Molecular and Cellular Endocrinology, 40 (1985) 33-40 33 Elsevier Scientific Publishers Ireland, Ltd.

MCE 01276

Specificity of the direct effect of an LHRH agonist on testicular 17-hydroxylase but not on 5a-reductase activity in hypophysectomized adult

rats

Y v e s T r e m b l a y , A l a i n B61anger * a n d B i a n c a M a r c h e t t i

Laboratory of Molecular Endocrinology, Le Centre Hospitalier de rUniversitb Laval, 2705 Boulevard Laurier, Sainte- Foy, Qubbec G1 V 4G2 (Canada)

(Received 25 September 1984; accepted 26 November 1984)

Keywords: steroidogenesis; direct effect; hypophysectomized; LHRH-A.

Summary

The direct effect of treatment with a potent LHRH agonist on testicular steroidogenesis was studied by incubation of radioactive steroids with a testicular homogenate or with a suspension of interstitial cells obtained following 7 days of treatment of adult hypophysectomized male rats. The animals received [D-Ser(tBu)6,des-Gly-NH12°]LHRH ethylamide (25/tg) administered 3 times a day, hCG (5 or 25 IU) once daily or a combination of both drugs. The metabolism of tritiated progesterone into Aa-metabolites by a suspension of interstitial cells was markedly reduced by treatment with the LH RH agonist (LHRH-A) alone or following combined treatment with hCG and LHRH-A. No formation of 5a-reduced steroids was detected in the medium following incubation with testicular homogenate or interstitial cells. Similar findings were obtained by measurement of testicular steroid content. The present data demonstrate that the direct effect of the LHRH agonist is limited to the Leydig cells on 17-hydroxylase activity. This inhibitory effect is reflected by an accumulation of testicular pregnenolone and progesterone content and a marked inhibition of progesterone metabolism into Aa-androgens. However, no stimulation of 5a-reductase, an enzyme localized in seminiferous tubules, could be detected. Such data show clear differences between the direct and the pituitary-mediated effects of treatment with LHRH agonists on testicular steroidogenesis. While the LHRH agonist administered at high doses in the rat can directly inhibit 17-hydroxylase activity, the stimulatory effect on 5a-reductase activity is regulated by another mechanism.

The inhibitory action of LHRH agonists on gonadal functions in male rats is well known (Labrie et al., 1978; B61anger et al., 1979, 1980c, 1983; Catt et al., 1979; Hsueh and Jones, 1981). Treatment with LHR H agonists inhibits testicular gonadotropin binding as well as testosterone secre- tion owing to a decrease in 17-hydroxylase and

* To whom reprint requests should be addressed.

17,20-desmolase activities in Leydig cells (Bdanger et al., 1980c; Dufau et al., 1979; Labrie et al., 1980) without loss of fertility (Labrie et al., 1978; Pelletier et al., 1980; Vickery et al., 1983). It has also been shown that administration of LH RH agonists causes a stimulation in 5a-reductase activ- ity present in non-Leydig cell elements (Carmichael et al., 1980; Wisner et al., 1980).

While the data obtained after treatment of adult male rats with low doses of an LH RH agonist

0303-7207/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland, Ltd.

34

(30-500 ng daily) indicate that the inhibitory ef- fects are a consequence of a pituitary-mediated mechanism (B61anger et aL 1980a; Labrie et al., 1978, 1980), studies using hypophysectomized ma- ture and immature rats indicate that when large doses of an L H R H agonist are administered (10-20 ~g daily), a direct effect at the testicular level is also involved in the mechanism of testicular desensitization (B61anger et al., 1980c; S6guin et al., 1982). In vitro as well as in vivo studies have shown that the direct gonadal effect of L H R H agonists can be associated with a reduction in LH receptor concentration and in the activity of the enzyme 17-hydroxylase. However, no information was available on the possible direct effect on 5c~- reductase activity.

In view of this, the following study was per- formed to investigate the effect of in vivo treat- ment with an L H R H agonist on testicular steroid content as well as on testicular 17-hydroxylase and 5a-reductase activities in hypophysectomized adult rats. To avoid the hypophysectomy-induced regression of testicular functions, the animals were treated with two different doses of hCG.

Materials and methods

Animals Hypophysectomized adult male rats obtained

from Charles River Canada Inc. (St. Constant, Qu6bec) were housed two per cage under con- trolled temperature (20-22°C) and lighting (14 h light, 10 h darkness, lights on at 0500 h) condi- tions. The animals were given food and liquid (5% glucose in 0.9% NaCI) ad libitum. All animals were killed by decapitation within 20 sec of re- moval from their cage.

Hormones The preparation of hCG (2960 I U / m g ) was a

gift from Roussel-UCLAF (Romainville, France). The L H R H analogue [D-Ser(tBu)6,des-Gly - NH~°]LHRH ethylamide was kindly supplied by J. Sandow, Hoechst AG (Frankfurt, F.R.G.).

Chemicals [1,2,6,7-3H]Progesterone obtained from New

England Nuclear was purified on LH-20 columns

(B~langer et al., 1980b) and by silica gel thin-layer chromatography. Non-radioactive steroids were purchased from Steraloids and Research Plus Steroid Laboratories Inc. Coated column packing for gas chromatography was obtained from Chro- matographic Specialties (Brockville, Canada).

Treatment Five days after hypophysectomy, animals (10

per group) were injected either 3 times a day with 25 /~g of the L H R H agonist [D-Ser(tBu)6,des-Gly - NH~°]LHRH ethylamide, once a day with 5 or 25 IU of hCG, or a combination of both drugs for 7 days. Control rats received the vehicle alone (0.15 M NaCI). The animals were sacrificed 24 h after the last injection. The testes and plasma were collected for interstitial cell dispersion and steroid radioimmunoassays, respectively.

Steroid assays Testicular and plasma steroids were extracted

with ether and separated on LH-20 columns. Chromatography and radioimmunoassays were performed as described (B61anger et al., 1980b). The intra-assay and inter-assay coefficients of variation are 10% and 15%, respectively.

Isolation of interstitial cells Interstitial tissue was isolated by a modification

of the method of Dufau et al. (1974). In brief, testes were decapsulated, pooled and incubated in 50 ml flasks containing 10 ml M-199 with 0.1% BSA and 0.18 mg collagenase (Worthington Type III) per ml at pH 7.4. The incubation was per- formed at 34°C with shaking at 200 cycles/min for about 9-10 min. The volume was then adjusted to 45 ml with incubation medium without col- lagenase and the tubes were inverted 10 times. After each tube had been allowed to stand verti- cally for 3 min, the turbid supernatant was filtered and centrifuged at 600 × g for 10 min. The sedi- mented cells were resuspended in 30 ml of M-199 containing 0.1% BSA, and 0.7 mg trypsin inhibitor per ml and centrifuged at 600 x g for 10 min. The sedimented cells were used as the interstitial cell preparation. This preparation contains 30-35% of Leydig cells and the viability test with trypan blue has been made.

Incubation procedure (a) Testicular homogenate incubation. Testes

were homogenized individually after weighing in 10 ml 0.25 M sucrose containing 0.001 M EDTA and pooled. 0.1 ml of buffer co-factor solution (0.3 M potassium phosphate, pH 7.4, 0.06 M nico- tinamide and 0.002 M MgCI2) containing the triti- ated progesterone (3 x 106 dpm) was introduced into 2 x 10 cm glass tubes. Each incubation was performed in triplicate. 0.4 ml of buffer co-factor solution containing 0.8 mg of NADPH was then added to each tube before the addition of homog- enate (equivalent to 20 mg of tissue) in 0.5 ml. The samples were incubated for the indicated time periods in a shaking water bath under an air atmosphere at 34°C. At the end of the incubation, the incubate was immediately acidified with 0.1 ml 1 N HC1 and placed in an ice-water bath. 0.1 ml of ethanol containing 10 /~g of the following carrier steroids (pregnenolone, 17-OH-pregnenolone, de- hydroepiandrosterone, androst-5-ene-3fl,17fl-diol, progesterone, dihydroprogesterone, pregnan-3a-ol- 20-one, pregnan-3f l -ol -20-one, 17 -OH-pro - gesterone, 17-OH-dihydroprogesterone, pregnane- 3c~,17a-diol-20-one, pregnane-3fl,17a-diol-20-one, androstenedione, androstanedione, androsterone, epiandrosterone, testosterone, dihydrotestosterone, androstane-3 a,17/3-diol, androstane-3fl,17fl-diol and estradiol) was then added. The reaction mix- tures were extracted 3 times with ether and the extracts were dried under a stream of nitrogen.

(b) Interstitial cell incubation. Triplicate in- cubations of interstitial cells (150000) were per- formed in polypropylene tubes containing 1 ml of M-199, 0.1% BSA. The incubation was started by the addition of [3H]progesterone (3 × 106 dpm) and was carried out at 34°C for the indicated time periods with continuous shaking under a constant stream of 95% 02, 5% CO 2. The incubation was stopped by the addition of 0.1 ml of 1 N HC1 and the tubes were placed in an ice-water bath at 4°C. The samples were processed as described above.

Identification of rnetabolites LH-20 columns were prepared as described pre-

viously (B61anger et al., 1980b). After packing and washing with 25 ml of benzene-methanol (85 : 15), the columns were washed with 20 ml of the first solvent system used for elution (isooctane-ben-

35

zene-methanol, 90 : 5 : 5). The residue obtained from the ether extraction was dissolved in 0.2 ml of the mixture i sooc tane-benzene-methanol (90 : 5 : 5) and then placed on LH-20 columns. The eluates were separated into the following fractions: (a) progesterone + dihydroprogesterone + pregnan-3e~-ol-20-one; (b) androstenedione + androstanedione + dihydrotestosterone + andros- terone + epiandrosterone + dehydroepiandroster- one + pregnenolone + pregnan-3fl-ol-20-one; (c) 17-OH-progesterone + testosterone + pregnane- 3a,17a-diol-20-one + androstane-3cd7fl-diol. The polarity of the solvent was then changed to 70 : 15 : 15 (isooctane-benzene-methanol) for the elution of fraction (d) (pregnane-3B,17a-diol-20- one + 17-OH-pregnenolone + androst-5-ene-3fl,- 17fl-diol + androstane-3fl,17fl-diol) while estradiol (fraction (e)) was recovered after changing the composition of the eluent to 60 : 20 : 20. An aliquot of each fraction was then counted and the per- centage of total radioactivity present in each zone was calculated. This percentage was distributed among those compounds which showed significant radioactivity, according to the proportion of each compound present as indicated by measurements in other chromatograms. The sum of the radioac- tivity in all fractions usually represented more than 90% of the radioactivity applied on the LH-20 column.

After evaporation of solvent, all fractions were acetylated with acetic anhydride in pyridine (2:3; v /v ) at room temperature overnight and re- chromatographed on 0.25 mm silica gel thin-layer plates (Merck Silica Gel (GF-254)). Fractions were chromatographed as follows: fraction (a), hexane / ether (1 : 3, v/v): progesterone, 0.59; dihydropro- gesterone, 0.78; pregnan-3a-ol-20-one, 0.90; frac- tion (b), hexane/petroleum ether /e ther ( 1 : 1 : 2 , v / v / v ) : androstenedione, 0.16; androstanedione, 0.40; dihydrotestosterone, 0.60; androsterone, 0.72; epiandrosterone, 0.72; dehydroepiandrosterone, 0.72; pregnenolone, 0.85; pregnan-3fl-ol-20-one, 0.85; fraction (c), petroleum ether /e thyl acetate (1 : 1; v / v ) : 1 7 - O H - p r o g e s t e r o n e , 0.38; testosterone, 0.61; pregnane-3a,17a-diol-20-one, 0.70; androstane-3a,17fl-diol, 0.80; fraction (d), petroleum ether /e thyl acetate (1:1; v/v): preg- nane-3fl,17e~-diol-20-one, 0.70; 17-OH-pregnen- olone, 0.70; androst-5-ene-3fl,17fl-diol, 0.80;

36

androstane-3fl,17/3-diol, 0.80; fraction (e), petro- leum ether /e thyl acetate (1:1, v/v) : estradiol, 0.55.

Radioactivity on silica gel plates was detected with a Berthold thin-layer scanner and radioactive zones were scraped off, eluted with methanol and analyzed by radio-gas chromatography (Varian model 3700 gas chromatograph equipped with a Packard gas proportional counter model 917). The steroids were analyzed as acetates on 1% SE-30 or 1% Hi-EFF8PB. Radioactive 17-hydroxypro- gesterone, androstenedione and testosterone formed during incubation of Leydig cells with tritiated progesterone were further analyzed by crystallization to constant specific activity. Preg- nan-3a-ol-20-one, pregnan-3fl-ol-20-one, preg- nane-3a,17a-diol-20-one and pregnane-3/3,17a- diol-20-one were isolated from incubation of tes- ticular homogenate with radioactive progesterone and were considered identified based on the same polarity on LH-20 columns, the same thin-layer chromatographic mobility and the same gas chro- matographic retention time as the reference com- pound with both stationary phases.

Calculations Statistical significance was measured according

to the multiple-range test of Duncan-Kramer (Kramer, 1956).

Results

As illustrated in Fig. 1A-B, daily administra- tion of 5 or 25 IU of hCG for 7 days markedly increases the testicular content of pregnenolone, progesterone, 17-hydroxyprogesterone, andros- tenedione and testosterone. In fact, the lowest dose of hCG induces testicular levels of pregnen- olone, progesterone and 17-hydroxyprogesterone comparable to those observed in intact rats (Chasalow et al., 1979; Carmichael et al., 1981) while the concentrations of the A4-androgen and 5a-reduced steroids are slightly higher (Fig. 2). The 25 IU dose of hCG leads, however, to levels of testicular steroids much higher than normal except for pregnenolone and progesterone which are less elevated.

In contrast with the observed inhibitory effect of LHRH agonist treatment in intact rats

(Carmichael et al., 1981), administration of the peptide to hypophysectomized animals does not change the basal testicular steroid content (Figs. 1 and 2). However, when testicular steroid content is stimulated by multiple injections of hCG, a marked effect of LHRH-A treatment on steroidogenesis is then detected. As seen in Fig. 1A, a marked accu- mulation of both pregnenolone and progesterone occurs after combined administration of hCG and LHRH-A. In fact, both doses of hCG induce a 300-400% increase in pregnenolone and a 100-200% increase in progesterone content, whereas testicular levels of 17-hydroxypro- gesterone remain similar to those found after the administration of hCG alone.

It can also be seen in Fig. 1B that the adminis- tration of the LHRH agonist induces a marked inhibition of testicular testosterone and andros- tenedione levels in hCG-treated animals. When animals are treated with 25 IU hCG, 60% and 80% inhibitions (p < 0.01) are observed for andros- tenedione and testosterone respectively following agonist peptide administration. As illustrated in Fig. 2, the effects of LHRH-A treatment observed on testicular A4-androgen levels are also reflected on the content of the testicular 5a-reduced steroids. An inhibition of 30-60% is observed for testicular dihydrotestosterone, androstane-3/3,17/3-diol and androstane-3a,17/3-diol levels after LHRH-A ad- ministration.

In agreement with the effects of LHRH-A on testicular testosterone and androstane-3a,17/3-diol levels, the plasma concentrations of these steroids are markedly inhibited after treatment with the LH RH agonist (data not shown).

In order to analyze further the inhibition of testicular 17-hydroxylase activity, tritiated pro- gesterone was incubated with suspensions of inter- stitial cells. As illustrated in Fig. 3, the rate of progesterone conversion is reduced in hypo- physectomized animals. In fact, approximately 40% of the [3H]progesterone precursor is converted into metabolites after 120 min of incubation with inter- stitial cells obtained from control hypophysecto- mized rats, while a marked inhibition is observed in LHRH-A-treated animals.

Administration of hCG alone results in the rapid conversion of [3H]progesterone into metabolites after 120 min of incubation (approx.

37

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15.

12.

9.

6.

[ ] S A L I N E

[ ] L H R H - A

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60

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0 w Z 600- O u.I

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S A L I N E S A L I N E h C G 51.U

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Fig. 1. Effect of chronic treatment for 7 days of adult rats with s.c. doses of the LHRH agonist (25 #g, 3 times a day) on the basal as well as stimulated levels (5 and 25 IU of hCG) of testicular prenenolone, progesterone, 17-hydroxyprogesterone (A), androstenedione and testosterone (B). Results are expressed as ng steroid per g testicular tissue and are the means_+ SEM of triplicate determinations

(10 animals/group), hCG was administered once daily.

70%), the rate of conversion being similar to that measured in intact rats (Carmichael et al., 1981). At both doses of hCG, the L H R H agonist exerts a potent inhibitory effect. It can also be seen in Fig. 3B that the rate of androstenedione, 17-hydroxy- progesterone and testosterone format ion is markedly inhibited in LHRH-A-treated rats in both the presence and absence of simultaneous treatment with hCG. It should be noticed that no formation of 5a-reduced steroids can be detected during incubation with interstitial cells.

The total percentage of formation of A 4- metabolites after 120 rain of incubation was de- creased from 30 + 1.0% to 1.6 + 0.02% ( p < 0.01) for the LHRH-A-treated rats, as illustrated in Fig. 4. In the presence of hCG, the total percentage of formation of zl4-metabolites was decreased from

65 + 5% to 25 + 5% ( p < 0.01). The present data clearly show a decrease of 17-hydroxylase activity in interstitial cells following administration of an L H R H agonist, the effect being clearly demon- strated on basal as well as hCG-stimulated steroi- dogenesis.

Since, in adult rats, testicular 5a-reductase ac- tivity is localized only in seminiferous tubules, the activity of this enzyme was measured in testicular homogenates using [3H]progesterone as precursor. As can be seen in Fig. 5, there is no change in 5a-reductase activity following L H R H agonist treatment. The total content of the two major 5a-reduced metabolites formed from progesterone, namely pregnan-3a-ol -20-one and pregnan- 3a,17a-diol-20-one, is increased following activa- tion by hCG of the 17-hydroxylase activity which

38

[ ] S A L I N E

[ ] L H R H - A

w

_z o O

(/)

I-

(5 <, 101 z ~r 0 ~

150

1 6o - 30

SAL INE hCG hCG 51U. 2 5 1 U

Fig. 2. Effect of chronic treatment for 7 days of adult rats with s.c. doses of the LHRH agonist (25 ~tg, 3 times a day) on the basal as well as stimulated levels (5 and 25 IU of hCG) of testicular dihydrotestosterone, 3fl-androstanediol and 3a- androstanediol. Results are expressed as ng steroid per g testic- ular tissue and are the means+ SEM of triplicate determina- tions (10 animals per group), hCG was administered once daily.

is able to convert pregnan-3a-ol-20-one into preg- nan-3a,17ct-diol-20-one. Moreover, we have not detected the presence in the medium of pregnan- 3fl-ol-20-one or pregnan-3/3,17a-diol-20-one.

D i s c u s s i o n

The present data clearly demonstrate that treat- ment with the L H R H agonist has no effect on 5a-reductase activity, either on basal levels or fol- lowing stimulation with hCG. These data are in agreement with the measurements of testicular steroid content in the same groups of animals. At the same time, the present data clearly show a direct action of the L H R H agonist at the gonadal level when high doses of the peptide are adminis-

>- I--

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3A PROGESTERONE METABOLISM

0...-~0 CONTROL H h C G (5 IU) I~---o hCG (25 IU) H L H R H - A z~---~hCG (5 IU) * LHRH-A -- :. hCG (25 |U) ~ LHRH-A

80 1 ~"" " ..................... o

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3B PROGESTERONE METABOLITES

o-----o ANDROSTENE DIONE = = 17-HYDROXYPROGESTERONE D---o TESTOSTERONE

100 CONTROL

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1OO LHRH-A hCG (5 IU)

80

0 80 120 180 0 80 120 180

hCG (25 [U) " LHRH-A

0 60 120 180

INCUBATION TIME (MIN)

Fig. 3. Effect of chronic treatment for 7 days with 25 /tg of [D-Ser(tBu)6,des-GIy-NH~°]LHRH ethylamide (3 times daily) of adult rats on [3H]progesterone metabolism (A) and the formation of androstenedione, 17-hydroxyprogesterone and testosterone (B) by testicular interstitial cells. Results are ex- pressed as the percentage of radioactivity present in the medium and are the means+SEM of triplicate determinations. The same animals received daily doses of 5 or 25 IU of hCG.

TOTAL A 4 -METABOLITES

O---.-.O CONTROL O~..QhCG (5 IU)

100- D- - -QhCG (251U) H LHRH-A

>" zx---.~, hCG (5 IU) ; LHRH-A F'- .L .t hCG (25 IU) * LHRH-A

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~_z 4o

z tu 20- O rr I.tJ a..

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0 ov 120 1

INCUBATION TIME (MIN)

Fig. 4. Effect of chronic treatment (7 days) with 25 #g (3 times daily) of [o-Ser(tBu)6,des-Gly-NH~°]LHRH ethylamide on the total formation of za4-metabolites from [3H]progesterone dur- ing incubation with testicular interstitial cells. The effect of the LHRH agonist was measured in the presence as well as the absence of simultaneous daily treatment with 5 or 25 IU of hCG.

PROGESTERONE 5a-REDUCED METABOLITES

o--...-,o PREGNAN-3a-OL-20-ONE = PREGNANE-3a. 17a-DIOL-20-ONE

100 CONTROL hCG (5 IU) hCG (25 IU)

F--

'<DO~O <0 "i""~'"] ..........

U.- t..LI O ~ 100 LHRH-A hCG (5 IU) hCG (25 IU)

~ ~ - .............. i 9 ......° o ~ ,o ...o t ..... " .............. ~ 20 ~ "'~ .........

0 , 0 2 0 , , o e o ; -204o60 0 ~ , , b ~o

INCUBATION TIME (MIN)

Fig. 5. Effect of chronic treatment (7 days) with 25 #g (3 times daily) of [D-Ser(tBu)6,des-GIy-NH~°]LHRH ethylamide on the formation of 5a-reduced metabolites from [3H]progesterone by total testicular homogenate. The effect of the LHRH agonist was measured in the presence as well as the absence of simulta- neous daily treatment with 5 or 25 1U of hCG.

39

tered in hypophysectomized rats. The inhibitory effect of LHRH-A is reflected

by the accumulation of testicular pregnenolone and progesterone and a decrease in A4-androgen concentration and furthermore by the demonstra- tion of a marked inhibition of progesterone metabolism by interstitial cells. The present find- ings of a direct inhibitory effect of the LH RH agonist in hypophysectomized rats are consistent with other reports (Rivier et al., 1979; Hsueh and Erickson, 1979; Bambino et al., 1980) and provide further information to our previous studies on the changes in steroidogenesis following L H R H agonist treatment (B61anger et al., 1980a, c; Carmichael et al., 1981; Labrie et al., 1978), which showed that administration of multiple high doses of LHRH in intact animals can inhibit testicular functions.

The most interesting finding is that LHRH-A treatment has no effect on testicular 5a-reductase activity in hypophysectomized rats while the administration of the same agonist in intact animals has a marked stimulatory effect on this parameter (Carmichael et al., 1980; Oshima et al., 1975). We also find that the effect of the L H R H agonist on 5a-reduced steroid formation cannot be detected even when testicular steroidogenesis is stimulated. While the results of this study provide additional support for a direct effect of LHRH agonists on testicular functions in the adult rat, it also illustrates basic differences between the direct and the pituitary-mediated effects of the agonist.

As mentioned earlier, the LH RH agonist binds to specific testicular sites present in Leydig cells (Lefebvre et al., 1980; Bourne et al., 1980; Clayton et al., 1980; S6guin et al., 1981, 1982) while histochemical (Pelletier et al., 1981) as well as receptor binding studies (S6guin et al., 1981, 1982) have demonstrated that the binding of iodinated [D-Ser(tBu)6,des-Gly-NH~°]LHRH ethylamide in seminiferous tubules is not significant. In the rat, 5a-reductase activity disappears in Leydig cells between the ages of 40 and 70 days while 5a-re- ductase activity in seminiferous tubules is ob- served only in the adult (Moger et al., 1974). Moreover, the increase in 5a-reductase activity in intact rats observed after LHRH-A administration occurs in non-Leydig cells (Carmichael et al., 1980). The present data indicate that the direct action of

40

LHRH agonists is localized in Leydig cells and results in a decreased conversion of progesterone. Such an effect results in an increase in testicular progesterone as well as in a decrease in A4-andro - gen concentration. Such data suggest that the elevation of testicular 5a-reductase after LHRH-A administration occurs through a pituitary-mediat- ed mechanism.

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