The Mode of Action of Carp Gonadotropin
on the Stimulation
of
Androgen
Production
by Carp Testis in Vitro
YEA-SHA CHANG AND FORE-LIEN HUANG’
‘Institute of Biological Chemistry, Academia Sinica, P.O. Box 23-106, Taipei, Taiwan
Accepted September 25, 1981
Carp gonadotropin (cGTH) can stimulate carp testis to produce androgen in vitro. This action can be mimicked by dibutyryl CAMP (Bt, CAMP) and prostaglandins (PG). Btp CAMP can enhance the androgen production when submaximal doses of cGTH are used but cannot do so when maximal dose of cGTH is used. PG F,,is more effective than PG E, and E,. Both the inhibitors of RNA and protein synthesis can interfere the stimulatory effect of cGTH. Actinomycin D has 87% while cycloheximide has 100% inhibition. The stimulatory effect of cGTH is much more dependent on de nova protein synthesis than on RNA synthesis. In addition, steroidogenesis inhibitors, glutethemide, metyrapone, and spirocyanoketone, can also inhibit the stimulatory effect of cGTH on androgen production.
Luteinizing
hormone (LH) can stimulate
testis to produce androgen in higher verte-
brates either in viva or in vitro (Cigorraga et
al., 1978; Cooke et al., 1975, 1979; Dufau et
al., 1971; Hafiez et al., 1972; Maung and
Follett,
1977; Mendelson
et al., 1975; Mul-
ler, 1977). The available evidences indicate
that cyclic adenosine 3’S’-monophosphate
(CAMP) is an intracellular
messenger of LH
on androgen production
(Cigorraga et al.,
1978; Cooke et al., 1979; Dufau et al., 1971;
Rommertz
et al., 1974). The stimulatory
effect of LH on androgen production
is de-
pendent on de wvo protein synthesis since
ribonucleic
acid (RNA) and protein synthe-
sis inhibitors
can inhibit the stimulatory
ef-
fect of LH (Cigorraga et al., 1978; Cooke et
al., 1975, 1979; Dufau et al., 1974; Mendel-
son et al., 1975; Moyle et al., 1971; Shin and
Sato, 1971). The effect of LH on androgen
production is mainly due to its promotion
of
the conversion of cholesterol
to pregneno-
lone (Bakker
et al., 1978; Cigorraga
et
al., 1978; Hall and Eik-Nes,
1964; Hall
and Young, 1968; Purvis et al., 1973; van
der Vusse et al., 1975).
Our previous results indicated
that fish
r To whom all correspondence should be sent.
gonadotropin
(GTH)
can stimulate
male
carp to produce androgen either in vivo
(Jean, 1980) or in vitro (Huang and Chang,
1980). The mode of action of fish GTH has
seldom been investigated.
Only a few lines
of evidence indicate that fish GTH can en-
hance the fish gonad to produce
CAMP
(Fontaine et al., 1970; Fontaine-Bertrand
et
al., 1978; Idler et al., 1975). In this study
attempts are made to investigate the mode
of action
of carp GTH
(cGTH)
on the
stimulation
of androgen production by carp
testis in vitro by the following approaches:
(1) whether dibutyryl
CAMP
(Btz CAMP)
and prostaglandins
(PG) can mimic the ac-
tion of cGTH, (2) whether the stimulatory
effect of cGTH
is dependent
on de novo
RNA and protein synthesis, and (3) wheth-
er the various
kinds of steroidogenesis
inhibitors
effective in mammalian
systems
can inhibit the stimulatory
effect of cGTH
on androgen production.
MATERIALS AND METHODS
Chemicals. Actinomycin D, bovine serum albumin (BSA), chick ovomucoid, cycloheximide, Bt, CAMP, PC E,, E, and F,,, and steroids were purchased from Sigma, St. Louis, Missouri. [3H]Testosterone was from the Radiochemical Center, Amersham, England. Ether was from Wako Pure Chemical Industries, 147
0016-6480/82/100147-07$01.00/O Copyright @ 1982 by Academic Press, Inc.
Japan. Glutethemide (3-phenyl-3-ethyl-2,6-dioxo- piperidin) and metyrapone (2-methyl-1,2-di-3-pyridyl- I-propanone) were the kind gifts from Dr. J . Y. Chang, Ciba Company, Basel. Spirocyanoketone (2a-cyano- 4,4-dimethyl-20-spirox-5-en-3-one) was the kind gift from Dr. G. H. Rasmusson, Merck, Sharp and Dohme Research Laboratories, New Jersey. The cGTH used in this study was prepared as previously described (Huang and Chang, 1980). For assay, actinomycin D, cycloheximide, BtZ CAMP, and glutethemide were dis- solved in incubation medium (see below) whereas metyrapone, PGs, and spirocyanoketone were dis- solved in absolute alcohol.
evaluation of the stimulatory effect of Btz CAMP and PGs, the amount of androgen produced was intrapo- lated into the dose-response curve of cGTH to get the equivalent amount of cGTH by which similar amount of androgen was produced. For evaluation of the in- hibitory effect of inhibitor on the action of cGTH, carp testis was incubated with 0.8 ,ug cGTH in the absence or presence of inhibitor. Similar intrapolation as that mentioned above was also made to get the equivalent amount of cGTH (in the unit of pg) in the presence of inhibitor and denoted as eq-CGTH,,,. The percentage of inhibition was calculated as (1 - eq-cGTI-&,,,/O.l) x 100%.
Androgen production by carp testis in vitro. The method for the assay of the effect of cGTH and other substances on androgen production by carp testis in vitro was the same as previously described (Huang and Chang, 1980). Mature male carps (Cyprinus carpio L.) were collected during breeding season (December to March). Their body weights and gonadosomatic in- dexes (testis wt/body wt X 100) were from 300 to 500 g and 15 to 25, respectively. Testes from 3 to 4 fishes were pooled and finely chopped. The chopped tissues were washed with washing medium (8/9 strength of Krebs-Ringer bicarbonate buffer, pH 7.4, sup- plemented with 0.1% BSA and 0.03% chick ovo- mucoid) until no whitish material was left in the supematant. The washed tissues were preincubated in washing medium under 95% O,-5% CO, for 15 min and then centrifuged at SOOOg for 10 min to remove excess medium. For assay, 200 mg of preincubated tissue were suspended in 2 ml of incubation medium (same as washing medium except 1 mM theophylline was supplemented). An aliquot containing a given quantity of cGTH or other substances was also added to the incubation medium. For those alcohol-soluble substances, a given quantity in 10 ~1 of absolute al- cohol was added. Vehicle alone was served as control. In this study, samples for all assays in a set of experi- ment (i.e., all assays included in a given figure) were derived from a same batch of tissue preparation unless otherwise indicated. Triplicate determinations were made for each assay.
RESULTS
The Stimulatory
Effect of cGTH
The cGTH
can stimulate
carp testis to
produce androgen in vitro. The range of
dose-response
curve is from 0.1 to 1.6 pug,
and in certain cases to 3.2 pg (Figs. 1 and
3). The time course of androgen production
under the stimulation
of cGTH is shown in
Fig. 4. The rate of androgen production
is
slower at the earlier than at the later period
of incubation.
The Effect of Bt, CAMP
The Btl CAMP can stimulate
carp testis
to produce androgen in vitro. The effect of
1, 5, 10, and 20 rnkf Bt, CAMP are equiva-
lent to 0.10, 0.20, 0.64, and 0.90 pg of
Incubation was performed at 25” under 95% O,-5% CO, with slow constant shaking for 4 hr and termi- nated by centrifugation (5OOOg for 10 min). The super- natant was extracted with ether and then the androgen content was measured by radioimmunoassay as previ- ously described. The antiserum used was raised against testosterone 17-hemisuccinate BSA conjugate. By using testosterone as reference (lOO%), the antise- rum had 153% and 6% cross-reactivity toward androstenedione and 1 I-ketotestosterone, respec-
tively.
O-El
DOSE OF GTH Cvg)Evaluation of the effect of Btz CAMP, PGs, and in- hibitors on androgen production. The dose-response curve of the stimulatory effect of cGTH on carp testis
to produce androgen is nonlinear (see Results). For triplicate determinations.
FIG. 1. The dose-response curve of cGTH on the
stimulation of androgen production by carp testis in
vitro. Each point and line indicate the mean ? SD of
cGTH
on the aspect of the stimulation
of
androgen production
(Fig. 2).
When Btz CAMP and cGTH
are added
together to the incubation
medium,
the ef-
fect is always higher than that when either
of them is present alone in the incubation
medium.
These results indicate
that Btp
CAMP can express its effect in the presence
of cGTH. However, the extent of the effect
of Bt, CAMP is dependent on the concen-
tration of coexisting
cGTH.
At the lower
concentration
of cGTH
(0.1-0.4
pg), the
effect of Btz CAMP and cGTH are additive.
When the concentration
of cGTH
is in-
creased up to 0.8 pg and more, the effect of
Bt, CAMP becomes progressively
less and
finally
disappears
(Fig. 3). Under
the
stimulation
of maximal
dose of cGTH,
the
amount of androgen produced is the same
whether Bt, CAMP is present or not. Thus
the effect of Btz CAMP
is completely
masked by maximal
dose of cGTH.
The Effect of PG
As shown in Fig. 2, PG El, Ez, and F,,
have stimulatory
effect on androgen pro-
duction. At the concentration
of 20 pg/ml,
200
1
GTH Bt2 CAMP E, E; F
FIG. 2. The stimulatory effects of Btt CAMP, PG El,
E$, and F,, on androgen production by carp testis in vitro, and the inhibitory effects of actinomycin D (AD) and cycloheximide (CH) on Btz CAMP. The doses of cGTH, BtZ CAMP, and PGs are shown in the figure whereas those of actinomycin D and cycloheximide are 0.1 m&f. Each bar and line indicate the mean and standard deviation of triplicate determinations.
0
.l .2 4 0 1.6 32
DOSE OF GTH Ivg/TUBE)
FIG. 3. The effects of BtZ CAMP and cGTH on the
stimulation of androgen production by carp testis in vitro. The doses of cGTH used are shown in the figure whereas that of Bt, CAMP is 10 mkf. 0, cGTH is pres- ent alone; 0, cGTH and Bt2 CAMP are coexisting. Each point and line indicate the mean 2 SD of tripli- cate determinations.
the effect of PG El, EP, and Fzo, are equiva-
lent to 0.33, 0.62, and 0.73 pg of cGTH,
respectively.
The Effect of Actinomycin
D
and Cycloheximide
When the concentration
of actinomycin
D is kept at 0.01 mM, the stimulatory
effect
of cGTH on androgen production
is inhib-
ited by 87% (Fig. 6). The inhibitory
effect of
actinomycin
D becomes progressively
less
marked as the interval between the addition
of cGTH
and actinomycin
D is increased.
When the time interval is increased up to 3
hr, actinomycin
D has no more effect (Fig.
4). In addition,
actinomycin
D can also in-
hibit the stimulatory
effect of Bt, CAMP on
androgen production
(Fig. 2).
When added simultaneously
with cGTH,
0.1 mM of cycloheximide
can completely
abolish
the stimulatory
effect of cGTH.
Furthermore,
the amount of androgen pro-
duced by the tissue in the coexistence
of
cycloheximide
and cGTH
is even lower
150
CHANGANDHUANGFIG. 4. The effect of actinomycin D added at vari- ous time intervals of incubation on the androgen pro- duction by carp testis in vitro under the stimulation of cGTH. The doses used for cGTH and actinomycin D are 0.8 @g and 0.01 mA4, respectively. 0 and lines, means rfr SD of triplicate determinations of control; 0 and lines, means and SD of triplicate determinations in which actinomycin D is added at various time intervals and incubation is allowed to continue up to 4 hr. **P < 0.05 and *P < 0.10 versus control of 4-hr incubation, respectively.
than that produced
by the control tissue
(significant at 10% but not at 5% level) (Fig.
6). In contrast with actinomycin
D, the in-
hibitory
effect of cycloheximide
on the
cGTH
does not become lessened as the
time interval between the addition of cGTH
and cycloheximide
is increased. Whenever
the cycloheximide
is added, the effect of
cGTH is abolished (Fig. 5). The statistical
analysis
indicates
that the amount
of
androgen produced by control group within
a given time interval is not different signifi-
cantly from that produced by the group in
which cycloheximide
is added at a given
time interval and incubation
is allowed to
continue for another time interval up to 4
hr. In addition,
cycloheximide
can also
abolish the stimulatory
effect of Bt, CAMP
on androgen production
(Fig. 2).
The Effect of Steroidogenesis
Inhibitors
As shown in Fig. 6, the three types of
inhibitors
effective in mammalian
systems
are also effective in carp testis. At the con-
centration of 2, 2, and 0.02 mM, glutethe-
mide, metyrapone,
and spirocyanoketone
have 95.8, 84.0, and 73.0% of inhibition
on the stimulatory
effect of cGTH
on an-
drogen production,
respectively.
DISCUSSION
The stimulation
of testis to produce
androgen is one of the main effects of LH in
higher vertebrates
(Cigorraga et al., 1978;
Cooke et al., 1979; Dufau et al., 1971;
Hafiez et al., 1972; Maung
and Follett,
1977; Mendelson et al., 1975; Mullet-, 1977).
The testis or its dispersed Leydig cells has
been used by many investigators as a model
system to probe the mode of action of LH
on steroidogenesis
(Bakker
et al., 1978;
Cigorraga et al., 1978; Cooke et al., 1975,
1979; Hall and Eik-Nes,
1964; Hall and
Young, 1968; Mendelson
et al., 1975; Pur-
vis et al., 1973; van der Vusse et al., 1975).
Recently,
we have demonstrated
that fish
GTH can stimulate
carp testis to produce
androgen in vitro (Huang and Chang, 1980),
therefore attempts have been made to use
this newly developed system as a model to
investigate the mode of action of fish GTH.
The action of LH on steroidogenesis
is
proposed to be via the CAMP as cellular
TlME’OF INC:t3ATION3,HR, L FIG. 5. The effect of cycloheximide added at vari- ous time intervals of incubation on the androgen pro- duction by carp testis in vitro under the stimulation of cGTH. The doses used for cGTH and cycloheximide are 0.8 pg and 0.1 mM, respectively. 0 and lines, means + SD deviations of triplicate determinations of control; 0 and lines, means and SD of triplicate de- terminations in which cycloheximide is added at vari- ous time intervals and incubation is allowed to con- tinue up to 4 hr. The same batch of tissue is used for assays of Figs. 4 and 5.
GTH AD CH G T Ml? SC FIG. 6. The effects of inhibitors of RNA and protein synthesis, and steroidogenesis on androgen production by carp testis in vitro under the stimulation of cGTH. The doses of cGTH used for dose-response curve are shown inside the bar whereas those for assay of the inhibitory effect of various inhibitors are 0.8 pg. The doses of inhibitors used are as follows: actinomycin D (AD), 0.01 mM; cycloheximide (CH), 0.1 mM; glutethemide (GT), 2 mM; metyrapone (MR), 2 n&f; spirocyanoketone (SC), 0.02 mM. Each bar and line indicate the mean and SD of triplicate determinations.
messenger (Cigorraga et al., 1978; Cooke
etal., 1979; Dufau et al., 1971; Rommertz
et
al., 1974). It is also demonstrated
that Btz
CAMP can stimulate
androgen production
by carp testis in the present study. This
finding seems to suggest that the action of
cGTH
may also be through CAMP as the
second messenger.
The effects of PG El, Ez, and FPa on
steroidogenesis
are variable depending
on
the type of steroid,
type of tissue and
species of animal
investigated
(Behrman
and Hichens,
1976; Erickson
and Ryan,
1975, 1976; Labhsetwar,
1974; Lindner
etal., 1974; McNatty
et al., 1975; Neal et al.,
1975; Patwardhan and Lanthier,
1977). The
present results provide additional
evidence
that PG El, Ez, and FZa can also stimulate
carp testis to produce androgen. Whether
the stimulation
of PGs on androgen pro-
duction in carp testis is through the stimu-
lation
of CAMP
formation
as found in
mouse ovary (Kuehl et al., 1970) remains to
be elucidated.
The present
results
indicate
that the
stimulatory
effect of cGTH
on androgen
production
by carp testis is dependent
on
de nova RNA and protein
synthesis be-
cause actinomycin
D and cycloheximide
can inhibit or abolish this effect. A similar
situation
is also found in the mammalian
system (Cigorragaet
al., 1978; Cooke et at.,
1979; Dufau et al., 1974; Mendelson
et al.,
1975; Moyle et al., 1971; Shin and Sato,
1971). In rat the dependence of stimulatory
effect of LH on de nova protein synthesis is
more than that on de lzovo RNA synthesis.
The inhibitory
effect of actinomycin
D be-
comes progressively
less marked
as the
time interval between the addition
of LH
and actinomycin
D is extended and finally
disappears if the interval is increased up to
2.5 hr. On the other hand, even when cy-
cloheximide
is added 2.5 hr following LH,
it can still affect the testosterone production
although
the extent is lessened relatively
(Mendelson
et al., 1975). In carp, the de-
pendence of stimulatory
effect of cGTH on
de 110~0 RNA synthesis becomes progres-
sively less marked and finally becomes in-
dependent if the time interval between the
addition
of cGTH
and actinomycin
D is
long enough. On the contrary, the stimula-
tory effect of cGTH
is stringently
depen-
dent on the de nova protein
synthesis.
Cycloheximide
can abolish
the effect of
cGTH whenever it is added to the incuba-
tion medium.
Our data suggest that cGTH
can induce the synthesis of regulatory pro-
teins including enzymes for steroidogenesis
by which androgen production
in carp testis
is stimulated.
These regulatory proteins are
very labile, therefore the stimulatory
effect
of cGTH is dependent on their continuous
synthesis. The finding that the inhibitory
effect of actinomycin
D becomes
less
marked or even totally
insignificant
may
suggest that the messenger RNAs for these
regulatory proteins are comparatively
more
stable and can be accumulated.
Other pos-
sibilities
are not excluded,
such as the
presence of lag period for actinomycin
D to
penetrate into the testicular tissue.
152
CHANG AND HUANGThe present results indicate that gluteth-
emide, an inhibitor
of cholesterol
20tr-hy-
droxylase
(Kahnt
and Neher,
1966), me-
tyrapone,
an inhibitor
of cytochrome
P-
450-linked
reactions
(Satre and Vignais,
1974) and spirocyanoketone,
an inhibitor
of
C19- and C2,-3P-hydroxysteroid
dehydroge-
nase (Goldman and Sheth, 1973) can inhibit
the stimulatory
effect of cGTH on androgen
production
by carp testis. The fact that the
stimulatory
effect of cGTH can be almost
completely
abolished by glutethemide
indi-
cates that the conversion of cholesterol
to
pregnenolone
is necessary for expression of
cGTH
effect on androgen synthesis. The
regulatory role of LH on the conversion of
cholesterol
to pregnenolone
has been well
demonstrated
in mammals
(Bakker et al.,
1978; Cigorraga et al., 1978; Hall and Eik-
Nes, 1964; Hall and Young, 1968; Purvis
etal., 1973; van der Vusse et al., 1975).
Whether
the conversion
of cholesterol
to
pregnenolone
in carp testis is also under the
control of cGTH is currently being investi-
gated in this laboratory.
ACKNOWLEDGMENTS
The authors express their sincere appreciation to Professor T. B. Lo, Dean of Science College, National Taiwan University, for his encouragement and advice. This work was financially supported by the National Science Council of the Republic of China (NSC-70- 0203-BOOl-03)
REFERENCES
Bakker, C. P., van der Plan-van Winsen, M. P. I., and van der Molen, J. J. (1978). Effect of cytosol frac- tions from lutropin-stimulated rat testes on preg- nenolone production by mitochondria from nor- mal rat testes. Biochim. Biophys. Acta 543, 235-242.
Behrman, H. R., and Hichens, M. (1976). Regulation of ovarian steroid secretion. Prostnglandins 12, 83-95.
Cigorraga, S. R., Dufau, M. L., and Catt, K. J. (1978). Regulation of luteinizing hormone receptors and steroidogenesis in gonadotropin-desensitized Leydig ceils. 1. Biol. Ciwm. 253, 4297-4304. Cooke, B. A., Janszen, F. H. A., Clotscher, W. F.,
and van der Molen, H. J. (1975). Effect of protein-synthesis inhibitors on testosterone pro- duction in rat testis interstitial tissue and Leydig- cell preparation. Biochem. J. 150, 413-418. Cooke, B. A., Lindh, L. M., Janszen, F. H. A., van
Driel, M. J. A., Bakker, C. P., van der Plank, M. P. I., and van der Molen, H. J. (1979). A Leydig cell tumor: A model for the study of lutro- pin action. Biochim. Biophys. Acfa 583,320-331. Dufau, M. L., Catt, K. J., and Tsuruhara, T. (1971).
Gonadotropin stimulation of testosterone produc- tion by the rat testis in vitro. B&him. Bioph.ys. Acta 252, 574-579.
Dufau, M. L., Mendelson, C., and Catt, K. J. (1974). A highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin: Testoster- one production by dispersed Leydig cells. J. C/in. Endocrinol. Meta. 39, 610-613.
Erickson, G. F., and Ryan, K. J. (1975). Effect of LI-FFSH, dibutyryl cyclic AMP and prostaglan- dins on the productions of estrogens by rabbit granulosa cells in vitro. Endocrinology 97, 108-113.
Erickson, G. F., and Ryan, K. J. (1976). Stimulation of testosterone production in isolated rabbit thecal tissue by LIVFSH, dibutyryl cyclic AMP, PG F,, and PG EP. Endocrinology 99,452-458.
Fontaine, Y. A., Burzawa-Gerard, E., and Delerue-Le Belle, N. (1970). Stimulation hormonale de l’acti- vite adenyl cyclasique de I’ovaire d’un poisson teleosteen, le cyprin (Carassius auratus L.). C.R. Acad. Sci. Paris Ser. D 271, 780-783.
Fontaine-Bertrand, E., Salmon, C., and Fontaine, Y. A. (1978). Effect d’hormones gonadotropes, in vitro, sur la concentration de I’adenosine mono- phosphate cyclique dans l’ovaire de l’anguille (Anguilla anguilla L.). Ann. Biol. Anim. Biochim. Biophys. 18, 805-811.
Goldman, A. S., and Sheth, K. (1973). Inhibitors of human placental C,,- and C,,-3P-hydroxysteroid dehydrogenases. Biochim. Biophys. Acta 315, 233-249.
HaEez, A. A., Lloyd, E. W., and Bartke, A. (1972). The role of prolactin in the regulation of testis function: The effects of prolactin and luteinizing hormone on the plasma levels of testosterone and androstenedione in hypophysectomized rat. J. Endocrinol. 52, 327-332.
Hall, P. F., and Eik-Nes, K. B. (1964). The effect of interstitial cell-stimulating hormone on the pro- duction of pregnenolone by rabbit testis in the presence of an inhibitor of 17a-hydroxylase. Biochim. Biophys. Acta 86,604-609.
Hall, P. F., and Young, D. G. (1968). Site of action of trophic hormones upon the biosynthetic pathways to steroid hormones. Endocrinology 82,559-568. Huang, F. L., and Chang, Y. S. (1980). The gonado-
tropic stimulation of androgen production on carp testis in vitro. Proc. Nat. Sci. Count. ROC 4, 392-400.
Idler, D. R., Hwang, S. J., and Basar, L. S. (1975). Fish gonadotropin(s). I. Bioassay of salmon go- nadotropin(s) in vitro with immature trout gonads. Endocrinol. Res. Commun. 2, 199213.
Jean, W. C. (1980). “The Effect of Pike Eel Gonado- tropin on the Content of Carp Plasma Androgen.” Masters thesis. Department of Zoology, National Taiwan Univ.
Kahnt, F. W., and Neher, R. (1966). Uber die adrenale steroid-biosynthese in vitro. III. Selektive hem- mung der nebennierenrinden-funktion. Helv. Chim. Acta 49, 725-732.
Kuehl, F. A. Jr., Humes, J. L., Tamoff, J., Cirillo, V. J., and Ham, E. A. (1970). Prostaglandin re- ceptor site: Evidence for an essential role in the action of luteinizing hormone. Science 169, 883-886.
Labhsetwar, A. P. (1974). Prostaglandins and the re- productive cycle. Fed. Proc. 33, 61-77.
Lindner, H. R., Tsafrii, A., Lieberman, H. E., Zor, U., Koch, Y., Bauminger, S., and Bamea, A. (1974). Gonadotropic action on cultured GrafIian follicles: Induction of maturation division of the mammalian oocytes and differentiation of the luteal cell. Rec. Prog. Horm. Res. 30, 79-138. Maung, Z. W., and Follett, B. K. (1977). Effects of
chicken and ovine luteinizing hormone on androgen release and cyclic AMP production by isolated cells from the quail testis. Gen. Camp.
Endocrinol. 33, 242-253.
McNatty, K. P., Henderson, K. M., and Sawers, R. S. (1975). Effects of prostaglandin Ftr, and E, on the production of progesterone by human granulosa cells in tissue culture. J. Endocrinol.
67, 231-240.
Mendelson, C., Dufau, M., and Catt, K. J. (1975). De- pendence of gonadotropins-induced steroidogen- esis upon RNA and protein synthesis in the inter- stitial cells of the rat testis. Biochim. Biophys. Acta 411, 222-230.
Moyle, W. R., Moudgal, N. R., and Creep, R. 0. (1971). Cessation of steroidogenesis in Leydig cell tumors after removal of luteinizing hormone and
adenosine cyclic 3’,5’-monophosphate. J. Biol. Chem. 246, 4978-4982.
Muller, C. H. (1977). Plasma 5dihydrotestosterone and testosterone in the bullfrog, Rana cates- beiana: Stimulation by bullfrog LH. Gen. Camp. Endocrinol. 33, 122- 132.
Neal, P., Baker, T. G., McNatty, K. P., and Scaramuzzi, R. J. (1975). Influence of prostaglan- dins and human chorionic gonadotropin on pro- gesterone concentration in organ culture. J. En- docrinol. 65, 19-25.
Patwardhan, U. V., and Lanthier, A. (1977). Effect of prostaglandin on the in vitro biosynthesis of es- trone, estradiol and progesterone by rabbit ovar- ian follicles. J. Steroid Biochem. 8, 777-780.
Purvis, L. J., Canick, J. A., Rosenbaum, J. H., Hologgitas, J., and Latif, S. A. (1973). Control of cytochrome P-450 in rat testis mitochondria by human chorionic gonadotropin. Arch. Biochem. Biophys. 159, 32-38.
Rommertz, F. F. G., Cooke, B. A., and van der Molen, J. J. (1974). The role of cyclic AMP in the regulation of steroid biosynthesis in testis tissue.
J. Steroid Biochem. 5, 279-285.
Satre, M., and Vignais, P. V. (1974). Steroid llp- hydroxylation in beef adrenal cortex mitochon- dria. Binding affinity and capacity for specific %-steroids and for 3H-metyrapol, an inhibitor of lip-hydroxylation. Biochemistry 13, 2201-2209. Shin, S., and Sato, G. H. (1971). Inhibition by ac-
tinomycin D, cycloheximide and puromycin of steroid synthesis induced by cyclic AMP in inter- stitial cells. Biochem. Biophys. Res. Commun. 45,
501-507.
Van der Vusse, G. J., Kalkman, M. L., and van der Molen, J. J. (1975). Endogenous steroid produc- tion in cellular and subcellular fractions of rat tes- tis after prolonged treatment with gonadotropins.