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Department of Physiology, University of Turku, 20520 Turku, Finland
Address all correspondence and requests for reprints to: Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllinkatu 10, 20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Introduction |
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LH plays a pivotal role in the control of LC development, both during normal puberty (16) and during EDS-induced repopulation (9, 15), thus implying that LH receptors (LHRs) are expressed in LC precursors. However, the current data on this point are still controversial. Shan and Hardy (17) identified LH binding sites, as well as LHR protein and messenger RNA (mRNA) in LCs (termed LC progenitors) isolated from prepubertal (21-day-old) rat testes. In contrast, Moore and Morris (18) were unable to detect human CG (hCG)-binding sites in LC precursors 4 days after EDS treatment. We previously reported the persistent expression of a truncated form [1.8 kilobase (kb)] of LHR mRNA, encoding regions of the extracellular domain of the receptor, in LC precursors 5 days after EDS treatment, a time point when neither other species of LHR mRNA nor hCG-binding could be detected (19). A similar finding was reported by Veldhuizen-Tsoerkan et al. (20) in testes of long-term hypophysectomized, EDS-treated rats, although weak expression of the longer LHR messages was also found. Whether the 1.8-kb LHR mRNA variant, highly expressed in the rat testis after EDS, has a functional role in the early LC precursors remains to be elucidated.
Considering the key role of LH in the differentiation process of LC precursors into mature LCs, we aimed to further characterize the pattern of LHR expression during LC development using the EDS-treated rat model. For this purpose, 1) testicular LHR mRNA levels were analyzed at different time points after administration of EDS in presence of high (EDS alone) or suppressed (EDS + T replacement) levels of LH, and 2) the timing in the onset of functional LHRs was mapped by evaluating the testicular responsiveness in vitro to human recombinant LH (recLH) in the same experimental paradigms. In addition, to elucidate the role of the truncated 1.8-kb LHR mRNA species in LC development, 3) the translational status of this form was evaluated in control and EDS testes by means of polysome association analysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Exp 1, detailed characterization of the temporal changes in the pattern of testicular LHR expression after EDS was carried out. Adult male rats (day 0) were injected ip with a single dose of EDS (75 mg/kg BW) or vehicle (dimethylsulfoxide-H2O, 1:3). In addition, to evaluate the modulatory role of LH in this event, male rats injected with EDS (day 0) were simultaneously implanted with a SILASTIC brand silicon tubing (Dow Corning, Midland, MI) elastomer (50 mm length; inner diameter, 0.062 cm; exterior diameter, 0.125 cm) containing testosterone (T), in a dose previously determined to suppress the endogenous rise in LH after EDS treatment (9). Groups of animals were sequentially killed 5, 15, 20, and 45 days after treatment. Trunk blood, testes, and the ventral prostate were collected from each animal, and the weights of the organs were recorded. Blood was allowed to clot overnight at 4 C, and the serum was separated by centrifugation, frozen, 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 LHR binding and RNA analyses.
In Exp 2, the translational status of the truncated (1.8-kb) form of the LHR mRNA, persistently expressed in rat testis after EDS treatment, was evaluated by polysome analysis. Adult male rats (day 0) were injected with EDS or vehicle and killed 5 days later. Trunk blood and testes were collected and processed as in Exp 1.
In Exp 3, the appearance of functional LHRs during LC repopulation after EDS treatment was assessed. Slices of testicular tissue from control, EDS-treated (5, 15, and 20 days after treatment) and EDS + T-treated (15 and 20 days after treatment) males were incubated in the presence or absence of human recLH, and the testicular responsiveness evaluated in terms of stimulation of cAMP and steroid production (see below).
All the experimental procedures were approved by the Turku University committee on laboratory animal care and were conducted in accordance with the European normative for care and use of experimental animals.
Hormone measurements
Serum LH levels were measured using a supersensitive
immunofluorometric assay, based on the Delfia principle (Wallac Oy,
Turku, Finland), as described by Haavisto et al. (21).
The sensitivity of the assay was 0.75 pg/tube, the intraassay
coeficient of variation (CV) was 7%, and the interassay CV was 10%.
The results were expressed in terms of the NIDDK (Bethesda, MD)
reference preparation LH-RP-2. Serum FSH levels were determined by a
double antibody RIA method, using kits supplied by NIDDK. The
sensitivity of the assay was 0.15 ng/tube, the intraassay CV was <8%,
and the interassay CV was <15%. The results were expressed in terms
of the reference preparation FSH-RP-2. Serum T levels were measured by
RIA, after diethyl ether extraction, as previously described (22). T
and progesterone levels in the tissue incubation media were assayed by
RIA (22, 23), without prior extraction of the samples.
125I-labeled T and progesterone (Orion-Farmos Diagnostica,
Turku, Finland) were used as tracers. Pregnenolone was measured from
diethyl ether extracts of incubation media by RIA, using
3H-labeled pregnenolone (Dupon NEN, Wallac Oy, Turku,
Finland) as tracer and a pregnenolone antiserum donated by Prof. R
Vihko (Department of Clinical Chemistry, University of Oulu, Finland)
(24).
Testicular LHR and PRL receptor (PRLR) binding measurements
Testicular LH and PRL receptors were measured as previously
described (25, 26, 27). Briefly, testes were homogenized in Dulbecco’s PBS
(10 ml/g tissue), and the homogenates centrifuged at 2000 x
g for 10 min. The supernatants were collected, and the
membrane fractions precipitated by centrifugation at 20,000 x
g for 20 min. Finally, the pellets were dissolved in
Dulbecco’s PBS + 0.1% BSA (Sigma Chemical Co., St. Louis, MO) (10
ml/g original tissue) and used for binding assays. Total testicular
[125I]iodo-hCG binding was measured by incubation of
100-µl aliquots of tissue homogenates in the presence of 150,000 cpm
(
3 ng) iodinated hCG. Nonspecific binding was assessed in matched
samples in the presence of a 1000-fold molar excess of unlabeled hCG
(Pregnyl, Organon, Oss, Netherlands). The incubation time was 16 h
at room temperature (23 C). Bound and free hormones were separated by
15-fold dilution with ice-cold Dulbecco’s PBS-BSA and centrifugation
of the samples. As the concentration of hCG used in the incubations was
near saturating (26), the binding assay gives a reliable estimate of
the LHR content. Measurement of testicular PRLRs was carried out
essentially as described above, using [125I]iodo-hGH as
tracer. Evaluation of nonspecific binding was achieved by incubation of
matched samples in presence of ovine PRL (Sigma, 5 µg/tube). Because
hGH specifically binds in rat tissues to lactogen receptors (28), the
hGH-binding assay allows accurate estimation of the number of PRLRs in
the samples analyzed.
Northern hybridization analysis
Total RNA was isolated from testicular samples using
the single-step acid guanidinium thiocyanate-phenol-chloroform
extraction method, as previously described (29). For Northern
hybridization analyses, RNA samples (20 µg/lane) were resolved on a
1.2% denaturing agarose gel and transferred onto Hybond-N+
nylon membranes (Amersham International, Aylesbury, UK) by the
capillary method (30). The membranes were prehybridized for 4 h at
64 C in a solution containing 50% deionized formamide (Sigma), 3x
SSC, 5x Denhardt’s solution, 0.1 g/liter heat-denatured calf thymus
DNA, 1% SDS, and 0.1 g/liter yeast transfer RNA. For hybridization, a
complementary RNA (cRNA) probe for the rat LHR was generated from a
fragment of the LHR complementary DNA (cDNA), spanning nucleotides
441–849 of its extracellular domain, subcloned into pGEM-4Z plasmid
(31). The 32P-labeled probe was generated using a Riboprobe
system II kit (Promega, Madison, WI), and the cDNA as template.
Hybridization was performed for 18–20 h at 66 C in the same
prehybridization solution after addition of the cRNA probe. After
hybridization, the membranes were washed in 2x SSC and 0.1% SDS at
room temperature for 15 min, and to remove nonspecific hybridization
treated with ribonuclease-A (3 mg/liter in 2x SSC) for 15 min at room
temperature, followed by two washes in 0.2x SSC and 0.1% SDS at 64 C
for 30 min. The filters were exposed to x-ray films (Kodak XAR-5,
Eastman Kodak, Rochester, NY) at -70 C for 7–14 days. Relative mRNA
levels of the different LHR splice variants were obtained by
densitometric scanning of the autoradiograms (TINA 2.0 package, Raytest
gbH, Straubenhardt, Germany), and the values normalized by the amount
of 18S ribosomal RNA transferred per lane, as estimated under ethidium
bromide staining. The molecular sizes of the mRNA species were
determined by comparison with the mobilities of the 18S and 28S
ribosomal RNAs.
Polysome analysis
Polysome-association analysis was performed as previously
described (32). Testicular samples from control and EDS-treated rats (5
days after treatment) were homogenized on ice in 1x HNM buffer [20
mmol/liter HEPES (pH 7.5), 100 mmol/liter NaCl, and 1.5 mmol/liter
MgCl2] containing 0.5% (vol/vol) Triton X-100, 3.5
mg/liter cycloheximide, 10 mmol/liter EGTA, 300,000 U/liter RNasin, and
3 mmol/liter ß-mercaptoethanol, and centrifuged at 8000 x
g for 5 min. The supernatants were collected and loaded onto
linear gradients of 10–35% (wt/vol) sucrose in 1x HNM buffer with
0.5-ml 60% (wt/vol) sucrose cushions. As control, equivalent samples
were processed as described above, using buffer (1x HNE) devoided of
MgCl2 and supplemented with 20 mmol/liter EDTA, to
dissociate the ribosomal complexes from RNA. The gradients were
centrifuged at 37,000 rpm for 2 h. Ten fractions of 1.05 ml were
collected, and made up to 5.0 ml with denaturing solution [4 mol/liter
guanidinium thyocianate (Fluka, Buchs, Switzerland), 25 mmol/liter
sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 mol/liter
ß-mercaptoethanol]. Total RNA was isolated from each fraction by the
single-step method (29). Thereafter, similar aliquots of each fraction
were resolved on denaturing gels and analyzed by Northern
hybridization, as described above. The relative mRNA levels per
fraction of the 6.8- and 1.8-kb forms were obtained by densitometric
scanning of the autoradiograms, and the results expressed as percentage
of the most abundant fraction (100%).
In vitro incubations
Testicular samples were obtained from control, EDS-treated (on
days 5, 15, and 20 after injection), and EDS + T-treated (on days 15
and 20 after treatment) male rats. For incubations, the testes were
removed immediately after decapitation, decapsulated, and cut into
pieces of approximately equal size. The testis slices were then
incubated in 2 ml DMEM/F12 medium (1:1) (Gibco BRL, Paisley, Scotland,
UK), supplemented with 10% FCS, 0.1 g/liter gentamicin (Biological
Industries, Bet-Haemek, Israel), and 0.1 mmol/liter
1-methyl-3-isobutylxanthine (Aldrich Chemie, Steinheim, Germany) in
a Dubnoff shaker (60 cycles/min) at 32 C under an atmosphere of 5%
CO2-95% O2. After a preincubation of 30 min,
the medium was replaced either by fresh medium (DMEM/F12) or medium
containing 100 µg/liter of human recLH (15,000 IU/mg, Ares Serono,
Geneva, Switzerland). The optimal incubation time was tested to be
4 h. At the end of this period, the testicular samples were
recovered and weighed, and the incubation media were collected for cAMP
and steroid measurements as described above. The levels cAMP, T,
progesterone, and pregnenolone in the media were expressed as
normalized values per 100 mg incubated tissue.
Statistical analysis
The data are expressed as 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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). In
EDS-treated rats, serum T significantly decreased to the range measured
in orchidectomized rats (<0.5 nmol/liter, data not shown) 5 and 15
days after EDS. A partial recovery was observed on day 20, and on day
45 the recovery was complete. Serum LH levels increased gradually 5,
15, and 20 days after EDS, returning to the control range by day 45
after EDS injection. Serum FSH levels already were increased 5 days
after EDS and reached their maximum (2-fold elevation) by day 20, but
remained higher than controls even 45 days after the treatment. In EDS
+ T rats, the serum T levels were significantly (2.5- to 3-fold;
P < 0.01) elevated throughout the experimental period.
Consequently, serum LH dropped to undetectable levels (<0.05 ng/liter,
by Delfia assay) at all the time points studied. In contrast, the serum
levels of FSH remained similar to those of control animals during the
whole study period. In summary, our data on endocrine profiles were in
concert with previous observations from us (19, 33) and others (9, 12, 34), thus supporting the validity of the results obtained
thereafter.
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). In EDS-treated rats, specific testicular binding of
[125I]iodo-hCG, as estimate of the total number of LHRs,
fell to undetectable levels 5 and 15 days after treatment, recovered
partially (10% of control levels) by day 20, and returned to
control levels 45 days after EDS, in accordance with previous results
(19). In EDS + T rats, a similar drop in LHR binding was detected on
days 5 and 15 after treatment. However, under the LH suppressed
conditions, no recovery of [125I]iodo-hCG binding was
detected on days 20 and 45. A similar pattern was identified in the
temporal changes of the testicular PRLR contents after EDS and EDS + T.
It has been shown that PRLRs are mainly expressed in LCs within the rat
testis (28, 35, 36). EDS administration induced a significant decrease
in [125I]iodo-hGH binding sites, already significant 5
days after EDS. Suppressed PRLR levels persisted 15 and 20 days after
EDS, whereas on day 45, the levels recovered to 65 ± 13% of
control values. As reported for LHR measurements, no recovery in the
number of PRLR sites was detected in the T-induced, LH-suppressed
conditions.
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The translational status of the 1.8-kb LHR mRNA transcript was
evaluated in control and EDS testes (5 days after treatment), by
assessing its pattern of polysome distribution (see Fig. 3). Because it
has been determined that the 6.8-kb variant of the LHR message is the
likely candidate to encode the functional LHR (39), we hypothesized
that this form has the maximal translational efficiency, and thus it
was used as positive control in the polysomal association study.
Northern hybridization of polysome fractions from control testis
demonstrated that the 6.8-kb transcript of the LHR mRNA was highly
associated with polysomes, showing increasing expression in fractions
with higher sucrose density, and reaching maximum intensity in the
fraction with highest sucrose content. After dissociation of the
mRNA-ribosome complexes by EDTA, the pattern of distribution of this
form shifted to fractions 3–7 with lower sucrose density. The profile
of polysome association of the 1.8-kb splice variant differed in
control testis from that of the 6.8-kb transcript. In this case, a
substantial proportion of the 1.8-kb message was localized in the
prepolysomal fractions, i.e. those with lower sucrose
density. Expression of this form was evident already in fraction 3,
reached its maximum in fraction 5, and declined thereafter. After EDTA,
the pattern of mRNA distribution resembled that of the longer variant,
being confined to fractions 2–7, and the shift in localization of this
transcript from non-EDTA condition was only minor. In polysomal
fractions from EDS rats, only the 1.8-kb variant was detected. The
polysome distribution of this form was similar to that of control
testis: the expression was first detectable in fraction 2, reaching the
maximum in fractions 3–5, and declining thereafter. Similarily, EDTA
treatment induced only a minor shift in the pattern of sedimentation of
this form.
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), because
LHR expression is confined to LCs. Incubation of testicular tissue from
control animals in the presence of human recLH (100 µg/liter
incubation medium) for 4 h induced a 35-fold increase in cAMP
concentration, as well as significant increases in the release of
pregnenolone, progesterone, and T to the medium. Five days after EDS
treatment, the basal steroid production in vitro was nearly
undetectable, with no significant responsiveness to human recLH, as
reflected by the negligible responses of cAMP, pregnenolone,
progesterone, and T. In contrast, 15 days after treatment, the basal
steroid production was increased, and LH was able to elicite a 3-fold
rise in cAMP levels in the incubation medium. This response was
associated with a significant release of progesterone and T to the
incubation media. Twenty days after EDS, the basal release of steroids
reached 99 ± 11%, 53 ± 3%, and 25 ± 1% of control
values in terms of pregnenolone, progesterone, and T secretion,
respectively. At this time point, LH induced a significant increase in
cAMP, pregnenolone, progesterone, and T release to the incubation
medium. Interestingly, the basal steroid production was nearly
undetectable, and no LH-induced steroid secretion was observed in
incubations of testicular tissue from the EDS + T rats on days 15 and
20 of the treatment. However, testicular responsiveness to human recLH,
in terms of stimulated cAMP release, was detected at both time points
(1.7- and 2.0-fold increase in cAMP levels after LH stimulation in 15
and 20 day EDS + T rats, respectively).
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Discussion |
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In EDS-treated rats, during the period of LC disappearance, specific LH/hCG binding of testis tissue dropped to undetectable levels, and the pattern of LHR mRNA expression was profoundly altered with persistent expression of the 1.8-kb mRNA form and disappearance of the longer species of the message, in agreement with our previous findings (19). As the repopulation process was taking place, LHR binding as well as expression of the longer forms of LHR mRNA (6.8, 4.2, and 2.7 kb) gradually returned to control levels. On the contrary, in EDS + T rats no recovery in testicular LHR content or in the pattern of LHR mRNA expression was detected. This shows that LC precursors, in the absence of LH, cannot differentiate into a state in which the LHR expression would be high enough to be detected by binding assay or Northern hybridization. It is worth noting that, in our experiments, T by itself did not promote LC development after EDS, as estimated by LHR binding and LHR mRNA expression analyses. This is in contrast to previous reports on the stimulatory role of androgens on the differentiation of LC progenitors isolated from immature (21-day-old) rats (45, 46, 47). A likely explanation for the apparent discrepancy is that the promoting actions of androgens on precursor development are exerted on a developmental step that has already acquired LH dependency. In fact, previous studies have firmly established that T-induced suppression of circulating LH is the main factor involved in the arrest of LC differentiation in the EDS rat treated with T (9, 15). Our results, suggesting a lack of direct stimulatory actions of T on early LC precursors, support this concept and validate the EDS + T rat as experimental model for the analyses carried out in the present study.
The question arising from the previous data is how does LH act on LC precursors to drive their differentiation into mature LHR expressing Leydig cells. Two possibilities were evaluated: the 1.8-kb form of LHR mRNA, persistently expressed after EDS, plays a role in targeting the actions of LH to early LC precursors; and/or functional LHR appears, at low level, in LC precursors during their differentiation in an LH-independent manner, thereby allowing the subsequent LH-dependent steps of maturation.
One of the most distinctive features of gonadotropin receptor gene expression is the presence of several splice variants of the coding mRNAs. Although only one fully functional LHR protein has been identified, four major forms of LHR mRNA, with molecular sizes of 1.8, 2.7, 4.2, and 6.8 kb, were shown in the rat testis (19, 37, 38, and present results). Among them, the 6.8-kb transcript is the likely candidate to encode the functional receptor (39). The 1.8-kb species has been previously identified as a truncated form, encoding regions of the extracellular domain of the receptor (37). Whether or not this variant, shorter than the full-length LHR-coding sequence, is provided with a function is still unknown. In this sense, it has been postulated, albeit not proven, that this form may encode a soluble LH-binding protein (48). One interesting feature of the 1.8-kb transcript is that it responds to various hormonal manipulations differently from the longer LHR messages. It has been shown that ligand-induced down-regulation of the LHR mRNAs does not involve a decrease in the level of expression of the 1.8-kb form (37). In addition, it was reported recently that unilateral cryptorchidism induced a marked increase in the level of expression of the 6.8-, 4.2-, and 2.7-kb transcripts, whereas the expression of the 1.8-kb form was reduced to nearly undetectable levels (49). Our findings on the pattern of LHR gene expression in the EDS-treated rat (19 and present results) are in concert with this concept.
The persistent expression of the truncated form of LHR message after EDS prompted us to analyze its potential role in mediating the actions of LH on early LC precursors. Assuming that this message must be translated to carry out a receptor-like function, we analyzed its translational status in control and EDS testes by means of polysome analysis. To our knowledge, no data on the pattern of polysome association of LHR transcripts have been previously reported. The distribution of the 1.8-kb variant clearly differed from that of the 6.8-kb transcript, with predominant localization in prepolysomal fractions, thus suggesting a low translational efficiency. The possibility still remains that the truncated LHR message could be partially translated. However, the present data, together with the absence of detectable testicular LH binding sites either in membrane preparations (see Results) or solubilized cell suspensions (data not shown), as well as the lack of LH-induced cAMP release 5 days after EDS, despite the high level of expression of the truncated message at this time point, cast doubts on its potential role in driving/modulating LH actions in early LC precursors. This transcript may constitute an early sign of LC differentiation, being constitutively expressed, as we have previously hypothesized on similar observations in connection with gonadal development during the fetal period (38, 50). Interestingly, in EDS + T rats, in which the absence of circulating LH induces an arrest of LC differentiation, and likely, accumulation of undifferentiated precursors, the expression of this variant was highly elevated. Alternatively, this form, translated at low rate, may participate in the early LH-independent stages of LC differentiation. We are currently studying this possibility.
To map the temporal pattern of reappearance of functional LHRs in rat testis after EDS and EDS + T treatments, the testicular in vitro responsiveness to LH as reflected by cAMP, T, progesterone, and pregnenolone secretion was evaluated at different time points. This approach is highly sensitive, thus enabling us to detect the presence of very low levels of functional LHRs. Five days after EDS, no testicular responsiveness to LH was detected, suggesting that at this early stage of differentiation LC precursors do not express functional LHRs, in accordance with previous reports (18, 19). However, LH-stimulated cAMP response was observed 15 and 20 days after EDS, regardless of the presence of high (EDS alone) or suppressed (EDS + T) LH in the donor animal. Hence, the appearance of low levels of functional LHRs can take place also in the absence of LH. Assuming that the 6.8-kb form encodes the functional, signal-transducting LHR, it is tempting to speculate that between 5 and 15 days after EDS a change in the splicing pattern of the LHR gene occurs in LH-independent manner, allowing expression of low levels of functional receptors and thus further LH-dependent precursor differentiation. The absence of any steroidogenic response to LH in testicular tissue from EDS + T rats indicates that LH action is necessary during LC differentiation for the acquisition of steroidogenesis. In addition, our results are in concert with a previous hypothesis pointing that the first stages of LC differentiation can take place in the absence of LH, but that LH stimulation is required for completion of differentiation of precursors into mature Leydig cells (14).
In conclusion, we have provided evidence for the concept that the appearance of low levels of functional LH receptors in LC precursors is an LH-independent developmental event, essential for the subsequent LH dependent maturational steps, including the onset of steroidogenesis and increased LHR expression. In addition, our results suggest that the truncated (1.8-kb) form of LHR mRNA, expressed at high level in the LC precursors, does not play a major functional role in LC differentiation.
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Acknowledgments |
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Footnotes |
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2 Supported by a postdoctoral grant from Direccion General de
Investigacion Cientifica y Tecnica (Ministry of Science, Spain).
Received February 4, 1997.
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) or EDS + T (
). Measurements were
carried out before (0) and 5, 15, 20, and 45 days after EDS injection
and application of T implants. **, P < 0.01 vs.controls; a, P < 0.01 vs. values
from EDS-treated rats 5 and 15 days after treatment (ANOVA followed by
Duncan’s new multiple range test).
