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Institut National de la Recherche Agronomique, Laboratoire Institut National de la Recherche Agronomique-Station Commune de Recherches en Ichtyophysiologie, Biodiversité et Environement, Campus de Beaulieu (M.G., H.M., Y.G.), 35042 Rennes Cedex, France; and National Diagnostics Center, BioResearch Ireland, National University of Ireland (O.M.M., T.J.S.), Galway, Ireland
Address all correspondence and requests for reprints to: Dr. Yann Guiguen, Laboratoire Institut National de la Recherche Agronomique-Station Commune de Recherches en Ichtyophysiologie, Biodiversité et Environement, Campus de Beaulieu, 35042 Rennes Cedex, France. E-mail: guiguen{at}beaulieu.rennes.inra.fr
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Abstract |
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Introduction |
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and ERß. Recently, it has been demonstrated that female mutant mice
homozygous for the targeted disruption of these two estrogen receptors
(ßERKO), exhibit some degree of morphological gonadal sex reversal
together with an increase in biochemical characteristics of Sertoli
cell differentiation (10). This strongly suggests that
even in mammals, the estrogen response can also to some extent lead to
perturbation in gonadal differentiation. Rainbow trout (Oncorhynchus mykiss) is characterized by a male heterogametic (XY) sex determination system, and new viable male genotypes (XX or YY) can be produced by hormonal phenotypic inversion and subsequent progeny testing (11). Using these new male genotypes we can either produce all male or all female populations, allowing us to work on a whole population of fish in which the genetic and phenotypic sex is known before gonadal sex differentiation. Using these populations we investigated the effect of a classical feminizing 17ß-estradiol (E2) treatment on the testicular messenger RNA (mRNA) levels of steroidogenic enzyme genes. We first looked at the effects of estrogen treatment on the differentiating testis and secondly studied the effect of a similar treatment in postdifferentiating immature males on both the testis and the interrenal (equivalent of the mammalian adrenal) to check whether this effect was specific to the differentiating testis. Because 11ß-hydroxylase (P45011ß) and aromatase (P450aro) have already been characterized as important enzymes expressed in a sexually dimorphic fashion during gonadal sex differentiation (1, 12, 13), we also compared the mRNA levels of these two genes in the differentiating testis after E2 treatment and, in some instances, in female differentiating gonads.
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Materials and Methods |
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Total RNA extraction
Total RNA was isolated from frozen gonads (-70 C) using the
TRIzol reagent (Life Technologies, Inc., Grand Island,
NY). TRIzol (1 ml) at 65 C was added to the gonads. The tissue was
drawn through a 26-gauge needle to ensure complete lysis. The TRIzol
mixture was cooled to room temperature, 200 µl chloroform were added,
and it was vortexed and centrifuged for 10 min at 14,000 rpm. The
aqueous phase containing the RNA was retained, and RNA was precipitated
with an equal volume of isopropanol. The RNA pellet was washed with
80% ethanol, dried, and resuspended in 50 µl
diethylpyrocarbonate-treated water. The concentration of RNA was
determined by spectrophotometry.
Semiquantitative RT-PCR
Semiquantification by RT-PCR was performed as previously
described (12). Primer sequences were: 11ß-hydroxylase
sense primer, 5'-T GAC GCC CAC AAG GCC CTG C-3'; 11ß-hydroxylase
antisense primer, 5'-GTG AGT TCA TTG AGA TTA CCT G-3'; aromatase sense
primer, 5'-CTC TCC TCT CAT ACC TCA GG-3'; aromatase antisense primer,
5'-CCA GAC TGA ACT CAT TGG GC-3'; ß-actin sense primer, 5'-AAA GAC
CCT GAG TTC ATC ATG C-3'; and ß-actin antisense primer, 5'-CCC AGT
CTC CAC TAA TCC CA-3'. To work in the exponential range of
amplification, the number of cycles was set at 20 for ß-actin and
aromatase and 30 for P45011ß. After PCR amplification, 1 µl of each
PCR reaction including appropriate controls (water only and genomic
DNA) was dotted on a nylon membrane (Hybond N+,
Amersham Pharmacia Biotech, Arlington Heights, IL). The
membrane was then denatured (3 min) and neutralized (5 min) by
capillary transfer with denaturation solution (0.5 M NaOH
and 1.5 M NaCl) and neutralization solution (0.5
M Tris-HCl and 1.5 M NaCl, pH 7), and finally
rinsed briefly in 2 x SCC (standard saline citrate) before UV
light cross-linking. Membranes were then hybridized with the
corresponding probe, i.e. P45011ß, P450aro, or ß-actin,
labeled by random priming with
[-32P]deoxy-CTP following the Ready-to-Go
Labeling Beads (Amersham Pharmacia Biotech) protocol.
Hybridization was carried out overnight at 65 C in 5 x SCC,
5 x Denhart’s, 0.5% SDS, and 20 µg/ml denatured calf thymus
DNA. Membranes were washed to high stringency in 0.1 x SSC/0.1%
SDS at 65 C and then quantitatively analyzed using an Instant Imager
(Packard, Downers Grove, IL). Data were expressed as logarithms of gene
of interest/ß-actin ratios. With these PCR conditions no
amplification was detected in either the water or the genomic DNA
control, and only a single band of the expected size was detected in
all other PCR reactions.
Northern blot and virtual Northern blot analysis
Northern blots were carried out on total RNA as previously
described (14) except for the use of ULTRAhyb
hybridization buffer (Ambion, Inc., Austin, TX). Reprobing
with 28S ribosomal RNA (rRNA) was used as an internal loading control,
and both specific and 28S rRNA signals were quantified using an Instant
imager (Packard). Because of the limiting amount of tissue available,
techniques such as classical Northern blot were not feasible on
differentiating gonads; thus, we used virtual Northern blots, as this
technique has been shown to be both very sensitive and quantitative
(15, 16). Virtual Northern blots were performed as
previously described (12), except that the complete PCR
reactions were precipitated with ammonium acetate (50 µl 4
M ammonium acetate and 375 µl 95% ethanol) and
resuspended in 20 µl sterile water. Ten to 20 µl of this amplified
complementary DNA (cDNA) solution were then loaded on a 1% Tris-borate
EDTA/agarose gel. After migration, the gel was denatured and
neutralized, and DNA was transferred to a nylon membrane (Hybond-N,
Amersham Pharmacia Biotech) by capillary Southern blotting
in 20 x SSC. DNA was fixed to the membrane by baking at 80 C for
2 h. The membrane was then hybridized overnight in ULTRAhyb
solution (Ambion, Inc.) at 42 C using a
[-32P]deoxy-CTP-labeled cDNA probe. The
membrane was washed in 0.2 x SSC/0.1% SDS at 65 C. The probe was
removed from the membrane by incubation for 15 min in 0.1% SDS at 95
C. To confirm loading of cDNA samples and estimate the relative
quantity of loaded cDNA between samples, the membrane was then reprobed
with rainbow trout ß-actin.
Rainbow trout P450scc (17) and 3ß-hydroxysteroid dehydrogenase (3ßHSD) (18) were obtained by cloning PCR-amplified fragments as previously described (19). A 17-hydroxylase/lyase (P450c17) probe (20) was obtained by RT-PCR using the oligonucleotides designed against the rainbow trout P450c17 sequence [17-hydroxylase sense, 5'-GGAAACCACGTCAACAGTCC-3' (bases 980–999); 17-hydroxylase antisense, 5'-TTACAAGGGCTGTCTCTGCG-3' (bases 3132–2151)]. This fragment was then subcloned, and its identity was confirmed by sequencing on both strands using an ABI prism 310 automatic sequencer (Perkin-Elmer Corp., Norwalk, CT). Rainbow trout P450aro (21) was provided by Prof. Y. Nagahama (Okasaki, Japan) and used as a full-length probe. Cloning and characterization of rainbow trout P45011ß (Guiguen, Y., et al., unpublished results; Accession No. AF217273) will be published elsewhere, and rainbow trout ß-actin is an unpublished sequence displaying more than 90% nucleotide identity with the ß-actin sequence from another salmonid, Salmo salar (Accession No. AF012125).
Statistics
All statistical differences were determined using ANOVA combined
with Newman-Keuls test and were calculated using Statistica
software (Statsoft, Tulsa, OK).
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Results |
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. Levels
of P450scc mRNA were not affected by E2
treatment, but 3ßHSD and P450c17 mRNAs were undetectable after
E2 treatment, as shown by their nondetection by
virtual Northern analysis in E2-treated male
gonads compared with the control male gonads (Fig. 1). The P45011ß
mRNA level was also investigated by virtual Northern analysis (Fig. 2A) and was detected in the control male
population, but was totally absent in the
E2-treated population. This noticeable decrease
after 10 days of E2 treatment was confirmed
(decrease by a factor of 40) by semiquantitative RT-PCR analysis (Fig. 2B), with P45011ß mRNA levels reduced to those observed during female
gonadal differentiation (13). P450aro mRNA was not
detectable in male gonads at that stage using virtual Northern
analysis; thus, only the semiquantification by RT-PCR analysis is shown
in Fig. 3. We detected, as previously
shown (12), a high mRNA level of P450aro in female
differentiating gonads compared with male gonads and no difference
between males and males treated with E2.
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. For each
steroidogenic enzyme gene we further quantified the ratio of each mRNA
over 28S rRNA, and this ratio for all genes studied is shown in Fig. 5. The P45011ß mRNA level in the testis
after E2 treatment (Fig. 4) decreased rapidly,
with the first detectable decrease occurring after 8 h (T8) of
E2 treatment. This decrease in testicular
P45011ß mRNA level in the E2-treated animals
was more pronounced after 1 and 2 days (T24 and T48) of treatment
(>50% decrease) and was statistically significant after 4 days (T96).
It reached less than 5% of the initial expression 8 days after the
beginning of the treatment (95% of inhibition at T192; significant
difference vs. T0, P < 0.025). In contrast,
the interrenal P45011ß mRNA level did not show any consistent or
significant variation after E2 treatment (Fig. 4). Interrenal 3ßHSD and P450c17 mRNAs also did not show any
significant variation after E2 treatment, but
interrenal P450scc mRNA did decrease slightly (30% inhibition) and
significantly (P < 0.05) after 8 days of treatment
(Fig. 5). In the testis, however, the 3ßHSD mRNA level was strongly
influenced by E2 treatment, with a decrease
similar to that in P45011ß mRNA (>50% decrease 8 h
posttreatment) and a final level reduced to less than 1% (>99%
decrease; P < 0.05) of the level detected before the
beginning of the treatment (Fig. 5). Testicular mRNA levels of P450scc
and P450c17 were also affected by E2 treatment,
but to a much lesser extent, i.e. 40–50% decrease in
P450scc (significantly different from T0, P < 0.05)
and 75% decrease in P450c17 (significantly different from T0,
P < 0.05), after 8 days of E2
treatment. For P450c17 a transient, but significant (P
< 0.001), increase was also detected after 24 h of
E2 treatment.
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Discussion |
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We demonstrated in this study that the short-term effect of this E2 treatment on steroidogenic enzyme genes during gonadal differentiation was a marked decrease in mRNA levels of all of the steroidogenic enzyme genes studied, except P450scc. This effect probably resulted in highly impaired gonadal steroidogenesis in these animals. Such a perturbation of gonadal steroidogenesis has been previously described in the differentiating gonad of rainbow trout after treatment of monosex populations with E2 (8). These researchers found that a 5 mg/kg diet E2 treatment was totally ineffective in feminizing an all male population, as at 2 yr of age, all of the E2-treated males had well developed testes. Despite the absence of effect on the resultant sex ratio, gonadal androgen production was significantly decreased in E2-treated males compared with that in normal males (8). The gonadal mRNA pattern of steroidogenic enzyme genes detected in our experiments during E2-induced female differentiation is very different from the natural ovarian differentiation in which 3ßHSD, P450c17, and P450aro are well expressed (25). The only similarity between E2-induced and normal ovarian differentiation is the noticeable decrease in gonadal P45011ß mRNA that is normally expressed only during testicular differentiation (13). The decrease in P45011ß mRNA together with the decrease in 3ßHSD and P450c17 probably result in complete inhibition of gonadal 11-oxygenated androgen production. These 11-oxygenated androgens have been shown to be naturally synthesized by the trout testis around the time of sex differentiation (1) and are also recognized as potent masculinizing steroids in several fish species (26, 27, 28, 29). The inhibition of their synthesis through the decrease in P45011ß mRNA may, therefore, be one of the crucial steps required in the active feminization of these genetic males by E2. As a result of this decrease in P45011ß mRNA, E2-induced feminization occurs at an elevated ratio of estrogens/11-oxygenated androgens.
E2 production has been previously detected in normal differentiating female gonads, but no gonadal E2 secretion was found in E2-treated males (8). This is completely consistent with our own results, as the P450aro mRNA level was not elevated by E2 treatment and thus did not reach the expression level found in normal differentiating ovaries (12). Conversely, in female Japanese flounders, Paralichthys olivaceus, inhibition of aromatase enzyme activity, and consequently E2 levels, has been shown to depress P450aro gene expression (30). The same results were found in the female chicken, where coadministration of the aromatase inhibitor with E2 restored P450aro mRNA to the levels found in normal differentiating ovaries (31). This suggests that aromatase gene expression may be under the positive control of estrogens in the female differentiating gonad. However, this does not appear to be the case in the differentiating male gonad, as E2 treatment did not increase the P450aro mRNA level (present data) or E2 synthesis (8).
The same experiment carried out on postdifferentiating males gave us the opportunity to analyze the kinetics of the same steroidogenic enzyme mRNAs after E2 treatment in two different steroidogenic organs, i.e. the testis and the interrenal. In the postdifferentiating testis, we found a similar pattern of mRNA levels as that observed during testis differentiation, with, after 10 days of E2 treatment, a large decrease in 3ßHSD, P450c17, and P45011ß mRNAs and a less marked decrease in P450scc mRNA. This decrease was detectable as early as 8 h postapplication of the E2 treatment for P45011ß and 3ßHSD. In the Atlantic croaker, Micropogonias undulatus, estrogens have been shown to inhibit gonadotropin-stimulated 11-ketotestosterone testicular production (32). This estrogen effect was very rapid (<5 min) and was further characterized as a nongenomic mechanism, probably mediated by a membrane receptor. The E2 effects found in our study reflect mRNA changes that are probably mediated at the transcriptional level through a classical genomic mechanism involving a nuclear steroid hormone receptor.
In the interrenal, only a slight decrease in the P450scc mRNA level was
detected after E2 treatment. The difference in
the pattern of expression of steroidogenic enzyme genes between testis
and interrenal may be related to their upstream transcriptional control
by the central nervous system with, respectively, ACTH and gonadotropin
hormones (Gths). In fish, Gths are well known to regulate gonadal
steroid hormone biosynthesis (33). However, estrogens have
also been shown to inhibit Gths-stimulated testicular steroidogenesis
in mammals (34, 35, 36), amphibians (37, 38), and
fish (39). Whatever the effect of E2
on steroidogenic enzyme mRNAs seen in postdifferentiated fish, its
effect may be slightly different, in terms of its upstream
transcriptional control, from the effect in differentiating fish. In
rainbow trout, Gth stimulation of steroid secretion occurs late,
i.e. 117 dpf (8), compared with the timing of
histological sex differentiation (
80 dpf in our study), but several
studies have also demonstrated that the hypothalamo-pituitary axis is
potentially active around the time of sex differentiation
(1). In that regard it should be noted that a much earlier
Gth stimulation of androstenedione synthesis in the interrenal has been
demonstrated in the rainbow trout (8). This interrenal
steroid production may be relevant with respect to gonadal
differentiation, as a hypothesis has been proposed involving the
participation of that tissue in the production of steroids potentially
acting on gonadal differentiation (40). The fact that
steroidogenic enzyme mRNA levels in the interrenal are only slightly
decreased by E2 after sex differentiation now
deserves more interest, and future investigations should concentrate on
the differentiation period. However, no effect of
E2 on interrenal steroid production could be
detected in rainbow trout during gonadal differentiation, and thus, it
has been concluded that this effect is gonad specific
(8).
Gth stimulation of steroid secretion only occurs after gonadal
differentiation in the rainbow trout (8); thus, the
E2 effects that we reported here in the
predifferentiating testis are probably induced independently from an
inhibition of Gth secretion. However, in postdifferentiating gonads the
question still remains as to whether E2 effects
are mediated either directly on the testis and/or through a feedback
inhibition of Gth secretion. Apart from these classical feedback
effects of estrogens there is some supporting evidence that estrogens
can have a direct action on the testis (41). For instance,
in the frog, Rana esculenta, E2 can
decrease in vitro testis androgen production
(38) and inhibit steroidogenesis (37). In the
rat, neonatal exposure to estrogen affects the expression levels of the
estrogen receptors, ER and ERß, and the androgen receptor
independently of an estrogen-induced suppression of Gths secretion
(42). Some direct inhibitory effect of estrogens on
testicular androgen production have also been found on human testis
in vitro (43), and in the rat, estrogens also
decrease testicular androgen production without a concomitant decrease
in serum LH (44). This assumption that estrogens can act
directly on the testis is also confirmed by the pattern of expression
of ERs, which are expressed in the testis (42). This is
further reinforced by the fact that transgenic mice in which the genes
for aromatase (45), ER (46), or both ER
and ERß (10) have been inactivated develop disorders of
spermatogenesis.
Many studies have reported an impaired steroidogenesis profile after estrogen treatment. However, only a few studies have investigated this effect at the level of the steroidogenic enzyme gene expression. In the rat, diethylstilbestrol (a synthetic estrogen) and 4-octylphenol (a xenoestrogen) both inhibit fetal testicular P450c17 gene expression (47). This has been shown to correlate with the inhibition of the steroidogenic factor-1 (SF1) gene by both diethylstilbestrol and 4-octylphenol (48). SF1 is a transcription factor that has been shown to be essential for both gonadal differentiation (49) and transcriptional control of most steroidogenic cytochrome P450 genes (49, 50) and also 3ßHSD (51). As such it has been postulated that the estrogen effects on the inhibition of fetal testicular P450c17 gene expression may be mediated via the alteration of SF1 gene expression (48). The same could also hold in our case, as the simultaneous decreases in 3ßHSD, P450c17, and P45011ß mRNAs produced by E2 treatment suggest a common transcriptional upstream control, and SF1 would be a good candidate for such a role. Future studies will focus on this transcriptional upstream control, but our present observations clearly suggest that estrogen action on steroidogenesis in the rainbow trout testis is mediated through changes in steroidogenic enzyme mRNA steady state levels and that these effects may explain the active feminization caused by estrogens in fish.
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Acknowledgments |
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Footnotes |
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2 Supported by a European Community project (PL 97–3796).
Received October 10, 2000.
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References |
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(Abstract)
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) and P450 arom messenger ribonucleic acid of gonads in
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and ß mRNA expression in
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-hydroxylase/C17–20 lyase in
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5-4
isomerase promoter by the transcription factor steroidogenic
factor-1/adrenal 4-binding protein and phorbol ester. J Biol Chem 272:7960–7967This article has been cited by other articles:
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HindIII) is shown on
the right of each figure. M, Male (pool of 785
differentiating gonads); M. E2, male treated with
E2 (20 mg/kg diet; pool of 752 differentiating gonads); P5,
pregnenolone; 17P5, 17-hydroxypregnenolone; P4, progesterone; 17P4,
17-hydroxyprogesterone; 
