount

amount

of

TABLE 3 TESTOSTERONE METABOLITES PRODUCED BY SERTOLI CELLS CULTURED PROM 36-DAY-OLD RATS. SUBSTRATE = [4- 14CI-TESTOSTERONE (57 qCi/mnol)-1.0 pCi

Steroid Testosterone c5) Androstenedione Dihydrotestosterone b-Androstane-3a,178-dial Estrone Estradiol-178 Unidentified radioactivity 5See Key to Appendix

Conversion (84.6) 3.0 1.0 2.3 ----------9.0

ng Steroid Formed (3662) 132 45 99

------389

the was of

dihydro-

testosterone being formed.

%

the

testos-

to androstenedione

proportionately

and

dihydro-

(Table 3). Considering

testosterone metabolism

liu-androstane-3o,178-dial

amount

and as ng

132 ng/mg protein. Androstenedione,

5a-androstane-3a,l78-diol

identified metabolites, the

in Table 3 as percent conversion mg

per

was 15.3% or

testosterone

TXlROXDb

xdmg Protein (722) 26 9 20 _----_----77

S

TBEOXDI

B. In the presence of FSH: Testosterone 3u,178diol

was metabolized

(Appendix,

Table

to androstenedione

17).

The

counts

and 5u-androstane14 C-dihydrotestos-

of

terone present were insufficient for recrystallization. samples

without

graphically constant

like

mean

the

estrone

specific

estrone-like during

FSH,

consecutive

activity

estrone

failed

to recrystallize 3

and

of

metabolites

estradiol-178

(Appendix,

consecutive

9-772

for

did

Table

compounds

recrystallizations.

for

metabolites

and

activity

17-69%

and

C-labeled

and estradiol-178-like

specific

estrone

14

behaving not

17).

The

decreased

recrystallizations

estradiol-17 8. The

chromato-

crystallize

to

14C-label

in

significantly

The percent deviation

from the

ranged

from

14 C-metabolites the

3 H-

as tracers did. Table 4 shows

the

to constant specific

H-estradiol-17 8 added

Similar to the

activity, while

formed as percent conversion and as ng substrate

converted

per mg protein.

TABLE 4 EFFECT OF FSH ON TESTOSTERONE METABOLITES PRODUCED BY SERTOLI CELLS CULTURED FROM 36-DAY-OLD RATS. SUBSTRATE = [4-14C]-TESTOSTERONE (57 mCi/wol) 1.0 $i

Steroid

% Conversion

Testosterone(5) (92.0) 2.9 Androstenedione Dihydrotestosterone(6) 0.0 5a-Androstane-3a,178-dial 0.4 _----Estrone ---_-Estradiol-178 Unidentified radioactivity 4.7 6Not crystallized

ng Steroid Formed (3979) 126 0 19 _------------_203

nglmg Protein (785) 25 0 4 _-------------40

S The total

substrate

represented

3.3%

terone

of

the a

metabolism

representing

to

substrate

identified

of

145

of

the

of

testosterone.

ng

the

was

or 36% of

was 8.0%

or

5o-androstan-3o,l7S-diol

41.5%

androstenedione

the substrate

steroids

and

or

metabolism

to

3% of

509

Andros t ened ione

representing

thus

metabolized

protein.

ndmg

29

TDEOXDI

total

metabolism, Testos-

predominant

total

metabolite

metabolism

(Table

4).

DISCUSSION In studies of

the

the

cell

it

our

shown

to

bind

cell

LH)

localization

cultures

yielded

cells,

Serto li

ce 11s.

germ cells

(

are attributed

(2,3,10,11).

the that

these

< 10%) their

study

of

with

Although

metabolism

Evidence

results

of to

suggests

testosterone the that

seminiferous Recently,

Sertoli the tubules

we

have

shown

we established

the

and

respond

to

to

cyto-

a small

in

these

(11).

These of

the

predominantly proportion

was low or absent various

FSH

Moreover,

contained

the

been

3’-5’

characteristics

activity to

have

endogenous

previously

contained

5o-reductase

free

oxidoreductase

cultures

cultures

prepared

(17,18,21).

morphologic the

are

cells

of

purity

steroid

Sertoli

levels

as

testicular

study

3S-hydroxysteroid

indicated

the

activity

(CAMP)

the

when determining

cells

(17,18)

monophosphate

negative

Thus,

I-FSH

increased

of

the

present

with

together

cultured

the

to

cells that

types,

thus

The cultured

125

specifically

chemical

parameters,

important In

cell

established,

Sertoli

preparations.

stimulation

testicular

be

of

extremely

adenos ine

cyclic

should

contamination.

of

not

is

cell

purity

(but

isolated

contribution

specific

Leydig

with

preparations

metabolism, of

dealing

steriods

of (9).

in

this

cells. observed may

steroid

reside

domonstrated

in that

metabolic the

enzyme

Sertoli

cultures

cells of

pure

S

510

Sertoli

cells

isolated

transforming Others rat

have

testes

testes

rat

testosterone

demonstrated

that

cultures

capable

of and

present are

adult

to

are

the

from

progesterone

dihydrotestosterone from

WIIEOXDM

to

and of

metabolizing

are

demonstrate

Sertoli

cells

testosterone

convert

dihydrotestosterone,

that

to

of

bio-

cells

testosterone

imoature

androstenedione, The

from to

5o-androstane-3a,178-dial

(11).

from

(12,131.

Sertoli

exogenous

capable

dihydrotestosterone

5a-androstane-3a,l7B-diol

study

able

testes

immature

andros

and

results rat

tenedione,

3 a-hydroxy-5

o-

androstan-17-one. Substrate reflects

conversion

the

cultered

activity

to

presence

of

indicates

that

influenced

by

metabolism,

study

two

specific

androgens

during

immature

been early

effect

:

show

untreated 3o,17B-diol

stages

of

effect

of

development substrate

conditions,

were

androgen

metabolism

cells

FSH on

are

androgen

5a-reductase, (12,13).

In

to by

3o-OR

the

conditions

used

the

evidence

Sertoli

FSH stimulates

saturating

a)

the

for

Strong

in

of

substrates

necessary

androgens.

of

ability

present b)

;

critically Sertoli

high inves-

cells

from

animals.

presence no

that

FSH on

One-way-analysis the

shown

substrate of

case

the

steroid

enzymes

enzymes

the

metabolites

Thus,

various

5o-reduced

In

approaches

the

steroidogenic

FSH (11-16).

activity

tigate

of and

intermediary

enzymes.

biotransform

steroidogenic

has

steroid

to

these

it

17S-OR

specific

steroidogenic

ce 11s

the

synthesis

and

of

Sertoli

points

to

and

of absence

significant except (Table

variance of

5),

the

the

FSH using

difference for

of

at ratio

indicating

of

saturating

the of

ratios

5% level

metabolites

formed

substrate

conditions

between

FSH-treated

in

and

dihydrotestosterone/5a-androstanethat

FSH may

have

stimulated

3a-OR

activity. the

when high

However,

ratios

absence

between

of

both of

strate

the

ratio

question

bioconversion.

Data

radioimnunoassay are

Sertoli

3a-OR

formed

in

that

were

the

activity.

FSH did

not

used,

presence

FSH significantly

(5%

and

level)

One-way-analysis for

testosterone/androstenedione

of

both ions,

significantly

at

crystallize

in

the

of

both

sub-

(5% level)

to

in each the

this

used

study

a

and

high

experiment, of

constant

specific

the

Sertoli

cells

estradiol-178-like

compound was not estradiol-176.

each

activity

have

or

as

compound

cons t ant

of

failed ratio.

estradiol-176

clearly

from

isolated

but

recovery

shown

careful

metabolite

radiolabeled

been

where

substrate

estradiol-17S

This

our

like

compound

this

activity.

Sertoli

However,

this

incubation

and

cross-reacted

FSH, showed that like

8 uch

finding

formation.

authentic

that

a similar

of

of

addition,

activity

specific the

In

specific

chromatographically cons t ant

suggest

that its

to

testosterone

(14-16).

results

without

termination

strongly

substance

and

capable

non-labeled

suggests

FSH stimulated

to

imnunoassayable

where

testosterone

en 2 ymes

of

crystallization

with

metabolize

metabolism

such

produced

behaved

crystallize

presence

were

9 aturat ing

testosterone

Meanwhile,

of

data

cells

studies

and where

examination

b ioconvers

the

techniques

cells

estradiol-176,

although

and

Sertoli

of

capable

radioimnunoassay

added

of

whether implies

to

that

indicates

estradiol-176

from

indicate

substrates

176-OR activity.

The

cells

5)

activity

metabolites

5crreductase

conditions

stimulate

various

FSH (Table

stimulated variance

the

specific

tracer

indicates to

produce

testosterone,

did that an this

S

512

'ELIEOXDI

TABLE 5 RATIO OF TESTOSTERONE METABOLITES FORMED BY SERTOLI CELLS IN CULTURE (%)

Age (days) Substrate Treatment

18 14C Control

Testosterone/androstenedione 16.5 Androstenedione/Sctreduced C-19 steroids 1.3 Androstenedione-Sa-androstane-3a,17&diol 2.2 Androstenedione/Sa-androstane-3a-,17&diol + 3ahydroxy-5cl-androstan-17-one 2.0 Dihydrotestosterone/501androstane-3a,17&diol Androstenedione!3a-hydroxy5a-androstan-17-one

0.6* 19.0

36 14c

FSH

3n Control

FSH

Control

11.4

11.2

10.9

27.8

31.6

1.2

0.9

0.6

0.9

6.6

2.0

2.5*

0.7

1.3

6.8

1.8

1.4*

0.7

-----

-----

0.2

1.0*

0.2

0.5

0.0

3.5

3.4*

0.7

_----

_-___

FSH

*Significant = 5% level (one way analysis of variance). The

data reported

in this

study

demonstrates

the presence

of

178-OR, Sa-reductase and Z&X-OR enzymes in Sertoli cells derived from imooature rats, and that FSH may influence the ability of Sertoli biotransform using

the

Furthermore, compounds

cells in the presence or absence of FSH to

testosterone various

do

to

estradiol-176

radiolabeled

was

metabolic

not

substantiated

techniques

indicated.

the results of this study indicate that irenunoreactive not

caution must

be

authenticity

of

necessarily

exercised the

when

compound

represent

is

steroids

and

data before

the

authentic

interpreting

identity of the estradiol-17B-like known.

their activity. However,

such

established.

At

present,

compound detected by RIA

the

is not

S

CPDEOfDrn

REFERENCES 1. Bass, J.J., Bell, J.B.G. and Lacy, D., J. ENDOCRINOLOGY a, 321, (1973). 2. Christensen, A.K. and Mason, N.R., ENDOCRINOLOGY 2, 646, (1964). 3. Hall, P.F., Irby, D.C. and deKretser, D.M., ENDOCRINOLOGY 84, 488, (1969). 4. Yamada, M., Yasue, S. and Matsumoto, K., ACTA ENDOCRINOL. 71, 393, (1972). 5. Folman, Y., Sowell, J.G. and Eik-Nes, K.B., Ahmad, N., ENDOCRINOLOGY 92, 41, (1972). 6. Rivarola, MA., Podesta, E.J. and Chemes, HE., ENDOCRINOLOGY 91, 537, (1972). 7. Ryvarola, M.A. and Podesta, E.J., ENDOCRINOLOGY 90, 618, (1972). 8. Dorrington, J.H. and Fritz, I.B., BIOCHEM. BIOPHYS. RES. COMM. 34, 1425, (1973). 9. Dorrington, J.H. and Fritz, I.B., ENDOCRINOLOGY E, 879, (1975). 10. Christensen, A. and Fawcett, D.W., J. BIOPHYS. BIOCHEM. CYT. 2, 563, (1961). 11. Tcholakian, R.K. and Steinberger, A., ENDOCRINOLOGY 103, 1335, (1978). 12. Welsh, M.J. and Wiebe, J.P., BIOCHBM. BIOPHYS. RES. COMM. 69, 936, (1976). 13. Welsh, M.J. and Wiebe, J.P, ENDOCRINOLOGY 103, 836, (1978). 14. Dorrington, J.H. and Armstrong, D.T., PROCEEDINGS OF NATIONAL ACADEMY OF SCIENCE, U.S. 72, 2677 (1975). 15. Armstrong, T.D., Moon, S.Y. Fritz, I.B. and Dorrington, J.H., CURRENT TOPICS IN MOLECULAR ENDOCRINOLOGY, Eds. French, Hansson, Ritzen and Nayfeh. Plenum Press, New York, p. 85, (1975). 16. Dorrington, J.H., Fritz, I.B. and Armstrong, T., BIOL REPROD. 3, 44, (1978). Lindsey, J.N., Elkington, 17. Steinberger, A,, Heindel, J.J., J.S.H., Sanborn, B.M. and Steinberger, E., ENDOCRINOL. RES. COMM. 2, 261, (1975). 18. Steinberger, A., Elkington, J.S.J., Sanborn, B.M., Steinberger, E Heindel J.J. and Lindsey, J.N., HORMONAL REGULATION OF S&MATOGENE&S, Eds. F.S. French, V. Hansson, E.M. Ritzen and S.N. Nayfeh, Plenum Press, New York, p. 399, (1975). 19. Layne, E., METHODS IN ENZYMOLOGY, Eds. S.P. Colowick and N.O. Kaplan, Academic Press, New York, P. 488, (1957). 20. Rodbard, D. and Lewald, J.E., ACTA. ENDOCR. (KBH) S~PPL. 147, 79, (1970). 21. Heindel, J.J., Rothenberg, R., Robison, G. and Steinberger, A., J. CY. NUCL. 1, 69 (1975).

s

514

TDDOXDrn

TO APPENDIX

KEY

Tables

No

6 -

17

= Sample number

C.W. = Crystal

weight

Mass = Steroid

added

(1)

Solvent

for

crystallization

pairs

A = Acetonefhexane B = Acetone/isooctane C = Acetone/heptane D = Dioxane/isooctane E = Tetrahydrofuran/isooctane

(2)

Substrate

derived

SA = Specific

qetabolites

activity

(3)

Recovery

tracers

added

(4)

Dev. % SA = Deviation activity

(5)

Unconverted

Substrate

from the mean specific

S

WXIEOXDI

515

TABLE 6

CRYSTALLIZATION OF 14C-TESTOSTERONE(ACETATE) AS UNCONVERTED SUBSTRATE FROM CULTURED SERTOLI CELLS PREPARED FROM l&DAY-OLD RATS SUBSTRATE = [4-14C]-TESTOSTERONE(57 mCi/mnol), 0.25,~ Ci./SAMPLE/lx lo6 CELLS

-No.

Treatment -No.

1

None

(3)

(2)

(1) MaSS

-S

cl&

14 C SA (dpm/mg)

Dev. ; SA (%)

3 HSA (dpm/mg)

(4) Dev. ii SA (x)

332 217 207

A E B

20

II III

15900 15500 16000

0 2 1

1650 1650 1700

1 1 2

I

2

None

I II III

229 251 225

A E B

20

18400 17400 17200

4 1 3

1750 1670 1690

3 2 1

3

None

I II III

223 227 224

A E B

20

21200 20800 20900

1 1 0

2000 2020 2050

1 0 2

4

None

I II III

249 199 223

A E B

20

17900 17400 17800

1 2 0

1730 1690 1730

1 2 1

5

FSH

I II III

268 198 247

A E B

20

12900 13100 13100

1 1 1

1140 1160 1180

2 0 2

6

FSH

I II III

212 194 214

A E B

20

16500 16600 16600

1 1 0

1500 1490 1470

1 0 1

FSH

I II III

194 258 276

A E B

20

20400 19600 19600

3 1 1

1770 1770 1760

0 0 0

FSH

I II III

255 196 262

A E B

20

11000 10600 11300

0 3 3

1000 989 999

1 1 0

lp2y3y4 See Key to Appendix.

s

516

WEIEOXDm

TABLE 7 CRYSTALLIZATIONOF 14C-ANDROSTENEDIONEPRODUCED BY CULTURED SERTOLI CELLS PREPARED FROM la-DAY-OLD RATS SUBSTRATE = [ 4-l4c I-TESTOSTERONE(57 mCi/mmol), 0.25 uCi/SAMPLE/l x lo6 CELLS

(2)

(1)

2NO

Treatment -No.

1

None

I II III

2

None

3

MaSS

14 C SA &m/mg)

(3) Dev. ii SA (%)

3 H SA (dpm/mg)

(4) Dev. < SA (%)

-S

w

270 189 258

A C A

20

310 332 322

4 3 0

522 517 500

2 1 3

I II III

259 184 217

A C A

20

497 515 480

0 4 4

641 669 683

4 1 3

None

I II III

233 248 122

A C A

20

385 403 384

2 3 2

660 690 678

2 2 0

4

None

I II III

276 179 318

A C A

20

779 728 782

2 5 3

569 599 572

2 3 1

5

FSH

I II III

173 233 196

A C A

20

617 630 634

2 1 1

767 742 741

2 1 1

6

FSH

I II III

254 167 198

A C A

20

786 783 749

2 1 3

600 629 632

3 1 2

7

FSH

I II III

223 205 204

A C A

20

497 516 495

1 3 2

699 696 681

1 1 2

8

FSH

I II III

241 322 306

A C A

20

340 350 330

0 3 3

365 363 357

1 0 1

1~2v3p4 See Key to Appendix.

S

TCBEOXDI

517

TABLE 8 CRYSTALLIZATION OF I4c-DI~YDR~TESTOSTERONE(ACETATE) PRODUCED BY ~LT~D SERTOLI CELLS PREP~D FROM 28-DAY-OLD RATS SUBSTRATE = [~-~~~]-~STOSTE~NE (57 mCibol), 0.25 ~i./SAMPLE/lx lo6 CELLS

(3)

(2)

(1)

14 C SA (dpm/mg)

Dev. x SA (x)

3 H SA (dpm/mg)

(4) DeV. ii SA (x)

C.W. $J&

-S

b&

I II III

159 401 248

A B A

10

904 956 936

3 3 0

264 288 272

4 5 1

None

I II III

178 151 251

A B A

10

956 920 956

1 3 1

320 304 336

0 5 5

None

I II III

306 533 547

A B A

10

992 1020 1060

3 :

344 352 368

3 1 4

-No.

Treatment -No.

1

None

2

3

MaSS

4

None

I II III

305 313 268

A B A

10

1000 1080 996

2 5 3

495 469 480

3 3 0

5

FSH

I II III

172 548 104

A B A

10

1030 1020 1030

1 1 0

350 356 340

0 2 3

6

FSH

I 11 III

213 489 404

A B A

10

872 864 888

0 1 2

319 316 326

0 1 2

7

FSH

I II III

326 113 179

A B A

10

1040 1050 1050

0 0 0

421 423 411

1 1 2

8

FSH

I II III

164 322 205

A B A

10

924 924 956

1 1 2

324 308 312

3 2 1

1,2,3,4 See Key to Appendix.

S

518

TEEOXDl

TABLE 9 CRYSTALLIZATION0~ 14C-5a-ANDROSTANE-3a,17B-~~~~ (DIACETATE) PRODUCED BY CULTURED SERTOLI CELLS PREPARED FROM 18-DAY-OLD RATS SUBSTRATE = [4-14C]-TESTOSTERONE(57 mCi/nmo .), 0.25 uCi/SAMPLE/lx 106 CELLS

(1)

NO. -

TreatNo. ment -

1

None

2

Mass

C.W.

(3)

(2) 14

C SA (dpm/mg)

Dev. ii SA (%)

3H SA (dpm/mg)

(4) Dev. X SA (%)

(Ile>

-S

iE!d

I II III

402 139 229

A C A

10

290 287 270

3 2 4

485 481 471

1 0 2

None

I II III

230 210 220

A C A

10

547 552 544

0 1 1

556 585 567

2 3 0

3

None

I II III

235 129 154

A C A

10

412 417 396

1 2 3

552 565 564

2 1 1

4

None

I II III

250 299 242

A C A

10

327 311 309

4 1 2

323 341 321

2 4 2

5

FSH

I II III

190 800 220

A C A

10

385 399 372

0 4 4

490 474 490

1 2 1

6

FSH

I II III

131 206 135

A C A

10

535 506 544

1 4 3

359 365 344

1 3 3

7

FSH

I II III

170 158 233

A C A

10

276 266 274

1 2 1

441 450 450

1 1 1

8

FSH

I II III

267 486 127

A C A

10

574 569 572

0 0 0

446 462 471

3 1 3

1,2,3,4 See

Key

t0

Appendix.

S

519

TBEOXDI

TABLE 10

CRYSTALLIZATION OF 14C-ESTRADIOL-17&LIKE METABOLITR ISOLATED FROM CULTURED SERTOLI CELLS PREPARED FROM 18-DAY-OLD RATS SUBSTRATE = [4-14C]-TESTOSTERONE(57 mCi/auuol) 0.25 ~Ci/SAMPLE/l x 106 CELLS

(1) No

Treatment -No.

1

None

2

None

2

6

7 6 8

C.W.

(3)

(2) MESS

14 C SA (dpm/mg)

Dev. i? SA (%)

3H SA (dpm/mg)

(4) Dev. f SA (%)

IEd

-S

(mg)

I II III

150 191 129

D E D

10

606 283 181

70 21 49

405 419 397

3 3 3

I II III

183 190 325

D E D

10

493

227 171

66 24 42

312 300 295

3 1 2

None

I II III

180 271 276

D E D

10

835 242 119

109 39 70

437 446 416

1 3 4

FSH

I II III

128 242 161

D E D

10

353 133 87

85 30 55

313 319 311

0

I II III

197 204 278

D E D

10

360 122 82

92

35 56

177 176 165

3 2 4

I II III

100 316 187

D E D

10

170 171 171

0 0 0

FSH

FSH

1y2,3,4 See Key to Appendix.

815 330 110

95

21 74

2 1

s

520

TIIEOXDI

TABLE 11 CRYSTALLIZATION OF 3H-TESTOSTERONE (ACETATE) AS UNCONVERTED SUBSTRATE FROM CULTURED SERTOLI CELLS PREPARED FROM 18-DAY-OLD RATS SUBSTRATE = [la, 2a-3H]-TESTOSTERONE(1 mCi/4.09 Vg) 1.25 &i/SAMPLE/l x lo6 CELLS

(1) -No.

Treatment -No.

C.W.

(2) MSSS

L&L

-S

m

3 HSA (dpmfmg)

(3) Dev. X SA (x)

14 C SA (dpm/mg)

(4) Dev. si SA

(x)

9

None

I II III

321 187 241

A E B

20

4380 4240 4170

3 1 2

110 108 101

3 2 5

10

None

I II III

291 332 239

A E B

20

18200 17200 17100

4 2 3

369 394 396

5 2 3

11

None

I II III

252 301 208

A E B

20

7680 7630 7480

1 0 2

173 163 177

5 4

I II III

294 174 372

A E B

20

4270 4220 4260

0 0 0

105 109 101

t

12

FSH

1

0

13

FSH

I II III

247 264 307

A E B

20

21800 20600 20900

3 3 1

537 541 524

1 1 2

14

FSH

I II III

235 212 219

A E B

20

16100 14800 15400

4 4 0

408 403 388

2 1 3

15

FSH

I II III

304 279 271

A E B

20

13900 13900 13600

1

378 387 364

0

1,2,

p4 See Key to Appendix.

1 2

3 3

S

TFPEOXD=

521

TABLE 12

CRYSTALLIZATIONOF 3H-ANDROSTENEDIONEPRODUCED BY CULTURED SERTOLI CELLS PREPARED FROM 18-DAY-OLD RATS SUBSTRATE = Llcr,2~-3H]-TESTOSTERONE(1 mCi/4.9 ug) 1.25 "Ci/SAMPLE/l x lo6 CELLS

(1) TreatNo qent No. --1.-

C.W.

!l!d

-S

(2) Mass w

3 H SA (dpm/mg)

Dev. x SA (%)

(3) l4 C SA (dpm/mg)

(4) Dev. x SA (%)

9

None

I II III

329 224 225

A C A

20

1800 1820 1840

1 0 1

28 25 27

5 6 1

10

None

I II III

352 160 245

A C A

20

687 661 684

1

99

2 1

93 105

0 6 6

11

None

I II III

298 237 291

A C A

20

900 929 921

2 1 1

53 48 48

7 3 3

12

FSH

I II III

147 174 184

A C A

20

238 229 233

2 2 0

24 25 24

1 3 1

13

FSH

I II III

348 230 109

A C A

20

997 995 963

1

1 2

122 117 119

2 2 0

I II III

240 275 266

A C A

20

1040 1040 1010

1 2

128 130 125

0 2 2

I II III

293 219 231

A C A

20

938 904 941

1 3 1

122 123 118

1 2 3

14

15

FSH

FSH

lp2p3y4 See Key to Appendix.

1

6L

522

TBEOXDl

TABLE 13

CRYSTALLIZATIONOF 31i-DIHYDROTESTOSTERONE (ACETATE) PRODUCED BY CULTURED SERTOLI CELLS PREPARED FROM 18-DAY-OLD RATS SUBSTRATE = Llcr,2cr3H]-TESTOSTERONE(1 mCi/4.9 pg) 1.25 pCi/SAMPLE/l x lo6 CELLS

(1) No. -

9

Treatment

None

C.W. c&_

No ;

None

A C A

10

946 994 966

2 3 0

207 258 255

A C A

10

820 822 822

0 0 0

333 347 190

A C A

10

II III

509 529

I II III

184 416 540

A C A

10

I

II II 11

12

None

FSH

Mass s(mp>

(4) Dev. X SA (4;)

252 160 161

I

II III 10

(2) 3 HSA (dpm/mg)

I

488

0

4 4

944

914 950

2 2

13

FSH

I II III

259 224 472

A C A

10

886 868 876

1 1 0

14

FSH

I II III

451 394 415

A C A

10

888 874 884

0

I II III

363 193 093

A C A

10

15

FSH

lF2,4 See Key to Appendix.

996 970 980

1

1 0

S

=J?mEOXDI

523

TABLE 14

CRYSTALLIZATION OF 3li-5ct-ANDROSTANE-3a,17f3-DIOL (DIACETATE) PRODUCED BY CULTURED SERTOLI CELLS PREPARED FROM la-DAY-OLD RATS SUBSTRATE = [la, 2u-%l]-TESTOSTERONE(1 mCi/4.9 up) 1.25 uCi/SAMPLE/l x lo6 CELLS

(1) No. 9

10

11

12

Treatment -None

None

None

FSH

C.W.

(2) MaSS

S

(mg)

167 191 181

A C A

10

I II III

193 194 407

A C A

10

I II III

162 313 194

A C A

10

I II III

131 172 182

A C A

10

No.

Q&l

I II III

-

3 HSA (dpm/mg)

928

857 865

(4) Dev. X SA

Testosterone (5)

I II III

278 256 247

A B A

31.8

36400 36300 36600

0 0 0

1330 1370 1380

2 1 1

Androstenedione

I II III

172 420 551

A B A

23.2

2970 2870 2900

2 1 1

883 873 863

1 0 1

I II III

1070 333 339

A

21.1

B A

368 369 351

2 0 2

727 716 714

1 0 1

Estrone

I II III

136 146 128

D E D

10.6

165 81 47

69 17 52

411 405 390

3 2 5

Estradiol-17S

I II III

223 229 321

D E D

10.0

476 309 64

68 9 77

1100 1040 1060

3 2 1

5a-Androstane-3a, 17@-diol

1, 2, 3, 4, 5 See Key to Appendix