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Dipartimento di Medicina Sperimentale-Sezione di Fisiologia Umana e Funzioni Biologiche Integrate "F. Bottazzi" (G.D.R., S.M.), Seconda Università degli Studi di Napoli-Via Costantinopoli, 16 80138 Napoli, Italy; and Laboratorio di Biochimica e Biologia Molecolare (F.A., M.B.), Stazione Zoologica "A. Dohrn"- Villa Comunale 121 80132 Napoli, Italy
Address all correspondence and requests for reprints to: Dr. Sergio Minucci, Dipartimento di Medicina Sperimentale-Sezione di Fisiologia Umana e Funzioni Biologiche Integrate "F. Bottazzi" Seconda Università degli Studi di Napoli-Via Costantinopoli, 16 80138 Napoli, Italy. E-mail: sergio.minucci{at}unina2.it
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Abstract |
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Introduction |
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Recently RLF has been isolated and characterized in other mammalian species (5, 6, 7, 8). Its sequence in all species shows the common B-C-A heteromeric structural organization in which the B and A domain are covalently linked by two intradomain disulfide bonds and in the B domain the highly conserved R-XXX-R relaxin receptor binding motif is present (9).
Several studies showed that RLF is expressed in fetal and adult testis (1, 3) and, in particular at a high level in the testicular Leydig cells of all mammalian species studied so far (9). It has also been detected at a low level in the placenta (10), theca cells, granulosa cells, corpus luteum, and ovarian stroma (11, 12). The expression of RLF in reproductive tissues suggests a potential involvement in reproduction; this is supported by the observation that transcription of the gene is mediated by steroidogenic factor I (SF-I) (13).
The functional role of the relaxin/insulin proteins in the testis is still not fully understood, although male mice with an inactive RLF gene show altered spermatogenesis (14) and male RLF-knockout mice exhibit bilateral cryptorchidism that results in abnormal spermatogenesis and infertility, probably due to a failure of the testis to descend properly during fetal development (15, 16).
No information about RLF expression is found in nonmammalian species. In the present study we report, for the first time, the cloning of a novel relaxin/insulin family member in the testis of an anuran, Rana esculenta, a seasonal breeder with a spermatogenic cycle regulated by endocrine and environmental factors and a cystic organization of its testis that favors the study of spermatogenesis (17).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of poly (A)+ RNA
Total RNA from the testes, HGs, kidneys, livers, brains,
muscles, ovary, and oviduct of the frog Rana esculenta were
prepared with a modification of Chomczynski and Sacchi procedure
(18). Poly (A)+ was purified by
oligo (dT) cellulose chromatography (19).
Isolation and sequencing of tissue-specific cDNA clones
The total cDNA inserts derived from 3 x
106 pfu from the two libraries prepared from poly
(A)+ messenger RNA (mRNA) extracted from the
testis and Harderian gland (HG) as described by Aniello et
al. (20) were rescued as Bluescript plasmids (SK+) by
a helper phage mediate in vivo excision as described by the
manufacturer (Stratagene, Cambridge, UK).
The maxiexcision of the HG cDNA library was used to prepare the probe to screen the maxiexcision of testis library.
In particular, 10 µg of plasmidic DNA obtained from HG cDNA library
maxiexcision were digested with cloning restriction enzyme
(EcoRI-XhoI) and the digestion product containing
insert of 0.5–2.5 kb was labeled with the random primer method using a
mixture of [
32P]-dCTP and
[32P]-dATP (Amersham Pharmacia Biotech, Aylesburg, UK).
A quantity of 2 x 104 testis library plasmidic clones (1000 colonies/plate) were plated on LB-ampicilline agar plates (50 µg/ml) and incubated overnight at 37 C. Colonies containing the pBluescript with the cloned DNA inserts were transferred on nylon filters (Colony/Plaque Screen NEN Life Science Products, Boston) and hybridized with the probe described above in 5x SSC, 5x Denhardt’s, 100 µg/ml salmon sperm DNA and 50 mM sodium phosphate at pH 7.0 overnight at 65 C. After the hybridization, plasmidic DNA was prepared from 14 negative clones as described (19). The directionated inserts (5'EcoRI- 3'XhoI) were sequenced on both strands by dideoxinucleotide procedure (21).
5'-RACE-PCR
The 5'-RACE-PCR was performed using 1 µg of total RNA prepared
from March testis according to the manufacturer’s instructions
(Life Technologies, Inc., Paisley, UK) employing the poly
(A)+ RNA from testis, the cDNA-specific primers
(test 1C: 5'-cacaggacatgacaactgtccgaata-3', test 1D:
5'-acatccatgccttggcaccaaaccg-3', test 1F: 5'-cggtatagccaacgtaaga-3')
and the universal primer (5'-ggccacgcgtcgactagtac-3', Life Technologies, Inc.). RACE-PCR was run for 35 cycles at an
annealing temperature of 68 C. The PCR product was separated on 1%
agarose gel, purified by QIAGEN gel extraction kit
(QIAGEN, Hilden, Germany) and cloned into the pGEM-T
vector (Promega Corp., Heidelberg, Germany). Sequence
analysis of PCR-amplified cDNA was performed as describe above.
Phylogenetic analysis
To trace the primary sequence relationships of fRLX with other
known RLF and relaxin, a phylogenetic analysis was performed using
amino acid sequence of seven RLFs and seven relaxins. Sequences were
managed and aligned using BioEdit version 4.5.8 software
(22). For the phylogenetic tree a Kimura 2p distance
matrix was obtained and the tree was constructed using UPGMA
(Unweighted Pair-group Method using Arithmetic Averages) clustering
procedure through the Neighbor joining method using the PHYLIP computer
package (23). The robustness of the phylogenetic
hypothesis was tested by bootstrapping. To assess branch reliability,
all bootstrap analyses of amino acids involved 500 replications of the
data.
Northern blot analysis
Total RNA (20 µg for each sample) was fractionated by
electrophoresis in 2.2 M formaldehyde on 1% agarose gel
and then transferred to a nitrocellulose membrane by overnight
capillary blotting. The filters were prehybridized for 5–6 h at 65 C
in 5x SSC, 5x Denhardt’s, 100 µg/ml salmon sperm DNA and 50
mM sodium phosphate at pH 7.0 and hybridized to test 1
probe (2 x 106 cpm/ml) at 65 C overnight.
The filters were washed twice for 30 min at 65 C in 0.2x SSC and 0.1%
SDS and finally were exposed to x-ray film (HR-H, Fuji Photo Film Co., Ltd., Tokyo, Japan).
RT-PCR
fRLX and frog heat-shock protein 90 (HSP90) mRNA levels were
measured by RT-PCR amplification.
First-strand cDNA was synthesized using 5 µg total RNA from a January ovary, oviduct and testis, 500 ng/µl oligo(dT)18 primer (Promega Corp., Heidelberg, Germany) and 200 U Superscript II RT enzyme (Life Technologies, Inc.) in a total volume of 20 µl according to the manufacturer’s instructions (Life Technologies, Inc.). Three microliters of this cDNA template was then used for the PCR (50 µl volume) with 1.5 mM MgCl2, 1 x PCR buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl, and 0.1% TritonX-100), 0.5 U Taq DNA polymerase (Promega Corp., Heidelberg, Germany) and 10 pmol oligonucleotide primers (fRLX forward primer: 5'-tgtatgcagagagccacat-3', fRLX reverse primer: 5'-gagtgcttgctctgcagac-3', HSP90 forward primer: 5'-tggctggacagctaacatg-3', HSP90 reverse primer: 5'-tcaggacatcacatactggc-3'). The RT-PCR product sizes were 265 bp for fRLX and 723 bp for HSP90.
An appropriate region of HSP90 cDNA was used as control. Amplifications, carried out for 30 cycles, were as follows: 94 C for 1 min, 53 C for 1 min, and 72 C for 1 min. Amplification products were electrophoresed on 1.5% agarose gel in 1x TAE buffer. Semiquantitative analysis of mRNA levels was carried out by the GEL DOC 1,000-UV fluorescent gel documentation system (Bio-Rad Laboratories, Inc., Hercules, CA).
In situ hybridization
Frog testes were perfusion fixed in Bouin’s fluid for 24 h
at room temperature. The tissues were dehydrated and embedded in
paraffin. Five-micrometer paraffin sections were dewaxed in xilol twice
for 10 min each time and transferred through descending grades of
ethanol to diethilpyrocarbonate-treated PBS for 5 min. The sections
were prefixed in 4% paraformaldehyde in 0.5 M NaCl, 0.1
M MOPS, pH 7.5, for 30 min at room temperature, then washed
in PBS. This was followed by incubation in 10 µg/ml proteinase K in
100 mM Tris-HCl, pH 7.2, and 1 mM EDTA for 10
min at room temperature. The sections were postfixed in 4%
paraformaldehyde in 0.5 M NaCl, 0.1 M MOPS, pH
7.5, for 30 min at room temperature. Slides were washed in PBS for 5
min, then transferred in 2 x SSC for 3 min. Before hybridization,
the sections were kept in Tris-glycine buffer for 45 min. The
hybridization solution comprised 40% deionized formamide, 5x SSC, 1x
Denhardt’s solution, 100 µg/ml sonicated salmon testes, 100 µg/ml
transfer RNA, and 80 ng digoxigenin-labeled complementary RNA (cRNA)
probe (see below). Eighty microliters of hybridization solution were
added on each slide. Hybridization proceeded overnight at 60 C in a
moist chamber. Slides were rinsed 3 times in 5 x SSC for 20 min
each, then washed in posthybridization buffer (0.5x SSC, 20%
deionized formamide) for 40 min at 60 C. Ribonuclease (RNase) A
digestion was then carried out to remove unspecifically bound
single-stranded cRNA probe. Sections were incubated for 30 min at 37 C
in RNase buffer (0.5 M NaCl, 10 mM Tris-HCl, pH
7, 1 mM EDTA) containing 100 µg/ml RNase A, and rinsed in
RNase buffer without the enzyme for 15 min at 37 C. The slides were
washed in posthybridization buffer for 30 min at 60 C and rinsed in
2 x SSC for 30 min at room temperature. The sections were
incubated in 1% blocking solution [1% blocking reagent
(Roche Diagnostics, Basel, Switzerland) in MBT buffer, 0.1
M maleic acid, 0.15 M NaCl, pH 7.5] for 10
min. The slides were incubated overnight at 4 C with an alkaline
phoshatase-conjugated sheep anti digoxigenin antibody
(Roche Diagnostics) diluted 1:2000 in MBT buffer. Sections
were rinsed 4 times in TBS for 10 min each and then in solution B
(0.1% Tween-20, 0.5 mg/ml levamisol) for 10 min before the color
detection substrate solution was applied [1 ml BM purple
(Roche Diagnostics), 10 µl 100x solution B per slide],
sections were incubated overnight in the dark at room temperature, and
the reaction was stopped by rinsing the slides in PBS, 1 mM
EDTA for 10 min at room temperature. Sections were dehydrated and
mounted. The 1222-bp test1 pBluescript II SK+ clone was linearized with
either EcoRI or XhoI to create antisense or sense
cRNA probes using T7 or T3 RNA polymerases, respectively. The sense
(control) and antisense cRNA probes were prepared by in
vitro transcription using (digoxigenin-UTP Roche
Diagnostics) exactly as recommended by the manufacturer.
Ethane 1,2-dimethane sulfonate (EDS) treatment
In February, adult frogs (n = 25) were divided into three
groups as follows: 1) 10 animals that received a single injection of
EDS [100 mg/kg body weight in dimethyl sulfoxide (DMSO)-water, 1:3
vol/vol]; 2) 10 animals that received a single injection of DMSO; 3) 5
animals were used as initial controls at the beginning of the
treatment. The EDS- and DMSO-injected frogs were killed on days 4 and
28 (5 animals/group/each time) after treatment and the testes were
removed and fixed for in situ hybridization.
Hypophysectomized (PDX) animals
In January, adult frogs (n = 25) were divided into two
groups as follows: 1) 20 animals were hypophysectomized
(24) and kept in captivity with food and meal ad
libitum for 30 days; 2) 5 animals were kept in captivity with food
and meal ad libitum for 30 days. At the end of the
experiment, the testes of all the animals were removed and fixed for
in situ hybridization.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The sequence following the
first methionine potentially encodes a protein of 155 amino acids
residues. The deduced amino acid sequence was compared with all
nonredundant GenBank entries and showed a low similarity to RLF and
relaxin from different species; for this reason, test 1 was named
"frog relaxin" (fRLX). The sequence shows a low identity at amino
acids level with RLF and relaxin known proteins (Table 1). Although the low value of identity,
fRLX prepolypeptide contains the classical B-C-A domain configuration
present in all the insulin/relaxin family proteins. Restricting the
comparison among the B and A domains, forming the mature proteins, the
values are higher (Table 1), showing that in all the members of the
family the cystein motifs present in the A and B domains were highly
conserved (Fig. 2). The A-domain motif is
CC X3 C X8 C where
XN represents the number of residues comprising
no amino acids other than cystein. The B-domain motif is LCG
X10 C (Fig. 2).
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, a single positive band is
present at high level exclusively in the testis. The size of this band,
estimated to be about 1.6 kb using as reference the 18S ribosomal RNA,
is very close to that of the total fRLX clone isolated. The amount of
RNA on each line was controlled using as a probe the frog ribosomal P1
cDNA (Accession No. AJ298875) (Fig. 4B).
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, a very low signal was evidenced both
in the ovary and oviduct (lanes 1 and 2, respectively) compared with
the strong signal observed in the testis (lane 3).
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Temporal expression of fRLX during spermatogenesis
To examine the expression of fRLX during spermatogenesis, a
Northern blot analysis was performed using total RNA from testes of
frogs collected twice a month during the annual cycle. fRLX mRNA was
detected at very high level during the months of January/February and
March/April, whereas it was detected at a low level in May/June and
July/August and at the lowest level in September/October (Fig. 6A). The amount of RNA on each line was
controlled using as a probe the frog ribosomal P1 cDNA (Fig. 6B).
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), whereas a weak interstitial signal
was present in the September testis (Fig. 7B). No signal was detected
in March sense-treated testis (Fig. 7C).
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) when Leydig cells are affected,
whereas a strong fRLX mRNA signal reappeared in the interstitial
compartment on day 28 when a new population of Leydig cells appear in
the interstitial compartment after 28 days (Fig. 8B).
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, A and B). The testis of PDX animals
shows the typical characteristic of hypophysectomy: the interstitial
compartment becomes hypertrophic, whereas the germinal compartment
showed signs of degeneration, such as seminiferous tubules without
spermatids (SPT), and very few secondary spermatogonia (II SPG),
whereas secondary spermatocytes (II SPC) were rare. Many degenerating
nests containing primary spermatocytes (I SPC) were observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fRLX protein, similar to the mammalian RLF and the other relaxin known proteins, contains a conserved amino acid motif, R-XXX-R, close to the first cystein of the B domain. Because this motif has been shown to be important for relaxin binding to its putative receptor in many tissues (29, 30, 31), it has been hypothesized that the relaxin receptor could be recognized by RLF with lower affinity (32). Alteration of basic residues in this binding motif completely abolished receptor interaction (29, 33, 34).
The phylogenetic tree for amino acid sequences related to RLF and relaxin proteins suggests that fRLX may be grouped with shark and mammalian relaxins. It is to be noted that fRLX, as the mammalian RLFs, is highly expressed in the testicular Leydig cells and lowly expressed in the ovary and in the female tract, whereas mammalian relaxins are classical endocrine hormones, produced by the ovary, and targeting the reproductive system to ripen the cervix, elongate the pubic symphysis and inhibit uterine contraction (35, 36).
Taken together, our results with phylogenetic analysis evidenced that fRLX sequence can represent an ancestral form of relaxin from which both modern mammalian relaxin and RLF might have evolved, respectively, for female and male functions.
The frog testis fRLX mRNA is a specific transcript found in the interstitial compartment and absent in the tubular compartment. Analyses of the Northern blot revealed a different expression of the transcript throughout the year. fRLX mRNA is more abundant in January and February. It reaches a peak in March-April then gradually declines from May onwards with a minimum in September-October. It is of interest to remember that circulating levels of androgens in Rana esculenta are relatively high when fRLX mRNA is more abundant (37). At the same time, androgens are necessary for spermatogonial proliferation and spermatid formation. Therefore, fRLX expression pattern correlates well with the frog steroidogenic and spermatogenetic wave.
It has been hypothesized that mouse RLF is a testicular factor that plays an essential role in testis descent, which is involved in gubernacolum development (15, 16) thus making the testis avail a slightly lower (compared with body) temperature threshold necessary for active spermatogenesis. These studies also mentioned that RLF knockout mice show abnormal spermatogenesis. However, normal spermatogenesis was observed in the surgically descended testis of RLF-deficient mice, strongly suggesting that in mice, RLF is not essential for germ cell development (15). In lower vertebrates, like the frog, where testicular descent does not occur, spermatogenesis is susceptible to seasonal thermal influence (17). The fact that the seasonal pattern of fRLX expression is consistent with seasonal testicular cycle strongly suggests a role of this factor in testicular activity.
In an attempt to obtain more information about the hypophysial influences on fRLX expression, if any, we investigated the presence of fRLX transcript in the testes of PDX animals after 30 days from surgical depletion. The fRLX expression was detected at a lower level in the interstitial compartment of the PDX frogs compared with that of the intact animals. This preliminary result seems to indicate that the fRLX expression could be under hypophysal control. A pituitary control of RLF expression 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 (38, 39, 40) in which RLF expression is totally absent, suggesting that Leydig cells seems to be arrested in a prepubertal state of differentiation (41).
In conclusion, our present study is the first report on the isolation and characterization of a new member of the relaxin/insulin family in a nonmammalian vertebrate, the frog Rana esculenta. EDS administration showed that Leydig cells are the specific interstitial cell type expressing fRLX. Its expression changes during the spermatogenetic cycle, suggesting that it might contribute to the efficiency of spermatogenesis in these species with seasonal breeding and it seems to be modulated by pituitary. In addiction, fRLX is expressed at a very low level also in the ovary and oviduct. fRLX may be considered a new marker for the study of the testicular activity in the frog Rana esculenta.
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Acknowledgments |
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Footnotes |
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2 All the authors contributed equally in the preparation of the
manuscript.
Received December 27, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This article has been cited by other articles:
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  G. De Rienzo, F. Aniello, M. Branno, G. Izzo, and S. Minucci The expression level of frog relaxin mRNA (fRLX), in the testis of Rana esculenta, is influenced by testosterone J. Exp. Biol., October 1, 2006; 209(19): 3806 - 3811. [Abstract] [Full Text] [PDF]
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 R. Ivell and R. A.D. Bathgate Reproductive Biology of the Relaxin-Like Factor (RLF/INSL3) Biol Reprod, September 1, 2002; 67(3): 699 - 705. [Abstract] [Full Text] [PDF]
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