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- and ßB-Subunit Messenger Ribonucleic Acid Expression in Rat Testis after Selective Leydig Cell Destruction by Ethylene Dimethane Sulfonate1
Departments of Physiology (M.T.-S., J.K., A.R., W.Y., I.H.) and Pediatrics (J.K., W.Y.), University of Turku, 20520 Turku, Finland
Address all correspondence and requests for reprints to: Dr. Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ßA, and -ßB messenger RNA
(mRNA) levels were assessed after ethylene dimethane sulfonate
(EDS)-induced destruction of Leydig cells (LC) in different animal
models: the intact rat, the rat treated with high doses of
testosterone, and the unilaterally cryptorchid rat. In intact rats, EDS
selectively eliminates the mature adult-type LCs, activating the
proliferation and differentiation of preexisting LC precursors into a
new population of functionally active LCs. In this model, a single dose
of EDS (75 mg/kg BW, ip) induced a significant increase in testicular
inhibin- and -ßB mRNA levels 5 days after treatment
(5.0- and 5.5-fold increases, respectively), whereas
inhibin-ßA mRNA remained undetectable upon Northern
hybridization in control and EDS-treated testes. Moreover, in
situ hybridization analysis demonstrated that the increased
expression of inhibin- and -ßB mRNAs observed 5 days
after EDS takes place mainly in Sertoli cells. Along with LC
repopulation, the expression level of inhibin- and -ßB
messages declined, and inhibin- mRNA returned to control values on
day 40 after EDS. This treatment, however, failed to alter the pattern
of testicular expression of FSH receptor and androgen-binding protein
mRNAs, thus suggesting selectivity for the above effects. In
EDS-treated rats supplemented with high doses of testosterone, the
preexisting mature LCs are destroyed, but, due to elevated testosterone
concentrations, disruption of spermatogenesis is attenuated, and the
post-EDS rise in serum gonadotropins is blocked; the latter prevents LC
regeneration. In this model, a 5.0-fold increase in inhibin- mRNA
levels, similar to that found in intact animals, was detected 5 days
after EDS administration, but the rise in inhibin-ßB
levels was partially delayed. In addition, the blockade of LC
repopulation resulted in permanent elevation of inhibin- and
-ßB messages throughout the study period. In unilaterally
cryptorchid rats, the abdominal testis shows disrupted spermatogenesis
and altered paracrine environment that expedites LC repopulation after
EDS treatment. In this model, the abdominal testes showed a significant
2.5-fold increase in inhibin- mRNA levels 5 days after EDS, but no
effect was found in those of inhibin-ßB. Further, the
faster rate of LC repopulation resulted in precocious decline of
inhibin- mRNA levels. Finally, the expression of inhibin/activin
subunit mRNAs was monitored during postnatal testicular development,
specifically at the time of regression of fetal-type LCs and appearance
of those of the adult type. High levels of expression of inhibin-
and -ßB mRNAs were detected in neonatal and infantile
testes. A sharp decline in both messages took place between days
15–20, i.e. at the time when fetal-type Leydig cells
are replaced by adult-type cells. From this time point onward,
inhibin- and -ßB mRNA levels remained low, ranging
between 15–30% of the maximum. In conclusion, our results suggest
that the adult-type LCs differentially modulate the expression of
inhibin/activin subunit genes and point to a major inhibitory role in
this cell type on expression of the inhibin- gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit linked to either
a ßA-subunit (inhibin A) or ßB-subunit
(inhibin B) (2, 3, 4, 5). Activins are homo- or heteromeric dimers of
ßA- and ßB-subunits, thus giving rise to
three possible forms: activin A, B, and AB (6, 7, 8). Inhibins and
activins are expressed in a variety of tissues during development and
in adult life, where they have been involved in multiple cellular
events (9, 10, 11, 12, 13, 14). However, despite their widespread pattern of expression
and different regulatory roles, inhibins and activins were first
identified as gonadal peptides with the ability to suppress or
stimulate, respectively, pituitary FSH secretion (4). Postnatally,
testicular Sertoli cells are the major source of circulating inhibin in
the male rat (15, 16, 17, 18, 19), although prominent expression of inhibin -
and ßB-subunits was detected in fetal-type Leydig cells
(18, 19). Testicular inhibin expression is under the control of a complex cellular network that involves Sertoli cells, Leydig cells (LCs), and germ cells. In recent years, this regulatory system has been assessed using different experimental approaches (20, 21, 22, 23, 24, 25). In the rat, administration of the cytotoxic drug ethylene dimethane sulfonate (EDS) induces selective and reversible elimination of mature adult-type LCs from testicular interstitium (26, 27), thus providing an optimal model to evaluate the specific contribution of this cell population to different testicular functions (for examples, see Refs. 28, 29). Indeed, a great deal of the physiological role of adult-type LCs in the control of inhibin secretion has been learned using the EDS-treated rat as an experimental paradigm (22, 23, 30). However, to our knowledge, no data are available on the molecular events involved in testicular inhibin/activin subunit messenger RNA (mRNA) expression in this animal model.
The present experiments were undertaken to characterize temporal
changes in the pattern of testicular inhibin/activin subunit mRNA
expression in response to selective elimination of adult-type LCs under
different endocrine/paracrine backgrounds. In addition, as our initial
results suggested a predominant inhibitory role of this cell type in
the regulation of inhibin- and -ßB messages,
testicular expression of inhibin/activin subunit mRNAs was monitored in
detail during the postnatal developmental phase when fetal-type LCs
regress and are replaced by adult-type LCs (31).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Exp 1, the pattern of testicular inhibin/activin subunit mRNA expression was studied in adult rats at different time points after EDS administration. Adult males (250–300 g BW) were injected ip (day 0) with a single dose of EDS (75 mg/kg BW) or vehicle (dimethylsulfoxide-H2O, 1:3, vol/vol). Groups of animals were sequentially killed 0, 5, 15, 20, and 40 days after EDS treatment. Trunk blood, testes, and ventral prostates were taken, and the weights of the organs were recorded. Sera were separated from blood samples and stored at -20 C until used for hormone measurements. After removal, the testes were immediately frozen in liquid nitrogen and stored at -70 C until used for RNA analyses.
In Exp 2, the time course of changes in testicular inhibin/activin subunit mRNA expression was assessed in EDS-treated rats supplemented with a high dose of testosterone (T). In this experimental model, exogenously administered T is able to prevent at least partially the disruption of spermatogenesis and to block the rise in serum gonadotropin levels observed after EDS administration (32, 33). Adult male rats injected ip (day 0) with a single dose of EDS (75 mg/kg BW) were simultaneously implanted with a SILASTIC brand elastomer (length, 55–60 mm; id, 0.062 cm; od, 0.125 cm; Dow Corning Corp., Midland, MI) containing T; this dose was previously demonstrated to suppress the serum LH rise after EDS administration (33). Groups of rats were sequentially killed 0, 5, 15, 20, and 40 days after EDS treatment. Trunk blood and testes were collected and processed as in Exp 1.
In Exp 3, testicular expression of the inhibin/activin subunit mRNAs was assessed in unilaterally cryptorchid rats treated with EDS. In this experimental paradigm, germ epithelium is highly disturbed due to abdominal location (34). Adult male rats were rendered unilaterally cryptorchid by anchoring the upper pole of the testis to the abdominal wall using a suture passing through the connective tissue of the caput epididymis, as previously described (35). One month after surgery, the animals were injected ip (day 0) with a single dose of EDS (75 mg/kg BW) or vehicle. Groups of animals were sequentially killed 0, 5, 15, 20, 30, and 40 days after EDS treatment. An additional sampling time point (day 30 after EDS) was included, given the expected faster rate of LC repopulation in this group (36, 37). Trunk blood and testes were collected and processed as in Exp 1.
In Exp 4, changes in the pattern of inhibin/activin subunit mRNA expression were monitored during postnatal testicular development. Specifically, as results from previous experiments suggested a modulatory role of adult-type LCs, the analysis was focused on the developmental frame in which regression of fetal-type LCs and the appearance of adult-type LCs take place (31). Hence, male rats were killed on days 1, 7, 15, 20, 30, and 60 of age. The testes were collected and processed as in Exp 1.
Northern hybridization analysis
Total RNA was isolated from testicular samples using the single
step acid guanidinium thiocyanate-phenol-chloroform extraction method,
as described previously (38). For Northern hybridization analyses, RNA
samples (20 µg/lane) were resolved on 1.2% denaturing agarose gels
and transferred onto Hybond-N+ nylon membranes
(Amersham International, Aylesbury, UK) using the
capillary method. The membranes were cross-linked by short wave UV
irradiation and prehybridized for 4–6 h at 64 C in a solution
containing 50% deionized formamide, 3 x SSC, 5 x
Denhardt’s solution, 0.1 g/liter heat-denatured calf thymus DNA, 1%
SDS, and 0.1 g/liter yeast transfer RNA. For hybridization,
32P-labeled complementary RNA (cRNA) probes specific for
the target genes were generated using the Riboprobe system II kit
(Promega Corp., Madison, WI) and the corresponding
complementary DNA (cDNA) templates (see below). Hybridizations were
carried out at 66 C for 20 h in the same prehybridization solution
after addition of the corresponding cRNA probe. After hybridization,
the membranes were washed in 2 x SSC-0.1% SDS at room
temperature for 20 min, 0.5 x SSC-0.1% SDS for 20 min at 65 C,
and three times in 0.1% SSC-0.1% SDS for 1 h at 65 C. The
filters were exposed to Kodak x-ray films
(Kodak XAR-5 and XLS 5, Eastman Kodak Co.,
Rochester, NY) at -70 C for 24–96 h. Relative mRNA levels were
obtained by densitometric scanning of the autoradiograms (TINA 2.0
package, Raytest GmbH, Straubenhardt, Germany), and the values were
normalized by the amount of 18S ribosomal RNA transferred per lane, as
estimated under ethidium bromide staining. In addition, as EDS
treatment induces a reduction in testicular volume without altering the
number of Sertoli cells (26, 39), for semiquantitative presentation
densitometric values were corrected by testicular weight, thus giving
an estimate of the total level of expression of each message per
testis. The molecular sizes of the mRNA species were estimated by
comparison with mobility of the 18S and 28S ribosomal RNAs. Reagents
for RNA analysis were obtained from Sigma Chemical Co.
(St. Louis, MO) unless otherwise stated.
In situ hybridization
Five-micron sections of testis tissue from control and
EDS-treated rats (5 days after EDS) were used for in situ
hybridization. Specific sense and antisense 35S-labeled
cRNA probes were synthesized using the Riboprobe system II kit
(Promega Corp.), [35S]UTP and the
corresponding cDNA templates (see below). Pretreatment of sections was
performed as described previously (40). Pretreated slides were
hybridized overnight at 50 C, and thereafter they were washed first in
2 x SSC, 50% formamide, and 10 nM dithiothreitol
(DTT; Roche Molecular Biochemicals, Ingelheim, Germany) at
50 C for 30 min and then in 0.2 x SSC, 50% formamide, and 10
nM DTT at 50 C for 10 min. The slides were rinsed with
1 x PBS and treated with 10 µg/ml ribonuclease A (Roche Molecular Biochemicals) in Tris-EDTA buffer and 0.5
M NaCl (pH 8.0) at 37 C for 30 min. After digestion,
washing with 2 x SSC, 50% formamide, and 10 nM DTT
was repeated. Finally, the slides were rinsed with 2 x SSC,
dehydrated in ethanol and processed for liquid emulsion autoradiography
using NTB-3 emulsion solution (Eastman Kodak Co.). The
slides were exposed at 4 C for 2 weeks and developed at 12 C by
treatment with D-19 solution (Eastman Kodak Co.) for 2.5
min. Thereafter, the samples were fixed for 5 min with Unifix
(Eastman Kodak Co.) and rinsed with distilled water for 5
min. Finally, the nuclei were fluorescence stained with Hoechst 33258
(Sigma Chemical Co.), and the slides were mounted with
Glycergel (DAKO Corp., Glostrup, Denmark; data not
shown).
Probes
For detection of inhibin subunit mRNAs, specific antisense cRNA
probes were generated using the following cDNA templates for in
vitro transcription: 1) BglII-linearized pGEM-4Z
plasmid (Promega Corp.) containing the full-length rat
inhibin- cDNA driven by T7 RNA polymerase promoter, 2)
StyI-linearized pGEM-4Z plasmid containing the full-length
rat inhibin ßA cDNA driven by T7 RNA polymerase promoter,
and 3) HindIII-linearized pGEM-4Z plasmid containing a
fragment of rat inhibin ßB cDNA comprising 120 bp of
precursor sequence and 230 bp from mature coding region under SP6 RNA
polymerase promoter (3, 41). In addition, for in situ
hybridization analysis, specific sense cRNA probes were generated
using, respectively, SP6 RNA polymerase and
HindIII-linearized rat inhibin- cDNA, and T7 RNA
polymerase and NheI-linearized rat inhibin ßB
cDNA in pGEM-4Z vector. For generation of FSH receptor (FSHR)
riboprobe, a template composed of a fragment of rat FSHR cDNA (spanning
bp 621-1031), subcloned into pGEM-4Z under T7 RNA polymerase promoter,
was used (42). Finally, antisense cRNA probe for androgen-binding
protein (ABP) mRNA analysis was produced using as template a fragment
of ABP cDNA (spanning bp 703-1235) subcloned into pBluescript II KS
under T7 RNA polymerase promoter (donated by Dr. G. L. Hammond,
London, Canada).
Hormone measurements
Serum FSH levels were assayed by a double antibody RIA using
kits supplied by the NIDDK (Bethesda, MD), and the results were
expressed in terms of reference preparation FSH-RP-2. The sensitivity
of the assay was 0.15 ng/tube, and the intra- and interassay variations
were below 8% and 15%, respectively. Serum LH levels were measured
using a supersensitive immunofluorometric assay (Wallac, Inc., Turku, Finland), based on the Delfia principle (43), and
the results were expressed in terms of the reference preparation
LH-RP-2 (NIDDK). The sensitivity of the assay was 0.75 pg/tube, the
intraassay coefficient of variation was 7%, and the interassay
coefficient of variation was 10%. Serum T levels were measured by RIA
after diethyl ether extraction of the samples, using
125I-labeled T (Orion-Farmos Diagnostica, Turku, Finland)
as tracer, and T antiserum donated by Dr. R Vihko (Department of
Clinical Chemistry, Oulu University, Oulu, Finland).
Statistics
The data are expressed as the mean ± SEM.
Statistically significant differences between groups were determined by
one-way ANOVA, followed by Duncan’s new multiple range test.
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Administration of a single dose of EDS (75 mg/kg BW) to intact rats
induced a drop in serum T levels to the range measured in
orchidectomized animals (<0.5 nmol/liter; data not shown) 5 and 15
days after EDS, followed by a gradual recovery of serum T along with LC
repopulation; a recovery that was complete by day 40 after EDS.
Inversely, serum LH (data not shown) and FSH levels increased gradually
up to day 20 after EDS administration and declined thereafter, although
serum FSH remained above control levels even 40 days after EDS
treatment. In addition, testis weight decreased significantly 15 and 20
days after EDS, but it was still similar to that in controls on day 5.
By day 40 after EDS treatment, a partial recovery of testicular weight
was detected, yet it remained lower than controls at this time
point.
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2.5-fold over control values; P < 0.01), and serum
LH levels fell to undetectable levels (data not shown) throughout the
experimental period. In addition, the rise in serum FSH after EDS
treatment was prevented, as FSH levels remained similar to control
values at all time points studied, and the drop in testicular weight
was significantly (P < 0.01) diminished on days 15 and
20 after EDS treatment. In UC rats, basal T, LH (data not shown), and
FSH levels were not significantly different from control values, but
cryptorchid testes showed reduced weight (50% of those of
contralateral scrotal testes). In this model, EDS administration
induced a reduction in serum T levels to values similar to those in
orchidectomized animals (see above) 5 days after treatment; this
response was followed by a precocious recovery of serum T values
compared with those in intact, EDS-treated rats. Accordingly, serum LH
(data not shown) and FSH levels significantly increased in early stages
after EDS administration and afterward showed a faster decline.
Interestingly, from day 30 after EDS treatment, FSH values had returned
to the basal range, in contrast to data from intact, EDS-treated rats.
Finally, weights of the abdominal testes remained unchanged at all time
points after EDS administration in UC rats.
Testicular inhibin/activin subunit mRNA expression was assessed in the
experimental paradigms presented above. For accurate comparison of the
semiquantitative values, relative steady state mRNA levels were
normalized by testicular weight, as EDS treatment induces a significant
shrinkage of testicular volume with no apparent alteration of Sertoli
cell number (26, 39). This allows reliable estimation of the total
level of expression of each message per testis. Administration of EDS
to intact rats induced a significant increase in testicular inhibin-
and inhibin-ßB mRNA levels 5 days after treatment (5.0-
and 5.5-fold increases, respectively; mRNA levels corrected per testis
weight; Fig. 2). Localization analysis by
means of in situ hybridization demonstrated that the
reported increased expression of inhibin- and -ßB
mRNAs at this time point took place mainly in Sertoli cells (Fig. 3). The level of expression of these
messages remained elevated on days 15 and 20 after EDS, declining
thereafter along with LC repopulation; on day 40 after EDS, inhibin-
mRNA levels, but not those of inhibin-ßB, had returned to
control values. Inhibin-ßA was undetectable upon Northern
hybridization in both control and intact, EDS-treated rat testes, nor
was it detectable in the other experimental paradigms (data not shown).
In addition, EDS treatment failed to significantly alter the pattern of
expression of FSHR and ABP messages, selectively expressed in Sertoli
cells, thus suggesting that the reported effects on inhibin/activin
subunit mRNAs observed after EDS administration were selective (Fig. 2).
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was detected 5 days
after EDS, but the rise in inhibin-ßB mRNA levels was
partially delayed (1.5- to 2.0-fold increase over controls on day 5;
4.0-fold increase on day 15 after EDS). In addition, the blockade of LC
repopulation by T (33) resulted in permanently elevated levels of both
messages throughout the study period (Fig. 4). In UC rats, temporal changes in the
patterns of expression of inhibin- and inhibin-ßB
mRNAs in contralateral scrotal testes mimicked those reported for
intact rats (data not shown). In cryptorchid testes, EDS administration
induced a significant 2.5-fold increase in inhibin- mRNA levels 5
days after treatment, but did not modify those of
inhibin-ßB. In addition, the expedited rate of LC
repopulation in abdominal testes (36, 37) resulted in a precocious
decline in the level of expression of inhibin- mRNA, that had
returned to control values on day 30 after EDS treatment (Fig. 5). The effects of EDS treatment on
testicular expression of inhibin- and -ßB mRNAs in the
different experimental paradigms are summarized in Fig. 6.
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, -ßA,
and -ßB messages was monitored in detail during the
developmental frame in which regression of fetal-type LCs and
appearance of adult-type LCs take place. For clear presentation of the
data, the maximum level of expression of each message during the study
period was taken as 100%, and the other values were normalized
accordingly, thus allowing semiquantitative comparison between
different time points. High levels of expression of inhibin- and
-ßB subunit mRNAs were detected in testes from neonatal
and infantile (1- to 15 day-old) rats, with maximum expression in
7-day-old animals. Between days 15 and 20 of age, a sharp decline was
noted in the expression of both messages. From this time point onward,
inhibin- and -ßB mRNA levels remained low, ranging
between 15–30% of maximum values of the infantile period. Similarly,
testicular inhibin-ßA expression was detected at high
levels in neonatal rat testis. However, a decline in expression of this
message was observed between days 7 and 15 of age, and it became
undetectable from day 30 onward, in keeping with our previous
observations in adult rat testes (see above). Finally, the
developmental pattern of expression of FSHR mRNA, another message
abundantly expressed in Sertoli cells, was clearly different from those
of inhibin/activin subunits, thus suggesting again that the reported
observations were not due to nonspecific factors (Fig. 7). A summary of the developmental
changes in the level of expression of inhibin-, -ßA,
and -ßB and FSHR mRNAs during postnatal testicular
development is presented in Fig. 8.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
-ßB mRNA levels was detected during the early stages
after EDS administration. In addition, although measurement of its
serum levels is still pending, these data suggest that inhibin B, the
physiological form of inhibin in the male (44), accounts for the
reported rise in serum immunoreactive inhibin after EDS treatment, as
inhibin-ßA mRNA was undetectable upon Northern
hybridization in both control and intact, EDS-treated rats.
The increase in the level of expression of testicular inhibin- and
-ßB mRNAs was observed in different experimental
paradigms after EDS-induced LC destruction. This observation together
with the fact that the rise in inhibin subunit mRNAs took place
regardless of the endocrine background, i.e. in the presence
of low and high FSH levels, strongly suggest that a short regulatory
loop involving mature adult-type LCs operates within the adult rat
testis for the control of inhibin- and -ßB gene
expression. In our study, relative steady state mRNA levels were
assessed by Northern hybridization of total RNA isolated from
individual testes at different time points after EDS administration.
Apparently, semiquantitative interpretation of the data could be
hampered by the fact that EDS treatment induces a significant shrinkage
of testicular volume without largely altering Sertoli cell number (26, 39). For this reason, quantitative comparison was carried out after
normalization of expression levels by testicular weight, a procedure
that gives a reliable estimate of the total level of expression of each
message per testis. In addition, several pieces of evidence indicate
that the reported increases in inhibin subunit mRNA levels were not an
artifact merely due to Sertoli cell enrichment after reduction in
testis weight. First, maximum increases in inhibin- and
-ßB levels were observed on day 5 after EDS, a time point
when no reduction in testicular volume is yet detected. Second, the
rise in inhibin subunit mRNA levels was observed even after partial
prevention of testicular shrinkage by simultaneous T treatment. Third,
temporal changes in inhibin- and -ßB levels appeared
tightly correlated with the fate of mature LCs, i.e.
elimination of LCs was associated with increases in inhibin subunit
mRNAs, whereas LC regeneration associated with a decrease in the
expression of both messages. Fourth, the pattern of expression of other
messages selectively expressed in Sertoli cells, as FSHR and ABP was
not similarly affected by EDS treatment, thus suggesting that the
reported effects on inhibin/activin subunit mRNAs were specific.
Finally, although no detailed quantitative analysis was performed, it
appeared evident from in situ hybridization data that an
increase in the level of expression of inhibin- and
-ßB mRNAs does take place in Sertoli cells at early
stages after EDS administration.
Despite the ability to assess inhibin- and -ßB subunit
mRNA expression, inhibin-ßA mRNA was undetectable by
Northern hybridization in both adult control and intact, EDS-treated
rat testes, nor was it detectable in the other experimental paradigms.
This apparent lack of expression may be related to the limited
sensitivity of this analytical technique, and more sensitive
procedures, such as quantitative RT-PCR, may be needed to fully unravel
changes in the pattern of expression of inhibin-ßA mRNA
after elimination of adult-type Leydig cells. However, our data from
Northern hybridizations clearly indicate that major differences exist
in the patterns of inhibin- and -ßB, and
inhibin-ßA mRNA expression, both in response to Leydig
cell withdrawal and in terms of basal expression along testicular
development (see below).
Compelling evidence indicates that germ cells influence testicular
inhibin expression. Inhibin- and -ßB subunit mRNAs
are expressed in rat testis in a stage-specific manner during the
seminiferous epithelial cycle (21, 25), and destruction of
spermatogenic cells by testicular irradiation increases inhibin
production by immature Sertoli cells (20). Further, removal of
pachytene spermatocytes resulted in elevated basal inhibin production
(24) and inhibin- mRNA levels (25). Thus, it is possible that the
reported effects of EDS administration on inhibin subunit mRNA levels
could be due to indirect disruption of spermatogenesis rather than
primary destruction of mature LCs. Some data, however, argue against
this possibility in the case of the inhibin- gene. First,
inhibin- mRNA levels rose with similar magnitude after EDS
administration in intact and T-supplemented animals. Presumably, the
dose of T used was unable to completely prevent the disturbance of
spermatogenesis, but it clearly delayed germ cell loss, as evidenced by
the reduced decline in testicular weight in this model. This moderate
dose of T was selected to avoid a suprapharmacological input on
testicular function, giving the conflicting results on the direct
effects of high doses of T on inhibin production (45, 46). Second,
elevation of inhibin- mRNA levels, albeit to a lesser extent, also
took place in cryptorchid testes, where spermatogenic cells were
destroyed before EDS administration due to abdominal testicular
location. Interestingly, whereas testicular inhibin- mRNA levels
increased in all experimental models after LC elimination, differences
were noted in the pattern of response of inhibin-ßB
message to EDS administration in intact, T-treated and UC rats. In this
sense, the rise in inhibin-ßB mRNA was significantly
delayed in EDS- plus T-treated animals and was completely absent in
abdominal testes of UC rats 5 days after treatment. Taken together, the
data presented herein strongly suggest that the increase in inhibin-
mRNA expression is at least partially directly caused by elimination of
inhibitory input(s) from mature LCs, whereas the effects of LC
withdrawal on inhibin-ßB mRNA expression might be carried
out mainly through an indirect loop that involves germ cell
degeneration. In addition, partial contribution of circulating factors
to the reported differences in the pattern of inhibin/activin subunit
mRNA expression among the experimental groups cannot be excluded.
Although long term cryptorchidism was shown previously to induce a
decrease in testicular inhibin production (47), basal expression of
inhibin- and -ßB mRNAs in abdominal testes of UC rats
was only slightly reduced (15–20% decrease from controls; mRNA levels
corrected per testis weight), and the rise in inhibin- was still
observed after EDS administration. The latter is consistent with
previous data showing that cryptorchid testes remain responsive to
stimuli of inhibin production (47).
Although Sertoli cells are the major source of testicular inhibin in
rat after puberty, prominent and ßB immunostaining is
detected in fetal-type LCs (18, 19). Thus, the possibility existed that
the rise in inhibin- and -ßB mRNA levels in adult rat
testes after EDS administration could be due to high levels of
expression of these messages in regenerating LCs. However, data from
in situ hybridization analysis clearly demonstrated that
expression of inhibin- and -ßB messages at early
stages after EDS administration is mainly located in Sertoli cells
within the seminiferous tubules. The low expression of inhibin- and
-ßB mRNAs in the interstitial space of adult, EDS-treated
rat testes, despite the presence of a high number of actively
proliferating LC precursors, is in keeping with previous data showing
that the expression of inhibin- and -ßB subunits, as
assessed by in situ hybridization and immunohistochemistry,
is very low in developing adult-type LCs (18, 48) and is in good
agreement with our own observation of the persistent increase in
inhibin- and -ßB mRNA expression after EDS
administration even when maturation of LC precursors was blocked by
administration of exogenous T.
The physiological role of inhibin in the male rat as a FSH-suppressing
signal declines with age. Immunoneutralization of endogenous inhibin
failed to alter FSH secretion after puberty (49, 50), and testicular
immunoreactive inhibin and inhibin- and -ßB mRNA
concentrations peaked before puberty and declined thereafter (50, 51).
The present data on the pattern of expression of inhibin/activin
subunit mRNAs during postnatal testicular development are in keeping
with those data. Further, when detailed analysis was carried out during
the period of regression of fetal-type LCs and appearance of adult-type
LCs (31), a sharp decline in the level of expression of inhibin- and
-ßB messages was detected between days 15 and 20 of age,
i.e. the time frame when fetal-type LCs are replaced by
adult-type cells. Although this relationship may not be causal, our
results together with the data from EDS-treated adult rats support the
possibility that, either through direct or indirect regulatory loops,
adult-type LCs have an inhibitory role in inhibin- and
-ßB mRNA expression. Interestingly,
inhibin-ßA mRNA levels were expressed at high levels in
infantile rat testes, but sharply declined between days 7 and 15 of
age, becoming undetectable from day 30 onward. This fact together with
the lack of expression of this message in adult testes after selective
LC destruction clearly indicate that different regulatory mechanisms
are responsible for the drop in expression of the different
inhibin/activin subunits in rat testis during development.
Presumably, our experimental data were limited to analysis of changes in the pattern of expression of inhibin/activin subunits at the mRNA level. Considering, however, that expression of these messages is just one step toward the synthesis of biologically active dimeric proteins (see introduction), it will be relevant to correlate our present findings with changes in the pattern of inhibin/activin subunit expression at the protein level to evaluate the physiological role of the proposed regulatory pathway. In addition, given the functional relationship among inhibins, activins, and follistatin (for a review, see Ref. 5), analysis of the pattern of follistatin gene expression in our experimental paradigms will help to fully characterize the paracrine regulatory network responsible for the control of biological actions of testicular inhibins and activins.
In conclusion, the results presented herein demonstrate that adult-type
LCs are involved in the regulatory mechanism(s) responsible for the
control of inhibin- and -ßB gene expression in rat
testis and point to a major inhibitory action of this cell type on the
regulation of inhibin- mRNA levels.
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Acknowledgments |
|---|
, inhibin ßA,
inhibin ßB, FSHR, and ABP cDNA templates. The skillful
technical assistance of Ms. Aila Metsävuori and Ms. Tarja Laiho
is gratefully acknowledged. M.T.-S. is indebted to Drs. E. Aguilar and
L. Pinilla for helpful discussions during preparation of this
manuscript. |
Footnotes |
|---|
2 Supported by a postdoctoral grant from DGICYT (Ministerio de
Educación y Cultura, Spain). Present address: Department of
Physiology, University of Cordoba, 14004 Cordoba, Spain.
Received April 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ßA and ßB subunits in
various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–251 and ßB subunits during
the cycle of the rat seminiferous epithelium. Endocrinology 124:987–991- and ß-subunit
complementary deoxyribonucleic acids and expression in the ovary. Mol
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, intact rats;
, EDS-treated rats supplemented with exogenous T; and 
, UC rats.
From the latter, only weights from abdominal testes are presented.
Data points with different superscripts are
significantly different from each other (by ANOVA followed by Duncan’s
test; mean ± SEM; four to eight samples per group).
