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First published online September 19, 2006
Journal of Experimental Biology 209, 3806-3811 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02424
The expression level of frog relaxin mRNA (fRLX), in the testis of Rana esculenta, is influenced by testosterone
1 Dipartimento di Medicina Sperimentale, Sez. Fisiologia Umana e Funzioni
Biologiche Integrate `F. Bottazzi', Seconda Università di Napoli, via
Costantinopoli 16, 80138 Napoli, Italy
2 Dipartimento di Biologia Strutturale e Funzionale, Università di
Napoli `Federico II', via Cinthia, 80126 Napoli, Italy
3 Laboratorio di Biochimica e Biologia Molecolare, Stazione Zoologica `A.
Dohrn', Villa Comunale, 80121 Napoli, Italy
* Author for correspondence (e-mail: sergio.minucci{at}unina2.it)
Accepted 3 July 2006
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Summary |
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Key words: relaxin, testosterone, cyproterone acetate, gene expression, Rana esculenta
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Introduction |
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TOPSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
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). All
the proteins of this family share similar tertiary structure, in fact, the
pro-prehormone peptide consists of a signal peptide, the common B-C-A
heteromeric structural organization in which the B and A domain are covalently
linked by two intradomain disulfide bonds. In the B domain the highly
conserved R-XXX-R relaxin receptor binding motif is present
(Bryant-Greenwood and Schwabe,
1994); a post transcriptional modification produces the removal of
the signal peptide and of the C domain
(Steiner, 1998).
In mammals, RLX is mainly secreted by the corpus luteum, a glandular
structure in the ovary, and is generally associated with the physiology of the
female reproductive tract (Sherwood,
1994) having a well-recognized role in parturition
(Bryant-Greenwood and Schwabe,
1994). Nevertheless, RLX is responsible for a wide range of
functions, with responsive tissues in the brain, heart, kidney and skin,
besides the more classical organs of the female reproductive system
(Bathgate et al., 2003).
By contrast, RLF is highly expressed in adult Leydig cells and at lower
levels in the theca cells of the corpus luteum, the trophoblast, breast, and a
variety of other tissues (Tashima et al.,
1994; Pusch et al.,
1996; Bathgate et al.,
1996; Balvers et al.,
1998; Spiess et al.,
1999). RLF knockout mice were cryptorchid suggesting that
RLF is involved in the gubernaculum formation necessary for the testis descent
in the scrotum (Zimmermann et al.,
1999; Nef and Parada,
1999). Recently, it has been demonstrated that, in the testis,
besides its action on the gubernaculums, RLX suppresses apoptosis and has a
paracrine action as survival factor for male germ cells
(Kawamura et al., 2004).
Frog relaxin (fRLX) is the first form of RLX molecularly
characterized in the testis of a non mammalian vertebrate
(De Rienzo et al., 2001). It is
highly expressed by the interstitial Leydig cells and the transcript levels
change during the annual reproductive cycle, suggesting its involvement in
spermatogenesis. Interestingly, although fRLX has a RLX structure,
its cellular localization is similar to that of the RLF. In addition,
phylogenetic analysis suggests that fRLX sequence can represent an
ancestral form of relaxin from which both modern mammalian relaxin and RLF
might have evolved, for female and male functions, respectively
(De Rienzo et al., 2001).
To date, the control of the expression of the RLX and RLF
genes is still unclear; the corpus luteum produces a small but consistent rise
in plasma RLX after the LH surge (Stewart
et al., 1990). However, the transcription of RLF is
mediated by steroidogenic factor I
(Zimmermann et al., 1998) and
it seems to be influenced by testosterone
(Paust et al., 2002;
Ivell et al., 2003).
Since fRLX transcript is more abundant when circulating levels of
androgens are relatively high and is differently expressed during the frog
reproductive cycle (De Rienzo et al.,
2001), investigated the effect(s) of the administration of
testosterone and its antagonist (cyproterone acetate; CPA) on fRLX
mRNA expression in the testis of the frog, Rana esculenta.
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Material and methods |
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This project was approved by the Italian Ministry of Education and Research (MIUR).
In vivo testosterone treatment
In May (when testosterone levels are low) adult frogs (N=35) were
divided into three groups as follows: five frogs, used as initial control,
were immediately killed; 15 animals were treated with a single injection of
0.2 mg of testosterone (T) in 100 µl of Krebs-Ringer solution at pH 7.4
(KRB); 15 animals were treated with a single injection of 0.2 mg of T plus 2
mg of cyproterone acetate (CPA) in 100 µl of KRB. The animals (5
animals/group/sample time) were sacrificed at different time points: 2, 8 and
24 h after the injection.
In vitro testosterone treatment
In May adult frogs (N=36) were killed and the testes were placed
in tubes (24 testes/tubes) containing: (1) KRB alone; (2) KRB plus T
(10-6 mol l-1) and (3) KRB plus T (10-6 mol
l-1) and CPA (10-5 mol l-1), for different
times (2, 8 and 24 h). At the end of the treatment the testes were removed
from the solution and frozen for RNA extraction. In addition, the testes
excised from four frogs were immediately frozen for extraction of RNA and used
as initial controls.
In vivo CPA treatment
In March (when testosterone levels are high) adult frogs (N=25)
were divided into three groups as follows: five frogs, used as initial
control, were immediately killed; 10 animals were injected on alternate days
with KRB alone; 10 animals were injected on alternate days with a dose of CPA
(0.33 mg 100 µl-1 KRB). The CPA- and KRB-injected frogs were
killed 15 days after the first injection and the testes were removed and
frozen for RNA extraction. In addition, three testes/group were fixed in
Bouin's fluid, and processed for in situ hybridization.
Preparation of total RNA and northern blot analysis
Total RNA from the testes of the frog Rana esculenta were prepared
with the procedure described (Sprenger et
al., 1995).
Total RNA (20 µg for each sample) was fractionated by electrophoresis on
a 1% agarose gel containing 2.2 mol l-1 formaldehyde and then
transferred to a nitrocellulose membrane by overnight capillary blotting
(HybondTM-N+; Amersham Pharmacia Biotech, Bucks, UK). The filters were
prehybridized for 5-6 h at 65°C in 5x SSC, 5x Denhardt's, 100
µg ml-1 salmon sperm DNA and 50 mmol l-1 sodium
phosphate at pH 7.0 and hybridized with a 32P-labelled probe
(2x106 c.p.m. ml-1) corresponding to the
fRLX cDNA (De Rienzo et al.,
2001) at 65°C overnight. The filters were washed twice for 30
min at 65°C in 0.2x SSC and 0.1% SDS and were then exposed to X-ray
film (HR-H, FUJI). In addition, the same filters were stripped in 0.1x
SSC and 0.1% SDS and rehybridized with a fP1cDNA probe as a positive
control with the same conditions as above.
RT-PCR
The levels of fRLX mRNA were determined by reverse
transcription-polymerase chain reaction technique (RT-PCR) using as control
the levels of fP1 mRNA.
First-strand cDNA was synthesized using 5 µg of total RNA from control and CPA-treated testes, 500 ng oligo(dT)18 primer (Promega, Heidelberg, Germany), 0.01 mol l-1 DDT and 200 i.u. Superscript II RT enzyme (Life Technologies, Paisley, UK) in a total volume of 20 µl according to the manufacturer's instructions (Life Technologies, Paisley, UK). Portions (1.5 µl) of the resultant cDNA were used to amplify, by PCR, a 265 bp fragment of fRLX (EMBL accession number: AJ298874) containing 211 bp of coding region and 54 bp of 3' UTR and a 356 bp fragment of fP1 (a Rana esculenta mRNA for acidic ribosomal protein 1; accession number: AJ298875). The reaction was performed in 1.5 mmol l-1 MgCl2, 10 mmol l-1 Tris-HCl pH 9.0, 50 mmol l-1 KCl and 0.1% Triton X-100, using 1 i.u. Taq DNA polymerase (Promega, Heidelberg, Germany) and 5 pmol oligonucleotide primers (fRLX forward primer: 5'-tgtatgcagagagccacat-3', fRLX reverse primer: 5'-gagtgcttgctctgcagac-3', fP1 forward primer: 5'-tggctggacagctaacatg-3', fP1 reverse primer: 5'-tcaggacatcacatactggc-3'). Amplifications, carried out for 25 cycles, were as follows: 94°C for 40 s, 56°C for 40 s and 72°C for 40 s. PCR products were separated on 1.5% agarose gel in 1x TAE buffer using ethidium bromide for visualization. The quantification of the bands was carried out with a Gel Doc 2000 (Bio-Rad, Hercules, CA, USA). The PCR products were sequenced to verify the specificity of the amplification.
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Results |
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A strong increase of fRLX mRNA expression was found in the testes of testosterone-treated frogs in all three groups as compared to that found in the testes of the control (Fig. 1A,C). By contrast, no differences of fRLX expression were found between the T plus CPA and the control testes (Fig. 1A,C).
In vitro testosterone treatment
To better understand the effect of testosterone treatment an in
vitro experiment was performed. The fRLX expression was
evaluated by northern blot analysis on total RNA extracted at different time
from frog testis incubated with vehicle alone, T, and T in combination with
CPA using fRLX cDNA as a specific probe
(Fig. 2A). The same filter was
rehybridized using fP1 cDNA (De
Rienzo et al., 2001) as control for RNA loading
(Fig. 2B). The single band
observed corresponds to a transcript of about 1.6 kb in length, which is the
size of the isolated cDNA clone (De Rienzo
et al., 2001).
No differences in fRLX mRNA expression were found in the testes incubated in vehicle alone at 2 and 8 h as compared with the control (Fig. 2A,C), whereas a drastically decrease was observed after 24 h of treatment (Fig. 2A).
By contrast, a strong increase in fRLX mRNA expression was found in the testes incubated in vehicle containing T at 2 and 8 h of treatment, whereas in T-incubated testes at 24 h fRLX expression showed a decrease, as compared to the control. Worthy of note is that the level of fRLX expression found after 24 h of testosterone treatment was higher than that observed in testes incubated in vehicle alone at the same time (Fig. 2A,C).
fRLX mRNA expression in testosterone plus CPA-treated testes at 2 and 8 h was lower than the control and the vehicle-treated testes at any time of incubation (Fig. 2A,C), whereas no differences were found between 24 h vehicle and T+CPA-treated testes (Fig. 2A,C).
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To ascertain the decrease in fRLX expression CPA-treated frogs at
15 days, in situ hybridization experiments were performed on control
and treated frog testis. A strong signal was detected in the interstitial
tissue around the germinal compartment (De
Rienzo et al., 2001) in control testes
(Fig. 3C), whereas the levels
of fRLX transcript were visibly reduced in the 15-day CPA-treated
testes (Fig. 3D).
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Discussion |
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TOPSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
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). It
should be noted that in lower vertebrates testicular descent does not occur
and spermatogenesis is susceptible to seasonal thermal changes
(Rastogi and Iela, 1992). In
this context, frog relaxin (fRLX), a member of the relaxin/insulin
gene family, has been found in the testis of Rana esculenta, and it
is specifically expressed by Leydig cells
(De Rienzo et al., 2001).
Interestingly, the expression of its transcript changes throughout the frog
annual cycle (De Rienzo et al.,
2001) strongly suggesting that it might contribute to the
efficiency of spermatogenesis in these seasonal breeders, possessing a
reproductive cycle regulated by endocrine and environmental factors and a
cystic organization of the testis that favours spermatogenesis
(Rastogi et al., 1978;
Rastogi and Iela, 1992). In
fact, fRLX transcript is more abundant when circulating levels of
androgens are relatively high and its expression pattern correlates well with
the frog steroidogenic and spermatogenetic wave
(De Rienzo et al., 2001).
It is worth remembering that, in the frog testis, androgens are produced by
the Leydig cells and are necessary for spermatogonial proliferation and
spermatid formation (Rastogi and Iela,
1992; Minucci et al.,
1992). Therefore, it is possible to hypothesize the existence of a
relationship between androgen production, RLX expression and
spermatogenesis.
In an attempt to obtain more information about the influences of androgens
on fRLX mRNA expression, if any, we evaluated the effect of
testosterone and its antagonist (cyproterone acetate; CPA), by in
vivo and in vitro experiments, in the testis of the frog
Rana esculenta. Experiments were performed in two different periods
of the frog reproductive cycle: May, when testicular testosterone and
fRLX mRNA levels are low, and March, when testicular testosterone and
fRLX mRNA are at their highest levels
(De Rienzo et al., 2001). It is
relevant to note that, in the testis of animals injected in May, testosterone
treatment strongly induced a significant increase in fRLX expression,
at all times post-injection, and this effect was counteracted by CPA,
supporting the existence of intratesticular (autocrine/paracrine) mechanisms
of action (Fig. 1). Our data
are also supported by the observation that fRLX expression increases
during the frog annual cycle in concomitance with the highest testosterone
concentration (De Rienzo et al.,
2001).
In addition, pieces of testis in May, incubated with testosterone, show a
significant increase in fRLX expression at 2 and 8 h, with these
effect counteracted by CPA (Fig.
2A). Interestingly, fRLX expression strongly decreased in
the testis of all the groups at 24 h of incubation, suggesting that factor(s)
other than testosterone may act(s) in controlling its expression. Moreover, it
has also been suggested that fRLX expression could be under
hypophysal control, in fact, fRLX transcript has been detected at a
low level in the interstitial compartment of hypophysectomized frogs 30 days
after surgical depletion (De Rienzo et al.,
2001). In mammals, other studies reported that LH/hCG is essential
to induce relaxin-like factor (RLF) expression in the adult Leydig cells of
the mouse (Balvers et al.,
1998). However, a pituitary control of RLF has been indicated,
using hypogonadic mice lacking an active pituitary-gonadal axis caused by a
deletion in the hypothalamically expressed gene for GnRH, with consequent
gonadotropin deficiency (Scott et al.,
1990; Cattenach et al.,
1997; Charlton et al.,
1983). Here, RLF expression is totally absent, suggesting
that Leydig cells seem to be arrested in a prepubertal state of
differentiation (Balvers et al.,
1998).
In addition, RT-PCR analysis performed on the testes of frogs in March (when testosterone levels are high), injected for 15 days with CPA, showed a strong decrease in fRLX expression (Fig. 3A) and this data was also confirmed by in situ hybridization. In fact, we observed a reduction of the hybridization signal from the interstitial Leydig cells, suggesting that CPA counteracts the effect(s) of endogenous testosterone on fRLX expression (Fig. 3C,D).
Furthermore, since GnRH and its long acting agonist, buserelin (GnRHa),
directly stimulate androgen production and spermatogonial multiplication in
the frog testis (Pierantoni et al.,
1984a; Pierantoni et al.,
1984b; Minucci et al.,
1986; Fasano et al.,
1990; Rastogi et al.,
1990), we performed experiments in which testis were incubated
with GnRHa in order to stimulate testosterone secretion in vitro.
Interestingly, fRLX expression progressively increases in testes
incubated with GnRHa and reaches a peak at 6 h (G. De Rienzo, G. Izzo, D.
Ferrara and S. Minucci, unpublished data), confirming the influence of
testosterone on its expression. Taken together, our results indicate that
testosterone probably acts directly in controlling fRLX expression in
the frog testis. In this context, consideration should be given to our recent
data which emphasize the fact that melatonin interferes with Leydig cell
activity, thereby inhibiting the GnRH-induced testosterone secretion in
vitro (d'Istria et al.,
2004) and induces the complete disappearance of fRLX
transcript from the testis of frogs injected with melatonin. This supports the
hypothesis that this hormone exerts an inhibitory effect on Leydig cells by
modifying their functional state.
In conclusion, our present study shows that fRLX expression, in the testis of the frog Rana esculenta, is regulated by testosterone both in vivo and in vitro, and this effect is counteracted by CPA. Lastly, fRLX together with testosterone may be considered a marker for the study of Leydig cells activity, in the testis of the frog Rana esculenta.
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Acknowledgments |
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