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BIOLOGY OF REPRODUCTION 48, 883-888 (1993)
Catecholamines Stimulate Testicular Steroidogenesis In Vitro in the Siberian Hamster,
Phodopus sungorus'
ARTUR MAYERHOFER, 3 ANDRZEJ BARTKE,2 and TIM BEGAN
Department of Physiology School of Medicine, Southern Illinois University, Carbondale, Illinois 62901-6512
ABSTRACT
We have examined direct effects of catecholamines on testicular testosterone production in a seasonally breeding species,
the Siberian hamster, Phodopus sungorus. Testicular parenchyma from gonadally active long photoperiod (LD)-exposed and
gonadally regressed short photoperiod (SD)-exposed animals was incubated for 6 h with norepinephrine, epinephrine, betaadrenoreceptor agonist isoproterenol, or alpha-adrenoreceptor agonist phenylephrine (all at 10 }IM), as well as with various
concentrations of norepinephrine (10 nM-10 iLM), and 10 M norepinephrine with or without hCG (0.7, 3.1, and 12.5 mIU/
ml). In addition, effects of alpha-adrenoreceptor antagonist prazosin and beta-adrenoreceptor antagonist propranolol (50 RM)
were tested in the incubations containing 10 I.M norepinephrine. In the incubations of testes from both LD and SD Siberian
hamsters, norepinephrine was most effective in stimulating testosterone production, followed by epinephrine and phenylephrine,
while isoproterenol failed to increase testosterone accumulation. The stimulatory effects of norepinephrine were dose-dependent
and were prevented by coincubation with prazosin, but not affected by coincubation with propranolol. In combination with
various doses of hCG, norepinephrine failed to stimulate testosterone production above the levels obtained with hCG alone.
These data indicate that the testicular receptors mediating the action of catecholamines on testicular steroidogenesis in Phodopus sungorus are of the alpha 1-subtype, a result in accordance with a previous study in the golden hamster. However, the
results of the present study are strikingly different from the findings obtained in the golden hamster in terms of the effects of
photoperiod on the responsiveness of testicular steroidogenesis to catecholamines. Thus, in the golden hamster, the seasonal
photoperiod-related transition from gonadal activity to quiescence is accompanied by an acquisition of responsiveness of testicular
steroidogenesis to catecholamines. In contrast, the testes of both LD-exposed and SD-exposed testes of Phodopus are equally
able to respond to catecholaminergic stimuli. However, this catecholamine effect may be of only minor importance in the LD
animal, in which hCG has a very strong stimulatory action on testosterone production with increases 70-90-fold over basal
levels, while the effects of catecholamines are an order of magnitude smaller. In contrast, in the incubations of testes from SD
animals, the stimulatory action of hCG (approximately 5-10 times basal values) was in the same range as the effect of norepinephrine (4 times basal production). Thus, the relative impact of catecholaminergic stimuli in the SD testis is by far greater than
in the LD testis. Perhaps catecholaminergic input in the testis could be viewed as a back-up system for LH during the time of
physiological suppression of LH release. Moreover, regardless of the functional state of the testis, catecholamines may participate
in the regulation of testosterone production in the Siberian hamster by exerting a constant "fine-tuning" effect on testicular
Leydig cells, which could be either direct or indirect via paracrine interactions with Sertoli cells or other testicular cell types.
INTRODUCTION
There is increasing evidence for a role of testicular innervation and/or peripheral catecholamines in the control
of male reproductive functions. Possible targets for catecholamines in the testes of several species (rat, mouse, pig)
have been identified by the presence of adrenergic receptors and include both Leydig cells and Sertoli cells [1-7].
In vitro, catecholamines modulate specific functions of these
cells, e.g., the production of cAMP and lactate by cultured
Sertoli cells [7, 8]. Leydig cells can be stimulated to produce
cAMP at the time of plating by adding catecholamines to
the culture medium, while catecholamines can stimulate the
production of testosterone by isolated Leydig cells only after several hours in culture [1, 5, 9].
In addition to in vitro findings, there is evidence that
catecholamines are involved in the regulation of testicular
function, especially testosterone production, in vivo. Thus,
in the adult rat, surgical denervation of the testis, or adrenalectomy, has been shown to abolish the acute increase
of testosterone levels in response to stressful stimuli [10].
Neuronal and/or catecholaminergic mechanisms may be
more important in the developing testis than in the fully
functional adult gonad. Denervation can interfere with both
normal testicular development and compensatory hypertrophy in immature animals [11, 12], and testicular catecholamine concentrations are higher in prepubertal than in adult
rats [13]. Moreover, catecholamines stimulate androgen
production in incubations of testicular tissue from fetal or
immature, but not from adult, mice or rats [3, 4,14].
In view of these observations and since the atrophic testis of a seasonal breeder shares functional characteristics
with the prepubertal testis [15], we previously [16] examined the effects of catecholamines on testosterone production by active and quiescent testes of seasonally breeding
golden hamsters (Mesocricetus auratus). We found striking
differences in the testosterone response of active and
quiescent testes to catecholamines. Thus, regressed testes
from short photoperiod (SD)-exposed animals responded
Accepted November 24, 1992.
Received February 27, 1992.
'Financial support was provided by Deutsche Forschungsgemeinschaft Ma 1080/
1-2 and NIH HD 20033.
2
Correspondence.
3
Current address: Artur Mayerhofer, Anatomie und Zellbiologie, Universitit Ulm,
PF 4066, D-7900 Ulm, Germany.
883
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MAYERHOFER ET AL.
to catecholamines by increased testosterone production,
while active testes from long photoperiod (LD)-exposed
hamsters did not. In the presence of hCG, both LD and SD
testes responded to catecholamines by an additional increase in testosterone production.
These results indicated a potential for significant involvement of catecholamines in the regulation of testicular steroidogenesis during stress and during the seasonal regression of the testes. To further test our hypothesis that
catecholamines are regulators of testicular function, the
present study was conducted in another seasonally breeding species, the Siberian hamster, Phodopussungorus [17,18].
stopped, and after centrifugation the media were frozen at
-20°C until determination of testosterone by RIA [16,19].
Results were calculated using an RIA-program [20]. Sensitivity of the testosterone assay was 5 ng/ml. Inter- and intraassay coefficients of variation were less than 6% and 9%,
respectively.
MATERIALS AND METHODS
RESULTS
Animals
Adult male Siberian hamsters were obtained from the
breeding colony at Southern Illinois University, Carbondale, IL, and were housed after weaning in groups of 2-4
per cage under standard conditions, with free access to food
and water. The animals were exposed to either a stimulatory long photoperiod (LD; 16L:8D) or to a short photoperiod (SD; 6L:18D), which causes testicular atrophy [17, 18].
Maximal testicular atrophy was achieved after 9-10 wk in
SD; therefore the animals were used for the experiments
at that time.
Testicular Incubations
The incubation procedure has been described in detail
previously [16]. In brief, the animals were decapitated; and
testes were immediately removed, decapsulated, and cut into
fragments of approximately equal size. The fragments were
preincubated for 30 min in Krebs'-Ringer bicarbonate buffer
containing 1% glucose, in a Dubnoff metabolic incubator
shaking at 100 rpm, at 32 ± 2C, in an atmosphere of 5%
CO2 :95% 02. Preincubation was performed in the anticipation that it would remove blood, interstitial fluid, and
preformed testosterone from the tissue. The fragments were
then transferred to media containing the catecholaminergic agents (-)-epinephrine-HCl, (-)-isoproterenol-HCl, (1)phenylephrine-HCl (these drugs were tested at 10 jiM), or
(-) norepinephrine (at 0, 10 nM, 100 nM, 1 M, 10 iM);
ascorbic acid (10 mM; to prevent break-down of catecholamines); and the phosphodiesterase inhibitor 3-isobutyl-1methyl-xanthine (MIX; 10 mM). As a positive control, fragments from each testis were incubated with hCG (3.1 mIU/
ml). Additional incubations contained 10 jiM norepinephrine and various concentrations of hCG (0.7, 3.1, or 12.5
mIU/ml). Incubations with 10 ,M norepinephrine and alpha-adrenoreceptor antagonist prazosin-HCl, or beta-adrenoreceptor antagonist (D/L)-propranolol-HCl (both at 50
jM) were also carried out. All chemicals were purchased
from Sigma Chemical Co. (St. Louis, MO), except hCG (from
Calbiochem, La Jolla, CA). After 6 h, the incubations were
Statistics
The data, expressed as production of testosterone per
milligram testicular parenchyma per 6 h, were analyzed by
ANOVA and post hoc tests (Scheffe test, Fisher's test) or
Student's t-test as specified.
Testes Weights
As expected, after 10 wk in SD, testes weights were significantly lower than the weights of testes from animals kept
under long photoperiod: average paired testes weight was
771. 2
46.3 mg (means + SEM) in LD animals and 42.7
± 2.1 mg in SD animals (p < 0.001; t-test).
Testicular Incubations
At concentrations of 10 piM, norepinephrine, epinephrine, and phenylephrine caused a significant increase in the
production of testosterone in the incubations of the testes
from both LD-exposed and SD-exposed animals (Figs. 1A
and 2A). Numerically, norepinephrine was the most potent
stimulus, followed closely by epinephrine (approximate 4fold increase over basal production) and by phenylephrine
(approximate 2-fold increase over basal production). No
stimulation was observed when isoproterenol was added
to the incubation media of SD or LD testes. In both SD and
LD incubations, norepinephrine stimulated testosterone
production in a dose-dependent manner (Figs. 1B and 2B).
Numerical increases became evident at doses as low as 10
nM, and at 1 M and at 10 M significant stimulation was
observed. Stimulatory effects of 10 jiM norepinephrine on
testosterone production in the incubations of SD and LD
testes were unaffected by addition of propranolol, but were
abolished by addition of prazosin (Figs. C and 2C).
Addition of hCG to the incubation media produced the
expected dose-related increases in testosterone production
of testes from LD-exposed and SD-exposed hamsters (Figs.
1D and 2D). Maximal doses of hCG (12.5 mIU/ml) caused
a much greater stimulation of testosterone in the incubations of LD testes (70-90-fold increase over production under basal conditions) than in the incubations of SD testes
(5-10-fold increase over basal production; Figs. 1D and 2D).
These effects of hCG were much greater than the effects of
norepinephrine in the same tissues (Figs. 1D and 2D and
data not shown). When norepinephrine, at 10 uIM, was added
together with various doses of hCG, no further significant
stimulation of testosterone production was observed in either
LD or SD incubations (Figs. 1D and 2D).
CATECHOLAMINERGIC STIMULATION OF TESTOSTERONE
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FIG. 1. A) In incubations of testicular fragments from gonadally active LD Phodopus, accumulation of testosterone in incubation media was increased by norepinephrine (NE), epinephrine (EPI), and phenylephrine (PHE) at 10 pM,
but not by isoproterenol (ISO). Asterisks over columns indicate statistically significant differences (means
SEM of
combined representative experiments; ANOVA; Scheffe test, p < 0.05). Numbers inside columns = animals per group.
B) In incubations of testicular fragments from LD Phodopus, NE caused a significant stimulation of testosterone at
concentrations of 10 IMand 1 M. Different letters over columns indicate statistically significant differences (means
± SEM of a representative experiment; ANOVA; Scheffe test, p < 0.05). Numbers inside columns = animals per group.
C) The stimulation of testosterone production by 10 M of NE in the incubations of testicular fragments from LD
animals was significantly inhibited by prazosin (Praz., 50 tiM), but unaffected by a similar dose of propranolol (Prop.).
Different letters over columns indicate statistically significant differences (means
SEM of a representative experiment; ANOVA; Fisher test, p < 0.05). Numbers inside columns = animals per group. D) Accumulation of testosterone
in media from an incubation from the testes of LD animals in response to stimulation by hCG (0, 0.7, 3.1, 12.5 mlU/
ml) in the absence or presence of NE (10 jiM). Asterisk denotes significant stimulation of testosterone by NE (means
+ SEM of a representative experiment; n = 6/treatment; ANOVA; Scheffe test, p < 0.05).
DISCUSSION
Results of the present study provide evidence for the existence of a direct stimulatory effect of catecholamines on
testosterone production by the testes of Siberian hamsters.
Similar stimulatory effects of catecholamines on testosterone production in vitro have been observed in other rodent species, the golden hamster [16] and the adult rat [21].
Similarly to our previous findings in the golden hamster
[16], the stimulatory effects of catecholamines in the Siberian hamster were mediated via alpha-1 adrenoreceptors.
To our knowledge, the existence and localization of this
catecholaminergic receptor type has not yet been reported
in the testis. To date, only beta-adrenergic receptors have
been identified on the Sertoli cell (beta-1 type; [7]) and on
the Leydig cell (beta-2 type; [4, 22]). However, beta receptors did not appear to be involved in mediating the effects
of catecholamines in the present study. Results obtained
previously in rats and mice have suggested that catecholamines act on Leydig cell testosterone production in vitro
via beta-2 adrenoreceptor, using cAMP as a second messenger [4, 22]. All of these studies, however, were conducted on cultured Leydig cells and agree that freshly isolated Leydig cells do not respond to catecholamines with
an increase in production of testosterone, but only with
production of cAMP. It has also been noted that Leydig cell
cultures acquire responsiveness of testosterone synthesis to
catecholaminergic stimuli with time in culture, with maximal responses recorded 24 h after plating [1, 4, 9]. This may
indicate that in vivo either the beta-adrenoreceptor-mediated effects on testosterone production are absent and/
or that steroidogenesis is uncoupled from stimulation of
beta-adrenoreceptors.
The few available reports of effects of catecholamines on
testicular function in vivo are conflicting and do not appear
886
MAYERHOFER ET AL.
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FIG. 2. A) As in incubations of LD testes, in incubations of testicular fragments from gonadally inactive, regressed
SD Phodopus, accumulation of testosterone in incubation media was increased by norepinephrine (NE), epinephrine
(EPI), and phenylephrine (PHE) at 10 IM, but not by isoproterenol (ISO). Asterisks over columns indicate statistically
significant differences (means ± SEM of combined representative experiments; ANOVA; Scheffe test, p < 0.05). Numbers inside columns = animals per group. B) In the incubations of testicular fragments of SD Phodopus, NE caused
a significant stimulation of testosterone at concentrations of 10 tiM and 1 .M. Different letters over columns indicate
statistically significant differences (means + SEM of a representative experiment; ANOVA; Fisher test, p < 0.05).
Numbers inside columns = animals per group. C) The stimulation of testosterone production by 10 p.M of NE in the
incubations of testicular fragments from SD animals was significantly inhibited by prazosin (Praz., 50 M), but unaffected by a similar dose of propranolol (Prop.). Different letters over columns indicate statistically significant differences (means - SEM of a representative experiment; ANOVA; Fisher tests, p < 0.05). Numbers inside columns =
animals per group. D) Accumulation of testosterone in media from incubation of testes from SD animals in response
to stimulation by hCG (0, 0.7, 3.1, 12.5 mlU/ml) in the absence or presence of NE (10 iLM). Asterisk denotes significant
stimulation of testosterone by NE (means - SEM of a representative experiment; n = 6/treatment; ANOVA; Scheffe
test, p < 0.05).
to be helpful in solving the question of the importance of
catecholamines. Thus isoproterenol injected directly into
the testicular artery of the dog increases testosterone production [23], whereas intravenous, subcutaneous, or intraarterial administration of epinephrine decreases testosterone levels in men [24] and rats [25, 26]. Species differences
and various side-effects of catecholamines may account for
these discrepancies and may be the crucial factors that until
now have hampered the study of effects of catecholamines
on the testis in vivo.
The present study as well as our previous study [16] has
used a different experimental approach, namely the shorttime incubation of testicular parenchyma. This in vitro
method eliminates the influence of the possible in vivo side
effects of catecholamines (for example, on testicular blood
flow). Compared to the typical isolation and culture procedures for Leydig cells, this method has the advantage that
the integrity of the testicular parenchyma is largely retained. Possible paracrine mechanisms are presumably not
disrupted, and therefore the incubation system used may
provide a more physiological method than Leydig cell culture. The presence of other testicular cells may well be the
reason for the observed effects of catecholamines on the
production of testosterone by the testes of the golden and
Siberian hamsters. These effects of catecholamines could
be due to indirect action on the Leydig cells mediated via
paracrine mechanisms [27].
In spite of the similarity of the receptor types involved
in mediating catecholamine effects in both the golden and
the Siberian hamster, there is a striking difference between
the responses in these two species. Thus, in the LD golden
hamster, similarly to the adult rat [21], catecholamines alone
were unable to stimulate testosterone production, but rather
acted to enhance the stimulatory effect of hCG. Only in the
CATECHOLAMINERGIC STIMULATION OF TESTOSTERONE
regressed gonads of SD-exposed golden hamsters did catecholamines stimulate testosterone production [16]. We have
speculated that this acquisition of responsiveness to catecholamines in the testes of SD golden hamsters might be
a consequence of the possible homologous up-regulation
of catecholaminergic receptors in the regressed testes. This
assumption was based on the interpretation of measurements of norepinephrine concentrations in SD and LD testes,
which indicated greater norepinephrine concentrations in
the interstitial spaces of LD testis [16]. However, when these
data were expressed as concentrations per gram of testis
weight, norepinephrine levels in LD gonads were not different from norepinephrine concentrations in SD testes [16].
Identical results of unchanged norepinephrine concentrations per gram of testis were previously reported in the
testes of SD and LD Phodopus [28], which might also indicate more norepinephrine to be present in the interstitial
testicular compartment of LD Siberian hamsters. However,
measurements of turnover of testicular norepinephrine and
determination of activation of the sympathetic nervous system, rather than determinations of norepinephrine content
alone, will be required to elucidate the mechanism(s) involved in the regulation of responsiveness to catecholamines.
In contrast to the situation in the golden hamster, epinephrine, norepinephrine, and phenylephrine alone stimulated testosterone production by the testes of both LDexposed and SD-exposed Phodopus to a similar extent (in
terms of stimulation over basal production). At present, we
are not able to provide an explanation for these differences
between the results obtained in Siberian and golden hamsters. Interestingly, catecholaminergic receptors on adipocytes of the golden hamster are under the control of photoperiod by a testosterone-dependent mechanism [29], and
golden hamsters gain weight when exposed to SD. In contrast, SD Siberian hamsters have lower body weight than
LD animals. This is an example of differences in the metabolic responses of these two seasonal breeders and leads
us to speculate that species differences in the regulation of
catecholaminergic receptors (and/or their function) could
account for the differences in responsiveness of testicular
cells to catecholamines. It should be stated at this point that
these species are taxonomically rather distant, even though
both are called hamsters. However, some similarities are
evident. Thus, in the SD-exposed golden hamster, catecholamines were able to stimulate testosterone production
to an extent comparable to the effects of hCG [16]. This is
similar to the stimulatory effect of norepinephrine in testes
from the SD Siberian hamster, which was of the same order
of magnitude (4 times basal production) as the stimulatory
effects of hCG in the regressed testes of this species (5-10
times basal production). In contrast, in testicular incubations of the LD Siberian hamster, stimulatory action of hCG
(70-90 times basal production) was dramatically greater than
the effects of catecholamines (4 times basal production).
887
Thus, in relative terms, catecholaminergic stimuli appear to
be potent stimulators of steroidogenesis in the regressed
testes during physiological suppression of gonadotropin secretion and may be regarded as a "backup system" for gonadotropin (LH) stimulation of testosterone production in
the Siberian [30] and golden [31] hamsters. Compared to
the situation in SD animals, stimulatory effects of catecholamine in the active LD testes are undetectable in the golden
hamster and appear to be of minor importance in the Siberian hamster. Nevertheless, they may be involved in "finetuning" of Phodopus Leydig cell function.
Potentiation of hCG effects by catecholamines was not
observed in the present study, in contrast to our previous
studies [16, 21]. In this conjunction, it should be noted that
in contrast to the golden hamster, hCG in LD Phodopus had
a very strong stimulatory action on testosterone production
(70-90 times basal production in LD Phodopus) compared
to the relatively weak stimulatory action of norepinephrine
(4 times basal production). Thus, we cannot rule out the
possibility that an additive effect of norepinephrine on testosterone production was masked by the overwhelming action of hCG. However, the fact that norepinephrine did not
augment the effects of hCG in SD Phodopus as well, cannot
be explained by a possible "overpowering" stimulation of
testosterone production by hCG, since the stimulatory action of hCG in SD incubations was much less pronounced
than in LD incubations. An alternative explanation could be
that the effect of hCG on testosterone, which is thought to
be mediated by cAMP and the phosphokinase A system, interferes with the alpha-1 receptor-mediated testosterone response to catecholamines, which is not likely to be coupled
to cAMP but rather to hydrolysis of membrane phosphoinositides, mobilization of calcium, and activation of protein kinase C [32]. Additional studies on possible receptor
"cross-talk" and post-receptor events will be required to
resolve these puzzling results.
Taken together, the results of the present study show
that the impact of catecholaminergic stimuli on steroidogenesis in the regressed testes of the Siberian (and the
golden) hamster is by far greater than in the active testes
of these seasonal breeders. This may indicate that catecholaminergic input via innervation of the testis [18, 33, 34]
or by catecholamines reaching the testis via blood flow can
be viewed as a back-up system for LH during the time of
physiological suppression of LH release [30]. The differences in the responses to catecholamines in the Siberian
and the golden hamster, the physiological significance of
catecholamine effects, the exact target cells for catecholamines in the testes, and the mechanism of catecholamine
action remain to be elucidated.
ACKNOWLEDGMENTS
We thank Dr. A. Amador for help in the statistical evaluation of the data and
Laura Israel for technical assistance.
888
MAYERHOFER ET AL.
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