771 IN VITRO

EFFECTS

STEROID METABOLIC STUDIES IN HUMAN TESTES I: OF ESTROGEN ON PROGESTERONE METABOLISM

Luis J. Rodriguez-Rlgau, Robert K. Tcholaklan, Keith D. Smith and Emil Steinberger Department of Reproductive Medicine and Biology The University of Texas Medical School at Houston Houston, Texas Received: l-31.-77

ABSTRACT

A technique of incubation of testicular tissue in vitro with radiolabeled precursors w IS applied in the investigation of the steroid biosynthesis by testes of four young men ~f.er long-term, high-dose estrogen treatment. A positive correlation between plasma and testicular steroid levels, and in vitro capacity of the testes to metabolize progesterone was demonstrated. Estrogen administration produced a very significant inhibition of plasma and testicular levels of testosterone. The in vitro synthesis of testosterone from progesterone was very severely especially 17cy-hydroxylation of progesterone. 2Ocu-hydroxyimpaired; steroid-dehydrogenase activity was found to be increased after estrogen treatment, both in viva and in vitro. These findings suggest that testicular 1‘la-hydroxylase activity (and possibly also 17-20 lyase ; ctivity) is either under gonadotropin regulation, or is directly suppressed by estrogen. This could result by decreased enzyme synthesis, direct enzyme inhibition or affectation of the cofactors or cytochromes necessary for the enzymatic activity. 20a-reduction of C2lsteroids would represent an alternative pathway for their catabolism, not regulated by gonadotropin or not affected by estrogen, that would be significant in situations with reduced 17cu-hydroxylase activity. INTRODUCTION With the use 01’in vitro techniques precursors,

considerable

information

biosynthetic

steps involved in androgen

of incubation has been formation

it became very difficult to obtain information Therefore,

most

orchiectomy, patients

in vitro

usually

from elderly

with testicular

(8) and Steinberger

feminization

in recent

years

on the

in human testes. For obvious reasons,

on “normal” human testicular metabolism.

studies patients

accumulated

were performed with carcinoma

in tissues

obtained

at

(1,2,3)

or

of the prostate

(4,5,6).

Before the studies by Danezis (7), Schoen

et al. (3) using testicular

biopsies of as little as 15 mg tissue weight,

almost no information

was available on the steroid biosynthetic

pathways of testis from

age. Steinberger et al. (3), (9) presented results of in vitro metabolic

men of reproductive

studies in normal volunteers They demonstrated

metabolic

of testicular tissue with radiolabeled

and in patients with various spontaneously

a correlation

of the pattern

occurring disorders.

of in vitro steroid metabolism

with the

histologic appearance of the testes, the clinical picture of the patient, and in some instances with the in vivo biochemical

parameters

Vokne

S

29,

Nwnber

6

of androgen

=EEI&OXDI

production.

It was clearly shown

June,

2977

S

772

-X-DEOTDI

extraction (3 x 5 ml) with ethyl-acetate. The extracts were washed three times with 1 ml of distilled water to eliminate all residues of pyridine. Purification: The dried extracts from acetylation were chromatographed again on silica-gel thin-layer chromatography in benzene:ethylacetate (4: 1 ,V/V), and radioactivity behaving like authentic testosterone-acetate, androstenedione, 17-hydroxyprogesterone and 20adihydroprogesterone-acetate was scraped, suspended in double-distilled water (OSml) and extracted with benzene (3 x 5 ml). Final identification and quantitation of metabolites: Authentic steroids (S-20 mg) were added to each of the residues derived from the last chromatography. The mixtures were then recrystallized to constant specific activity and 3H/14C ratio from different solvent combinations. The amounts of progesterone substrate converted to specific metabolites, expressed as percent conversions, were calculated from the crystallization data, by the use of the formula: 3H dpm crystal x 14C dpm added % conversion = x 100 14C dpm crystal x 3H dpm substrate Posteriorly, the data were expressed as picomoles per mg protein, determined by the method of Lowry et al. (38). The amounts of unconverted substrate and unidentified metabolites were calculated from the radioactivity in the first chromatogram. Chemicals and reagents: Nanograde solvents (Mallinckrodt) were used, and all evaporations were done in an atmosphere of nitrogen. Non-radioactive steroids were obtained from Steraloids, Inc., or Sigma Chemical Co. In addition to the substrate (3H-pro esterone), the following radioactive steroids were purchased from Amersham Searle Co.: 4- Ig4C-progesterone (61 mCi/mmol), 414C-1 7hydroxy-progesterone (50 mCi/mmol), 4-14C-androstenedione (60 mCi/mmol), 4-14C-testosterone (59 mCi/mmol), 4-14C-estrone (58 mCi/mmol) and 414Cestradiol (SO mCi/mmol). All isotopes were checked for radioactive purity by subjecting aliquots to paper- and thin-layer chromatography. Acetic anhydride and pyridine used in acetylation reactions were distilled over fused sodium acetate and borum oxide, respectively, and stored in a dessicator over anhydrous calcium chloride. NADP and glucose-6-phosphate were stored in a dessicator at -20°C. Nicotinamide and glucose&phosphate dehydrogenate were stored at 4°C. Measurement of radioactivity: Radioactivity in samples was measured with a liquid scintillation spectrometer (Packard Model B2450) in a toluene-PPO-POPOP system. The efficiency in simultaneous 3H and 14C counting was 59% and 0.02% for 3H, and 69% and 16% for 14~ in the respective channels. RESULTS

A.

Histology The testicular

biopsies obtained

in patient

of spermatogenic

arrest at the primary spermatocyte

a uniform

picture

peritubular

fibrosis and mature

After treatment, the same changes:

D.A. prior to estrogen treatment

Leydig cells in the interstitial

microscopic

the diameter

and there was heavy hyalinization

examination

showed

level, moderate

area.

of the testes of all four patients showed

of the seminiferous

tubules

was markedly

and fibrosis. Most tubules contained

some had few germinal stem cells, and very few occasional

primary

diminished,

only Sertoli cells; spermatocytes.

The

S that

testicular

especially

tissue of younger

773

men had higher content

Syndrome,

testicular steroidogenesis

hypogonadotropic testosterone

with marked elevations was demonstrated,

17-hydroxyprogesterone

being

formed

hypogonadism

(6)

with considerable amounts of testosterone from

abnormality

feminization

demonstrated

progesterone.

active

cases

of progesterone

of

prepuberal

were converted

to

gonadotropins

production

steroid

in a prompt

increase in sperm count resulted

is still controversial

AS early as 1956, Samuels and Helmreich testicular

with gonadotropin.

et ~2. (14), Acevedo (15X Carstensen

(20)

Steinberger

Steinberger et al. (24) demonstrated and rat testicular of gonadotropin

steroidogenesis

at the

(16), Huseby

the

enzymes following more extensive a progressive in

dehydrogenase

Stimulation

of 17-20 lyase and

has been reported repeatedly:

by hypophysectomy

the 17fl-hydroxy-steroid-dehydrogenase

with addition

as well as

in the process

(13) demonstrated

As-3 p-hydroxy-steroid-

17or-hydroxylase in the testis by gonadotropin

Menon

with

and fragmentary.

or as result of their removal are apparently

treated

etc. Removal of gonadotropin

and motility,

However, changes in steroidogenic

than

rats

of this patient

of

possibly by specifically stimulating

with gonadotropin

of

Very small amounts

direct evidence that LH stimulates

of cholesterol.

activity

man with an

from this.

stimulation

hypophysectomized

17P-hydroxy-

to the role of gonadotropins

side-chain cleavage of cholesterol to pregnenolone,

in

and

Although this has been investigated extensively, especially

this information

Hall (1 l), (12) provided

pathway.

were formed. Treatment

our attention

of testicular steroid biosynthesis.

2Ocy-hydroxylation

17-20-lyase

on a case of an oligospermic

biosynthetic

rate. A pregnancy

All these findings brought

in lower mammals,

17a-hydroxylase,

and testosterone

resulted

testosterone

recent studies by Bell et al. (4), (5), and

et al. (10) reported

in the testicular

17-hydroxyprogesterone

of human

In

and

in these testes in vitro.

In 1974 Steinberger

enzymes:

of the prostate. In cases

of FSH and LH levels, a very active

small amounts

with testicular

steroid-dehydrogenase

increase

enzymes,

and 17-hydroxyprogesterone.

In patients

that.

of steroidogenic

17-20 lyase, than testis of older men with carcinoma

of Klinefelter

Schindler

TElROIDS

Dominguez

(17), Schoen (19), Hagerman (19) suppresses the activity of these two

(21), (22). Shikita and Hall (23) demonstrated

that

of rat testis is also suppressed by hypophysectomy. a progressive decrease of androgen synthesis by cultures

tissues with time in culture, and were able to stimulate to the culture.

They concluded

it

that the effect of culture

r%

774

on steroidogenesis

WDROXDC3

was due to the sudden removal of gonadotropin

stimulation,

rather

than a gradual loss of tissue viability. Very few studies have been reported the steroid biosynthetic

pathways

dealing with the effect of gonadotropins

of human

testicular

Most studies related to this topic were performed treated

with estrogens,

effect secondary Price

(26),

under the assumption

to negative

etc.).

Slaunwhite

steroid-dehydrogenase restoration

feedback

activity

of this activity

tissue. with testicular

that estrogens suppress gonadotropins

demonstrated

in tissues

of men

treated

with

of

estrogen.

Schoen

(8) concluded

and

No effect on

a suppression

but no change in 17@-reductase activity

with

17p-hydroxy-

diethylstilbestrol

administered.

the 17-20 lyase was observed. Tamaoki et al. (28) demonstrated

by

axis (Greep (25)

suppression

when H.C.G. was concomitantly

lyase and 17~hydroxylase,

tissues of patients

on the hypothalamus-pituitary

(27)

on

of 17-20

in testicular

tissue

of a patient

treated

gonadotropins

are essential for the activity of the 17p-reductase. In 1974 Oshima (2) for

the first time compared the in vitro biosynthetic with estrogen, concluding

Payne (29) demonstrated treatment.

that

activity of testes before and after therapy

that the 17~hydroxylase

affected by the treatment,

in a similar study

and the 17-20 lyase were maximally

and no definite effect could be observed on the 17p-reductase. inhibition

of As-3p hydroxy-steroid-

dehydrogenase after estrogen

This was confirmed by Fan (30). However, previous studies by Carstensen (16)

and Oshima (2) were in disagreement

with these findings.

All these studies clearly indicated

that the observation

the human testicular

testis. Gonadotropins steroidogenesis

pregnenolone.

the human

seem to play an important

beyond

the

step

of

However, while in lower mammalian

that gonadotropins

stimulate

activity

of a number

testis are in far less agreement.

were performed

on lower mammals apply to

cholesterol

role in the regulation side-chain

cleavage

of to

species most studies seemed to indicate of specific enzymes,

It should be remembered

on testes of aged men with carcinoma

the studies on

that all these studies

of the prostate,

that most likely

the clinical state of these individuals was not uniform, nor was the form, dose or duration of the hormonal

treatment.

This by itself could explain some of the discrepancies in results

reported.

On the other hand, except for one study (2), no comparison

function

before and after treatment

Consequently,

the purpose

vitro steroid biosynthesis

of the testicular

with estrogen was made.

of the present study was to compare the pattern

by testicular tissue of men of reproductive

of in

age, before and after

S long-term

estrogen

therapy,

hormonal

and histologic

TXIROXDS

and to attempt

to correlate

775 these results with the clinical,

findings.

MATERIALS

AND METHODS

A.

Patients Four male transsexuals: D.A., A.C., T.M. and G.S., ages 26,23, 28 and 46 respectively, were treated with ethinyl-estradiol (1-2 mg daily) for at least twelve months prior to orchiectomy and sex-change surgery. Hormonal studies were conducted at frequent intervals during the entire period of estrogen administration. At the time of surgery testicular tissue was obtained from histology studies, determination of testicular steroid concentrations, and for in vitro steroid metabolic studies. Patient D.A. was studied prior to and after initiation of therapy. Plasma levels of hormones were determined, and a bilateral testicular biopsy obtained. The remaining three patients had already been on estrogen therapy at the time of the first visit. Therefore, only results obtained while on estrogen therapy will be reported on these patients. B.

Hormonal Studies Serum FSH and LH were measured by double antibody radioimmunoassay, as previously described [Smith et aE. 1974 (31)]. Coefficients of variation were 15.8% for LH and 6.42% for FSH between assays and 9.2% for LH and 6.79% for FSH within assays. Plasma and testicular concentrations of testosterone were measured by our modification (3 1) of the radioimmunoassay technique described by Nieschlag and Loriaux (32) as previously reported. Interassay and intraassay variations were 5.18% and 3.93% respectively, as calculated from analysis of different concentrations of standards and from a plasma pool. Plasma estradiol was measured by our modification (Smith et al., (33) of a method described by Hotchkiss et al. (34); inter- and intraassay variations were 8 and 6% respectively. Testicular progesterone concentration was measured by our modification (Tcholakian et al., unpublished) of Thomeycroft and Stone’s (35) radioligand immunoassay. Testicular 20a-dihydroprogesterone concentration was determined by radioimmunoassay, using similar methodology as for testosterone and progesterone, as developed in our laboratory. Interassay and intraassay variations in all cases were within 8 and 6% respectively. C.

Histology Fresh testicular tissues were fixed in Cleland’s or Bouin’s fixatives and processed by standard histologic methods. Four micron sections were stained with periodic acid-Schiff (P.A.S.) or by the Mason’s-Trichromic method. D.

In Vitro Steroid Biosynthetic Studies The method utilized in the incubations of testicular tissue with radiolabeled precursors is a modification of those previously reported: Steinberger and Fisher (36) and Tcholakian and Eik-Nes (37). Tissue preparation and incubation. Immediately after orchiectomy or testicular biopsy, the tissue was placed in 0.25 M sucrose, 0.01 Tris buffer, pH 7.4 at ice temperature. Upon arrival in the laboratory, within 30 minutes of collection, the tunica albuginea was removed, and an approximately 50 mg fragment of testicular tissue excised, weighed and immediately placed in an incubation flask containing 1.5 ml of freshly prepared Krebs-Ringer bicarbonate buffer, pH 7.4 (at 37” C), and the radioactive substrate, which had been dissolved in a drop of absolute ethanol just prior to the addition of the buffer.

776

S

'PIIEOXDI

Following preincubation at 37” C for 5 minutes, 1.5 ml of NADP fortified Krebs-Ringer bicarbonate buffer was added to each flask. Composition of incubation medium (for total amount of 3 ml per flask): 3 ml of Krebs-Ringer bicarbonate buffer, pH 7.4 (at 37°C) 0.78 mg NADP 3.5 1 mg glucose-6-phosphate 11 mg nicotinamide 0.92 mg MgC12 a crystal of glucose-6-phosphate-dehydrogenase 6 mg of glucose Substrate: la, 2a3H-progesterone (Amersham TRK 341, batch 7), supplied by the manufacturers at specific activity of 47 Ci/mmol was reduced to specific activity of 0.33 nmol/,uCi by addition of unlabeled progesterone. Its purity was checked by paper and thin-layer chromatography. Incubations were carried out with IOpCi = 3.3 nmols of this substrate per flask. Incubation conditions: The flasks were incubated in a Dubnoff shaker in an atmosphere of 95% 02:5% CO2 at 37°C for 3 hours, at which time the reaction was terminated by the addition of 0.1 ml of 1N HCl. At this point 10 I.cg of the following unlabeled carrier steroids were added to each flask to facilitate detection on chromatograms: progesterone, 20ol_dihydroprogesterone, 17-hydroxyprogesterone, androstenedione, testosterone, 5a-androstane3a, 17P-diol, dihydrotestosterone, estradiol and estrone. The material was then frozen until further processing. Together with the samples, tissue-less controls were run. No 3H-labeled material besides the substrate was detected in any of these flasks. Extraction procedure: After thawing, the following 14C-labeled steroids were added to all flasks for correction of procedural losses (approximately 50,000 Dpm of each per flask): progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, estrone and estradiol. The samples were then extracted repeatedly with ether:chloroform (80:20, V/V), until background count was achieved in the incubation flasks, as well as in the aqueous phases in the extraction tubes. Initial Separation: The dry residues obtained from the ether:chloroform extraction were chromatographed on 50 cm-long paper strips (Whatman No. 1) in hexane:formamide to the front. The hexane was allowed to evaporate, and the paper was then rechromatographed in hcxane:benzene (50:50, V/V):formamide to the front. The chromatograms were dried at 45°C overnight. Carrier steroids on the paper were visualized under ultraviolet light (253 run), and their mobility compared to that of authentic standards. For the scanning of radioactivity a Packard Radiochromatogram scanner (Model 7200) was used. Radioactive areas were compared carefully with those of carrier steroids. The paper strips were then cut according to the radioactive peaks, and eluted separately with methanol (8 x 10 ml). The eluates were evaporated to dryness. Counts of radioactivity at this point yielded more than 90% of the initial radioactivity. Radioactive materials behaving chromatographically like authentic testosterone, androstenedione, 17-hydroxyprogesterone and 20adihydroprogesterone were subjected to silica-gel thin-layer chromatography in benzene:ethyl acetate (4: I, V/V). The steroids were located on the plates as dark zones using a short-wave ultraviolet lamp, and these areas were carefully compared with the radioactive peaks obtained by scanning with a Berthold Radiochromatogram scanner LB 2723, and the mobility of authentic standards. These areas were then scraped from the plates, extracted with methanol (5 x 5 ml) and the dry extracts were acetylated. Acetylation procedure: The dry material was dissolved in 0.4 ml pyridine, to which 0.1 ml acetic anhydride was added. This mixture was left overnight at room temperature, in the dark. 0.5 ml of distilled water was added to stop the reaction, followed by rapid

S interstitial

area contained

no recognizable

with PAS positive material

777

TDICOXDS

mature

Leydig cells. Numerous macrophages

were seen. These findings are similar to those described in et al. (2)l.

the literature

for estrogen

treated

men-[Oshima

Electron

microscopic

studies

were performed

reported B.

[ Lu and Steinberger,

elsewhere

in all these testicular

tissues, and

(39)l.

Hormonal Studies Phsma levels: The circulating levels of FSH, LH, estradiol and testosterone

D.A., prior to the initiation

of the estrogen therapy were in the normal range for normal

adult males. During treatment levels were markedly Testicular

both gonadotropins

suppressed,

levels:

20a-dihydroprogesterone

in patient

The

and estradiol

were undetectable, elevated (Table

concentrations

of

plasma testosterone

1).

testosterone,

progesterone,

and estradiol in the testes of the four estrogen-treated

men are

shown in Table 2. We measured the testicular levels of the same steroids in orchiectomy specimens from two prostatic cancer patients, who received no hormonal medication, whose circulating laboratory.

gonadotropin

of control),

progesterone

levels were significantly

(8.1%) and 20a-dihydroprogesterone seems to be different

suppressed

HORMONAL

Testosterone (ng %) Estradiol (ng %) FSH (mIU/ml) LH (mIU/ml)

in Table 2.

resulted in a significant depression of testicular testosterone

The rate of the depression

TABLE 1:

and estrogen levels were within the normal range for our

These values are labeled as “controls”

Estrogen treatment

and

(14.5%) levels (Table 2).

for each steroid. Testicular

in three of the four estrogen-treated

men.

LEVELS (BLOOD)

Before treatment x + s. e.

During treatment x f s. e.

361.5 3.2 2.1 2.6

73.9 26.1 not not

f 25.5 f 0.6 f 0.1 4 0.4

(4.57%

+ 23.0 f 6.4 detectable detectable

estradiol

S

778

TABLE 2:

TIIEOIDS

TESTICULAR STEROID CONCENTRATIONS

Progesterone

Testosterone

Controls rig/g

F.B. D*G.

201.14 144.09

x + S.D. f

2OorDHP

Ektradiol

17.09 13.07

4.80 2.58

15.08 2.84

3.69 + 1.57

186.2 94.2

172.62 40.34

140.2 65.0

f

f

Estrogen treated rig/g

D.A.

4.65

7.8

1.87

0.42

+.$. G.S.

10.61 5.87 10.41

14.9

3.00 1.60 2.24

0.14 0.37 2.94

7.88 zk 3.07 4.57

11.35 f 4.04 8.1%

2.18 kO.61 14.5%

x +_ S.D.

% of control

TABLE 3:

I

0.97 1.98 ---

METABOLISM OF PROGESTERONE BY TESTICULAR TISSUE OF PATIENT D.A.BEFORE ESTROGEN TREATMENT*

Metabolites

% Conversion

pmols/mg protein

Polar steroids 17-hydroxy-progesterone Androstenedione Testosterone 20ar-dihydroprogesterone Other steroids Progesterone (unconverted substrate)

12.91 52.41 0.84 1.10 1.85 0.19 30.70

190.2 772.1 12.4 16.2 27.3 2.8 452.3

Total conversion

69.30

1021.0

“50 mg tissue incubated

with 3H-progesterone

(10 p Ci = 3.3 nmol)

C.

In vitro Steroid Biosynthetic

Studies

Metabolism of progesterone prior to estrogen treatment: The results are summarized in Table 3. The major metabolite pmols/mg protein), of incubation, steroids.

formed was 17-hydroxyprogesterone

30.70% of the substrate (or 1.01 nmols) was unconverted

69.30% (or 1.021 pmols/mg protein)

Other

identified

dihydroprogesterone.

(52.4% or 772

metabolites

were

was actively metabolized

testosterone,

Highly polar material accounted

after 3 hours to various

androstenedione

and

20~

for 13% of the total radioactivity

of the substrate. Metabolism conversion

of progesterone 3H-progesterone

of

estrogen-treated metabolized amounts

by testes of estrogen-treated to various

(over 70% was left unconverted

sum of these three metabolites

The

by

testicular

tissue

of four

men, expressed as % conversions of the substrate. The substrate was poorly

of 17-hydroxyprogesterone,

substrate,

metabolites

men: Table 4 shows the

as compared major

after 3 hours of incubation).

androstenedione

represented

or testosterone

were formed. The

less than 2% of the total radioactivity

formed

was

20a-dihydroprogesterone

approximately

50% of the total substrate

TABLE 4:

METABOLISM OF PROGESTERONE TREATED MEN (% CONVERSION)

conversion

D.A.

Patients A.C. T.M.

BY TESTES OF ESTROGEN-

G.S.

x f S.D.

-

Total conversion

1.12 0.60 0.07 0.04 11.06 4.85 81.45

18.55

(5.3-13.2%),

(2.7% before treatment).

-.

Polar metabolites 17-hydroxy-progesterone Androstenedione Testosterone 20ar-dihydroprogesterone Other steroids Progesterone (unconverted substrate)

of the

to 54% before treatment.

metabolite

Metabolites

Very small

3.68 1.62 0.07 0.07 13.18 8.40 71.29

2.75 0.16 0.05 0.05 5.61 5.93 84.82

0.86 0.16 0.07 0.04 5.32 5.26 87.91

2.10 0.63 0.065 0.05 8.80 6.10 81.37

28.71

15.18

12.09

18.63 * 6.26

f f f f f + f

1.16 0.60 0.008 0.012 3.39 1.42 6.21

The amount treatment

they

chromatography,

of unidentified represented tentative

progesterone

non-polar

only

0.2%

identification

(Sa-pregnane-3,20-dione)

metabolites

of

mg of testicular

TABLE 5:

total

radioactivity).

of these peaks as Se-reduced and

5ar-pregnan-3 one) was made. No crystallizations Table 5 summarized

the

was large: 4.8-8.4%

20or-dihydroprogesterone of these materials

(before

By sequential metabolites

(20ol-hydroxy-

were carried out.

these data expressed as pmols of each metabolite

formed per

protein.

METABOLISM OF PROGESTERONE BY TESTES OF ESTROGENTREATED MEN (pmols/mg testes protein)

Metabohtes

Polar metabolites 17-hydroxy-progesterone Androstenedione Testosterone 20u-dihydroprogesterone Other steroids

D.A.

44.0 23.6 2.8 1.6 434.5 190.5

Patients T.M. AC.

35.9 15.8 0.7 0.7 128.7 82.0

68.5 4.0 1.3 1.3 139.8 147.8

G.S.

x * S.D.

22.0 42.6 4.1 11.9 1.8 1.6 1.0 1.1 136.1 209.8 134.5 138.7

of

f 19.5 + 9.6 f 0.9 ?r 0.4 2149.9 f 46.6

a hundred-fold

gradient

between

levels in the estrogen-treated presently

testis and plasma. The increase of circulating

men probably

represents

peripheral

estradiol

conversion.

This is

being investigated.

As controls

for the intratesticular

prostatic

cancer patients

measured

concentration

with normal of

To our knowledge,

estradiol

and 20a-dihydroprogesterone concentration

circulating

testosterone,

estradiol.

testosterone

levels we used orchiectomy

progesterone,

no previous

reports

and androgen levels. We

20a-dihydroprogesterone

on concentrations

tissue from “normal”

and

of progesterone,

in the testis have been published.

in testicular

6. Our values are considerably

gonadotropin

specimens from two

Reports

of

men are shown in Table

lower. It appears, therefore, that testosterone

concentrations

in the testes of elderly men with carcinoma of the prostate are inferior to those of younger men.

This would support

previous observations

by Axelrod

(42) who demonstrated

a

deficiency of the 17-20 cleaving enzyme in testicular tissue of a man with prostatic cancer, when compared Steinberger TABLE 6:

to the activity

et al. (3)

of testes of a younger

who confirmed

TESTOSTERONE “NORMAL”

Author

man, and by Murota (1) and

this observation.

CONCENTRATIONS

IN TESTICULAR

TISSUE OF

MEN

Number

of Subjects

Mean Testicular

Testosterone

nglgm

Ruokonen et al. (40) Morse and Heller (41) Steinberger et al. (9) This report

6

550

6

553

1

560

2

173

Therapy with estrogen resulted in a significant suppression of testicular testosterone concentrations

(4.57% of control).

Progesterone

suppressed, but to lesser degree than testosterone The ratio progesterone:

testosterone

testes. This suggests that in addition

and 20a-dihydroprogesterone

were also

(8.1% of control and 14.5%, respectively).

was 0.8 for control testes and 1.4 for estrogen-treated to the suppression

of the steroidogenic

pathway to

S progesterone,

there is a further inhibition

involved in the conversion 20o-dihydroprogesterone indicating

that

suppressed,

in the activity of the steroidogenic

of progesterone

to testosterone.

the activity

but actually

increased reflects

by estrogen only

men or laboratory

prior to estrogen treatment

concentration

of intratesticular

depression

of progesterone, steroid concentrations

animals have been reported

hormonal

is not only not

The absolute

of the in

to date.

studies in testicular biopsies of patient D.A.

are very similar to those reported

of men with normal circulating

in the literature

for testes

levels: Sharma et aZ. (43) in a man with breast

et aZ. (3) in patients

in four young volunteers

treatment.

the lower

The results of the in vitro biosynthetic

cancer, Steinberger

Similarly, the progesterone:

of the 20cr-hydroxy-steroid-dehydrogenase

for the enzyme. No determinations

estrogen-treated

enzymes

ratio was 0.3 for the controls and 5.2 for the estrogen treated,

20ct-dihydroprogesterone substrate

TDROXDls

with prostatic

cancer, and Steinberger et al. (9)

(Table 7). In the four studies the total conversion of the substrate

was 60% or higher, and 17-hydroxyprogesterone

was the metabolite

produced in greater

quantities. TABLE 7:

IN VITRO METABOLISM OF PROGESTERONE TISSUES OF “NORMAL” MEN”

Sharma et al. (43)

Metabolites

BY TESTICULAR

Steinberger et al. (3)

Steinberger et al. (9)

Present Study 12.90% 52.41% 0.84% 1.10% 1.85% 30-70%

Polar steroids 17-hydroxy-progesterone Androstenedione Testosterone 20a-dihydroprogesterone Progesterone (unconverted substrate)

29.6% 1.O% 2.6% 4.3% 35.5%

13.0-23.0% 8.0-50.0% 2.0- 3.0% 0.3- 2.0% 6.0-I 3.0% 21.0-37.0%

48.5% 14.0-2&O% 2.7% 1.7- 6.0% 7.0% 7.0-2&O%

Total Conversion

64.5%

63.0-79.0%

74.0-93.0%

*All values expressed After

estrogen

progesterone

as % conversions treatment

to testosterone

substrate

was metabolized

severely

affected

by

17-hydroxyprogesterone,

the

of the substrate

a very significant was demonstrated

(70% before estrogen,

androstenedione

69.3%

of the metabolism

in all four subjects.

treatment). as

suppression

17~hydroxylase

demonstrated

and testosterone

by

the

Only

18% of the

activity small

of

was very

amounts

formed from progesterone.

of Due

S to the small quantities

of 17-hydroxyprogesterone

17-20 lyase and 17-hydroxy-steroid-dehydrogenase studies using proximal

precursors

substrates

by

two

gonadotropins,

of 17~~hydroxylase mechanisms:

in order to clarify this point, as

in order to assess the effect of estrogen

activity

system. by the estrogen

1) this enzymatic

activity

(23) and Steinberger

of 17~hydroxylase

blocked

is directly

via gonadotropin

postulated

steroidogenic

enzymes,

by the estrogen,

et aZ. (47), Machino

possibly

through

influence

on the synthesis

17ar-hydroxylase

et al. (48)].

on DNA metabolism. requires NADPH and molecular

Menard and Purvis (49) suggested that the levels of cells is controlled

by gonadotropin

of 17or-hydroxylase demonstrated

from decreased enzyme synthesis, direct enzyme inhibition, necessary

The effect

for the enzymatic

of estrogen

treatment

activity deserves some attention.

an induction

of this enzyme

on effect

Steinberger

or more likely,

(20a-reduction

and gonadotropin

and decreased 20a-reductase et al. (24)

demonstrated

Treatment activity. a drop

pathway

This is supported by previous treatment

et a2. (21), (22)].

synthesis.

it would be a

as an alternative

on metabolism

of

20o-dihydroprogesterone

increased greatly after hypophysectomy,

a progressive decrease in androgen

tissue,

an increase of the activity of

men, both in vivo and in vitro. This could represent

tissue [Steinberger

from progesterone

of the cofactors

20&-hydroxysteroid-dehydrogenase

not affected by the estrogen treatment).

by testicular

synthesis

on testicular

inhibition

of hypophysectomy

in this study could result

activity.

by the estrogen,

of the 17~hydroxylase

for progesterone,

androgen

and consequently

or affectation

Our findings demonstrate

this enzyme in testes of estrogen-treated

formation

[Inane

as well.

Therefore, the suppression

or cytochromes

of testicular

P-450 as the site of molecular oxygen activation

P-450 in interstitial

progesterone

by

being the effect not mediated

that estrogen would act directly

oxygen, and involves cytochrom

reports

regulated

et al. (21), (22), or 2). the activity

On the other hand, it is known that 17~hydroxylase

consequence

is directly

can be

This was suggested by Samuels et al. (44), (45), (46). These

suppression.

investigators

either

treatment

as suggested by Dominguez (14), Acevedo (15), Huseby (17), Schoen (18),

Menon (20), Shikita and Hall

cytochrome

it is not clear whether

activities were affected as well. Further

isomerase/3P_hydroxy-steroid-dehydrogenase

This suppression explained

synthesized,

are being conducted

well as studies using A53-hydroxy on the A54

783

TPEIROIDS

simultaneously

with gonadotropins

with

stimulated

In cultures of human testicular in 17-hydroxyprogesterone

and

testosterone

synthesis,with

of gonadotropin progesterone

time in culture, and postulated this to be due to sudden removal

stimulation.

in culture.

20a-dihydroprogesterone

This was confirmed

was the major metabolite

from

by Tence et al. (50).

It appears, therefore, that unlike 17cu-hydroxylase, 20a-hydroxysteroid-dehydrogenase is not under

gonadotropin

is low (hypophysectomy, becomes an important

regulation,

and in situations

culture, estrogen treatment, pathway

for the catabolism

where 17a-hydroxylase

immature testes, etc.), 20a-reduction of progesterone.

In summary, a positive correlation between testicular steroid concentrations of in vitro steroid biosynthetic

It was shown that estrogen

production,

not only by inhibiting

but also on specific enzymatic view of recent reports gonadotropin Johnson

(53), Tcholakian

(57), etc.],

our findings

steroidogenesis: inhibition,

the ratios between specific individual

exert a suppressive effect on testicular

the cholesterol

[Chowdhury

of testicular

suggest two mechanisms

1) an immediate

androgen

et al. (51), Danutra

et al. (54), Chowdhury

androgen

side-chain cleavage to pregnenolone,

activities of the pathway progesterone

of rapid suppression

suppression

and results

studies was observed in this study. Similar conclusions

could be drawn using both methods by calculating steroids.

activity

-* testosterone. production

In

without

et al. (52), Mallampati

and

et al. (55), Sholiton et al. (56) Moger of action

of estrogen on testicular

direct effect on the Leydig cell, resulting

and 2) an effect mediated via suppression of gonadotropins

in enzyme

that would require

longer time and higher doses. Acknowledgments: This work was supported Ford Foundation. determination

in part by NIH Center Grant HD 08338 and a grant from the

We wish to thank Dr. Barbara Sanborn

and Dr. Mridula Chowdhury

for her assistance in protein

in radioimmunoassay

of gonadotropins.

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