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Regular Article |
a Dipartimento di Medicina Sperimentale, Sez."F.BOTTAZZI," II Università di Napoli, 80138 Napoli, Italy
b Dipartimento di Biologia "L.GORINI," Università di Milano, 20133 Milano, Italy
c Unité INSERM 421, Faculté de Médicine, 94010 Créteil, France
d Dipartimento di Biotecnologie e Bioscienze, Università Bicocca, Milano, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and reduced intratesticular androgen levels. However, androgen treatment did not affect MSJ-1 protein levels in either frogs or mice. In conclusion, our data in Rana esculenta and the wobbler mouse demonstrate a tight correlation between MSJ-1 protein expression and postmeiotic stages. In particular, the findings in the wobbler testis suggest a role for this protein in acrosomogenesis.
developmental biology, fertilization, sperm maturation, testis, Wobbler mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The msj-1 (mouse sperm cell-specific DnaJ first homologue) gene was isolated by molecular cloning of a mouse spermatogenetic cDNA library. Its transcripts are specifically expressed in germ cells at haploid stages [6], and MSJ-1 protein appears in round spermatid cytoplasm and accumulates in the periacrosomal region of elongating spermatids. Immature spermatozoa display MSJ-1 immunoreactivity [7, 8].
The aim of this work was to investigate on the role played by MSJ-1 during spermatogenetic processes focusing, in particular, on spermiogenesis events. We used 2 experimental models: the anuran amphibian, Rana esculenta, and the wobbler (wr) mutant mouse. The frog, Rana esculenta, is a very suitable model for spermatogenesis studies. It is a seasonal breeder, characterized by a slow progression of spermatogenesis throughout a year. During the annual reproductive cycle, spermatogenesis arises in spring, with proliferation of spermatogonia; proceeds actively in summer; declines in autumn, when there is an active spermiogenesis; and reaches a quiescent stage in winter months [9].
Wobbler mice are known as a model of hereditary spinal atrophy. In addition to causing neurologic disorders, the wobbler mutation causes male infertility. Wobbler is an autosomal recessive mutation of unknown identity mapping to the proximal region of chromosome 11 [10]. Homozygous wr/wr mice display a loss of 20%–40% of their spinal and brainstem motor neurons [11]. Clinical signs of the illness appear 3–4 wk after birth, with mutant mice displaying muscle atrophy and impaired movement. Wobbler mouse infertility is due to a defect in spermiogenesis; sperm cells have a rounded head and a reduced motility. Ultrastructural studies show that wobbler spermatozoa lack a real acrosome, even if several little acrosomic vesicles are dispersed in round spermatid cytoplasm [12]. The spermatozoa tail has an unstable axonemal complex and deficiencies in the outer microtubular doublets [13].
Here we report the MSJ-1 protein expression pattern in both Rana esculenta testes, during the annual reproductive cycle, and in wobbler juvenile and adult mouse testes. For the first time in a lower vertebrate, we demonstrate that MSJ-1 protein expression correlates with the appearance of postmeiotic cells, suggesting a highly conserved role for this protein in spermiogenesis. The data obtained in wobbler mice, an acrosome-negative model, strongly suggest that MSJ-1 plays a role in acrosome formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Intact male frogs, Rana esculenta, were captured in the vicinity of Naples. The animals were anesthetized with MS222 (Sigma Chemical Co., St. Louis, MO), and the testes were immediately removed and stored at -80°C until processed for protein analysis.
Wild-type (+/+), heterozygous (+/wr), and wobbler (wr/wr) mice were deeply anesthetized with sodium pentobarbitone, and their testes were rapidly removed and either prepared for immunocytochemical analysis or frozen on dry ice and stored at -80°C before being used for RIA of androgen levels, RNA extraction, or protein extraction.
To allow prediction of the mutation before the onset of the disease, a polymorphic genetic marker, the microsatellite of the glutamine synthetase gene (G, g), was introduced close to the wr gene by crossing C57B1/6J mice (+/wr, g/g) with New Zealand Black mice (+/+, G/G) as previously described [14]. The heterozygotes carrying both G and g alleles are phenotypically normal. Mice carrying only the G allele correspond to wild-type mice (+/+), whereas mice carrying only the g allele correspond to the wobbler mutant (wr/wr) and display the clinical signs of the disease. The DNA was extracted from the mouse tail, and the alleles were analyzed by polymerase chain reaction (PCR) amplification as described previously [14]. The primers used amplify a microsatellite DNA located in the 5' noncoding region of the glutamine synthetase gene that contains a variable number of GT repeats each determining g and G allele. The primer sequences were as follows: forward primer: 5'-AGCTTTGGAGACAACAATTAGATC-3'; reverse primer: 5'-GGATGGGGAAATGGTGGTACA-3'.
RNA Extraction
Total RNA was extracted from the testes of juvenile (20-day-old) and adult (90-day-old) mice (+/+, +/wr, and wr/wr) according to the method of Chomczynski and Sacchi [15].
Northern Blot Analysis
Total RNA (30 µg per lane) from the testes of 20-day-old mice (+/+, +/wr, and wr/wr) was electrophoresed after denaturation with glyoxal and dimethylsulfoxide on 1.4% agarose gel [16] and transferred to nylon membranes (Nytran; Amersham Pharmacia Biotech, Buckingamshire, U.K.). MSJ-1 clone B cDNA [7] was labeled with [32P]dCTP using the Redi Prime II labeling kit (Amersham Pharmacia Biotech). Hybridization was performed in Church buffer (0.5 M PBS pH 7.4, 7% SDS, 1 mM EDTA, 10 mg/ml sonicated salmon sperm DNA) at 65°C overnight. The membranes were washed twice in 2x saline-sodium citrate, 0.2% SDS at 65°C for 15 min and exposed for a suitable time. The 28S ribosomal RNA signal was of similar intensity in all lanes when the gels were stained with ethidium bromide (data not shown).
MSJ-1 mRNA Detection by Reverse Transcription-PCR
A reverse transcription (RT)-PCR assay was carried out to check for the presence of msj-1 transcripts in +/+, +/wr, and wr/wr mouse testes. In brief, 2 µg of total RNA from testes of 20- and 90-day-old mice were extracted and reverse-transcribed to prepare cDNA as described previously. Total RNA from each sample that was not treated with reverse transcriptase was used as a negative control. The resulting cDNA mixture was used in the PCR assay. The normalization has been carried out using two primers specific for ß-actin (mouse ß-actin control Amplimer Set; Clontech, Palo Alto, CA). Msj-1 transcripts were identified using two primers designed to amplify a 484-base pair (bp) fragment corresponding to nucleotides 272–756 of the MSJ-1 cDNA: the sense primer was BA1 (5'-GCTGCGGCGAAGTGGGCG-3'; nucleotides 272–289), and the antisense primer was NB (5'-CAGGGACTTTAACTCTCCATC-3'; nucleotides 735–756). Amplification was carried out in the presence of 2 µl of the respective cDNA and 1.25 units of Taq polymerase (Boehringer Mannheim, Mannheim, Germany) in a final volume of 50 µl according to the manufacturer's instructions. The thermal cycle profile was 95°C for 2 min, 63°C for 30 sec, 72°C for 1 min for five cycles, and then 95°C for 2 min, 62°C for 30 sec, and 72°C for 1 min for 25 cycles. The resulting products were electrophoresed on a 1.2% agarose gel.
RT-PCR Detection of Transcripts of Genes Associated with Testicular Activity
Total RNA from 20-day-old mice that was not treated with reverse transcriptase was used as a negative control. The positive control for estrogen receptor (ER) and dosage-sensitive sex reversal, adrenal hypoplasia congenita, X-linked (DAX-1) was mouse ovary RNA from adults, whereas the positive control for androgen receptor (AR) and c-fos was both adult mouse ovary and testis RNA. The presence in mouse testes of transcripts of genes (ER [17]; AR [18]; dax-1 [19]; c-fos [20]; hypoxanthine-guanine phosphoribosyltransferase [HPRT] [21]) was demonstrated by amplifying specific DNA sequences with PCR according to the manufacturer's instructions (GenAmp, Perkin Elmer, Milano, Italy).
Two micrograms of total RNA was reverse transcribed to prepare cDNA. The reaction conditions for reverse transcription used were 0.05 µg/µl oligo(dT), 1 mM dNTP, 5 mM MgCl2, 20 units RNase inhibitor, and 50 units reverse transcriptase for 30 min at 42°C. To stop the reaction, 30 µl of distilled water was added to the reaction mixture.
The PCR was performed on 1 µl of the cDNA product with 0.2 pmol specific oligonucleotide primers, Taq (Thermus aquaticus) DNA polymerase, and Q solution (Quiagen; Promega Corp., Madison, WI). The primer sequences and predicted sizes of the reaction products are shown in Table 1.
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Thirty cycles of amplification were performed with a Celbio thermocycler apparatus (Celbio Italia, Milano, Italy) as follows: 90°C for 5 min, 95°C for 1 min, specific annealing temperature (Table 1), 72°C for 1 min, and 72°C for 5 min. The amplified products were analyzed on an ethidium bromide agarose gel. RT-PCR experiments were performed 3 times, independently from each other.
Cytosolic Protein Extraction and Western Blot
Frog testes (collected monthly from January to December) and testes from mice (+/+, +/wr, and wr/wr) of various ages (10, 20, 30, and 90 days of age) were thawed and gently homogenized in seven volumes of buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA pH 8.1, 0.5 mM spermidine, 12% glycerol, 0.5 mM dithiothreitol, 0.5 mM PMSF, 4 µg/ml leupeptin, 4 µg/ml chymostatin, 4 µg/ml pepstatin A, and 5 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone). Large particulates, such as intact cells and nuclei, were removed by centrifugation at 800 x g for 30 min at 4°C. The supernatant fraction was then stratified on glycerol and centrifuged at 100 000 x g for 1 h at 4°C. Cleared cytosolic extract was removed and stored at -80°C until used. The positive control consisted of adult mouse testis, and the negative control consisted of frog heart and muscle protein extracts. Protein concentrations were measured according to the method of Lowry et al. [22].
Twenty-five micrograms per lane of frog and mouse cytosolic extracts were separated using 0.1% SDS, 12% polyacrylamide gel [23, 24]. After electrophoresis, the proteins were blotted to nitrocellulose membranes (Amersham Pharmacia Biotech) for 2.5 h at 280 mA at 4°C in a mini trans blot cell apparatus (BioRad Laboratories Inc., Hercules, CA). After the transfer, the membranes were rinsed for 10 min in PBS pH 7.6 (20 mM NaH2PO4, 80 mM Na2HPO4, 100 mM NaCl) and then treated for 2 h with blocking solution (5% nonfat powdered milk in TBS-T pH 7.6 (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.25% Tween 20) to prevent nonspecific adsorption.
An MSJ-1 antibody has been raised against a recombinant protein glutathione S-transferase (GST)/MSJ-1 (amino acids 149–242) corresponding to a fusion between GST and the C-terminal portion of MSJ-1 that does not contain the highly homologous J domain [7]. To develop MSJ-1 antibody, the antiserum was affinity-purified using a GST-coupled Affigel-10 column (BioRad Laboratories) [8].
Hybridization was performed overnight at 4°C in 3% nonfat powdered milk, PBS pH 7.6 containing the antibody anti-MSJ diluted 1:3000 for frog samples and 1:5000 for mouse samples. The membranes were then washed 3 times for 10 min each in TBS-T and once for 10 min in TBS and then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (DAKO, Glostrup, Denmark). After the washing, the immunocomplexes were detected using the Electro-Chemi-Luminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The same membranes were stripped for 30 min at 60°C in 62.5 mM Tris-HCl pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol. After incubation, they were rinsed several times in PBS pH 7.6. Antibody specificity was checked by incubating the stripped membranes with a large excess (10-6 M) of the respective antigen.
Immunocytochemistry
Testes of +/+ and wr/wr mice (40 days of age) were fixed at 4°C in buffered paraformaldehyde and then embedded in paraffin. Deparaffinized sections (7 µm) were immunostained with MSJ-1 affinity-purified antibody (1:100 in 3% BSA in Tris-buffered saline), followed by 1:200 rhodamine-conjugated anti-rabbit IgG (Sigma) as a secondary antibody. To test the specificity of the reaction, in control samples, the primary antibody step was omitted. The sections were examined under an Olympus epifluorescence microscope (Olympus Italia, Milano, Italy) equipped with standard filters for red fluorescence.
RIA of Intratesticular Androgen Content in Mice
Intratesticular androgens were measured by RIA as described elsewhere [25]. Since the antiserum was specific for both testosterone and 5-dihydrotestosterone, data are expressed as androgen data. The interassay and intraassay coefficients of variation were 8% and 5%, respectively. The sensitivity was 2 pg per tube.
In Vivo Experiment in Thermally Treated Frogs
Fifty male frogs captured in October were kept at 4°C for a prolonged period of time (approximately 2 mo) and injected on alternate days with pituitary homogenate (one third of a pituitary per frog per injection). Periodically, two animals were killed to check, by routine histology, for the total absence of all spermatogenic stages except spermatogonia. After 2 mo, 10 animals were killed, and samples from these animals were stored at -80°C; the remaining animals were kept at 4°C for an additional 2 wk.
In Vitro Incubation of Testis Fragments from Frogs and Mice
Ninety male frogs, captured in June, were anesthetized, and their testes were rapidly removed, cut in half, and immersed in 0.5 ml of Krebs-Ringer buffer (KRB) for amphibia for the control group or in KRB for amphibia containing 10-6 M testosterone or containing both 10-6 M testosterone and 10-4 M flutamide (Sigma) at 22°C for 1 and 6 h. The same experimental procedure was used for mouse testes, except that tissues were incubated in KRB for mammals at 37°C.
Statistics
The significance of differences was evaluated by ANOVA followed by the Duncan test or the Student t-test, where appropriate.
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RESULTS |
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Western blot analysis carried out on cytosolic preparation of testicular proteins show the presence of a specific MSJ-1 signal of 30 kDa in frog testis (Fig. 1). The size of the band was identical to that observed in mouse testis protein extracts.
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The specificity was determined by preabsorption of the antibody with a large excess (10-6 M) of the antigen. Disappearance of the 30-kDa band was used as the criterion for signal specificity to identify MSJ-1 protein.
During the annual seasonal cycle (Fig. 1), MSJ-1 protein was detected at high levels in Rana esculenta testis from April–May until December, a time period characterized by the presence of postmeiotic stages. Conversely, a very scanty MSJ-1 signal was observed from January until March, when spermatogonia constitute the main cellular population of the testis. In fact, during this period, the frog testis undergoes degeneration of meiotic and postmeiotic stages and depletion of spermatozoa, an event due to spermiation that occurs during the breeding season (late February and March).
To further confirm the association of MSJ-1 expression with the appearance of postmeiotic stages, the frogs were kept at 4°C and treated with pituitary homogenates. This treatment is known to produce testes enriched in spermatogonia and to result in the progressive time-dependent disappearance of postmeiotic stages [26]. As expected, pituitary homogenate treatment resulted in the progressive disappearance of the MSJ-1-specific signal (Fig. 2). Disappearance of postmeiotic stages was confirmed by histologic findings in controls (data not shown).
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Northern Blot Analysis of msj-1 mRNA in Juvenile Mice
Northern blot analysis was carried out on total RNA extracted from testes of juvenile (20-day-old) mice (+/+, +/wr, and wr/wr) to detect the presence of msj-1 mRNA. Two bands of about 1 and 1.2 kilobase (kb) were observed in the +/+ and +/wr testes, but a signal was barely detectable in the wr/wr testes (Fig. 3).
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RT-PCR Amplification of msj-1 Transcripts from Juvenile and Adult Mouse Testis
To confirm the detection of msj-1 mRNA and to quantify the differences between mice with different genotypes, we carried out RT-PCR analysis on cDNA derived from testis mRNA from 20- and 90-day-old +/+, +/wr, and wr/wr mice. The levels of the msj-1 transcripts, detected as a 484-bp cDNA fragment, were similar in 20-day-old +/+, +/wr, and wr/wr mice (Fig. 4, lanes 1–6). In contrast, a reduction in levels of msj-1 transcripts was observed in 90-day-old +/wr (lane 9) and wr/wr (lane 11) mice as compared with +/+ mice (lane 7), with the decrease particularly marked in wr/wr mice (lane 11).
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MSJ-1 Protein Detection by Western Blot in Wild-Type, Heterozygous, and Wobbler Mice and Its Localization by Immunocytochemistry
No MSJ-1 immunoreactive signal was observed in testes from 10-day-old mice, regardless of their genotype (Fig. 5). In contrast, at 20 days of age, a specific MSJ-1-immunopositive band of 30 kDa was detected with similar intensity in both wild-type and heterozygous mouse testes, whereas a very scanty signal was observed in wobbler mouse testes (Fig. 5). In 30- and 90-day-old mice, the MSJ-1 signal was also decreased in heterozygous mice, showing an intermediate intensity between that of +/+ and wr/wr mice at 90 days of age. Preabsorption of the antibody with its antigen abolished the 30-kDa immunoreactive signal, thus confirming the antibody specificity. Failure related to the disappearance of the upper band indicates nonspecificity.
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Immunofluorescence analysis carried out in testis sections of sexually mature (40-day-old) mice revealed the presence of MSJ-1 protein within the seminiferous epithelium of +/+, +/wr, and wr/wr mice (Fig. 6). MSJ-1 immunoreactivity was restricted to the spermatid population in mice of all genotypes, but the number of MSJ-1-immunoreactive spermatids appeared to be reduced in +/wr mice and markedly reduced in wr/wr testes (Fig. 6, left column). As could be appreciated in the respective phase-contrast images (Fig. 6, right column), spermatogenesis progressed regularly from mitotic spermatogonia, at the periphery of the seminiferous tubule, to postmeiotic spermatids in the adluminal region. Spermatozoa were present in the lumen in +/+ and +/wr testes. In wr/wr testes, the cytoarchitecture of the spermatid layers is rather disarranged because of the presence of roundish and vacuolized cells and the sperm cells in the lumen lacking the typical flattened shape of normal mouse spermatozoa. A total extinction of the reaction was obtained by omitting the primary antibody step (Fig. 6, upper panel, left column).
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Characterization of Wobbler Mouse Testicular Activity
Reduced MSJ-1 expression in the mutant testis prompted us to examine whether additional defects were present, especially regarding androgen metabolism. PCR amplification of the chosen genes was used to characterize testicular activity of 20-day-old +/+, +/wr, and wr/wr mice at a stage just before the appearance of the first neurologic signs of the disease, when postmeiotic stages are available in the testes.
A 440-bp amplified ER band was observed in both +/+ and +/wr mouse testis, whereas this band was barely detectable in the testes of wr/wr mice (Fig. 7). Conversely, the results for AR, dax-1, and c-fos did not differ between +/+ and wr/wr testes, and the amplified fragments were of 593, 290, and 238 bp, respectively (Fig. 7). Amplification of the HPRT gene was used to check the cDNA quality and eventual procedural losses.
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Intratesticular androgen content was evaluated by RIA in +/+, +/wr, and wr/wr mice at 10, 20, 30, and 90 days of age. Androgens were undetectable at 10 and 20 days of age in all groups considered (data not shown).
At 30 days of age, the intratesticular androgen content of wr/wr mice was significantly reduced (P < 0.01) as compared with that of +/+ and +/wr mice (Fig. 8). At 90 days of age, the intratesticular androgen content was comparable in +/wr and wr/wr mice, and both were significantly reduced as compared with that of +/+ animals (P < 0.01).
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Effect of In Vitro Androgen Treatments on Testicular Fragments from Both May Frogs and Normal, Heterozygous, and Wobbler Mice
Frog and mouse (+/+, +/wr, and wr/wr) testes were incubated with testosterone alone or in combination with an antiandrogen, flutamide, to evaluate the possible link between androgens and MSJ-1 expression. Western blot analysis did not reveal any change in MSJ-1 protein content in response to the androgen treatment in either frogs or mice (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we report that MSJ-1, a member of the DnaJ family, specifically expressed in rodent postmeiotic germ cells during the differentiation of round spermatids into mature spermatozoa [7, 8], is expressed in the frog, Rana esculenta, testis. MSJ-1 expression during the annual reproductive cycle coincides, as in mice, with spermiogenesis events. In addition, the results obtained on an acrosome-lacking model, the wobbler mutant mouse, favor a role for MSJ-1 in spermatozoa maturation.
Patterns of MSJ-1 expression in both frogs and mice were closely associated with the end of meiosis and the onset of spermatid maturation. Experimental induction of the quiescence of testis activity with thermal treatments in frogs [27] confirmed this association: depletion of frog testis of the postmeiotic stages induced decreased MSJ-1 expression.
Comparison of phylogenetically distant species is a basic approach for detecting highly conserved molecules. If the molecules are also localized in the same cell types, they may have similar function and are likely to be related to a master (fundamental) system. Thus, for example, in cellular differentiation and development, homeodomains have been characterized from invertebrates to humans [28] or, in the hypothalamus-pituitary-gonadal axis regulation, releasing hormones, gonadotropins, and steroids are present in all vertebrates [29]. Therefore, the use of the Rana esculenta model was justified to determine if MSJ-1 protein is conserved. Accordingly, conservation of MSJ-1 from frogs to mice suggests that MSJ-1 plays a central role during spermiogenesis. We therefore carried out further investigation of MSJ-1 function in the testis using mutant mice that are defective in spermiogenesis. Previous localization of MSJ-1 protein in the periacrosomal region [8] raised the possibility that MSJ-1 plays a role in acrosomogenesis. We therefore chose wobbler mutant mice to investigate the possible role of MSJ-1.
Testicular total RNA from +/+, +/wr, and wr/wr mice aged 20 days were processed, and the results showed two bands of about 1 and 1.2 kb. The concentration of msj-1 mRNA was markedly higher in +/+ and +/wr mice as compared with wr/wr mice. These data confirm previous results obtained in +/+ mice [7]. Moreover, we give evidence that wr/wr mice have very scanty expression of the msj-1 gene starting from 20 days of age, when haploid spermatogenic stages, except sperm cells, appear [30]. The interesting point is the presence of msj-1 mRNA in +/wr animals, confirming that heterozygotes do not manifest the disease. To extend results on the scanty expression of the gene in wr/wr mice, PCR amplification was performed in animals (+/+, +/wr, and wr/wr) aged 20 and 90 days. The results show that in 90-day-old mice, only wild-type animals had a clear band, whereas heterozygotes possess a very scanty expression that was further decreased in wr/wr animals.
The MSJ-1 protein in wr/wr testes is expressed at very low levels when compared with that in +/+ testes. In 10-day-old animals, the protein is completely absent in all samples, according to the lack of postmeiotic cells at this stage of development [30]. We consistently detected MSJ-1 protein in +/+ testes starting from 20 days of age, thus further confirming that its expression is linked to the appearance of haploid stages, as secondary spermatocytes and round spermatids are increasing in numbers by 20 days of age [30]. Wobbler mice show a scanty appearance of MSJ-1 from 20 days of age onward and, interestingly, +/wr animals also show a decrease in MSJ-1 protein signal, but starting at 30 days of age. In +/wr animals, the MSJ-1 protein signal appears at low levels also at 90 days of age, but the intensity of the immunoreaction is stronger than that in wr/wr mice. Similar results were obtained using immunocytochemistry, which also showed localization of MSJ-1 in postmeiotic stages. Therefore, Western blot analysis and immunocytochemistry results parallel the results obtained using Northern blot and PCR, demonstrating that the enormous decrease of MSJ-1 expression in wr/wr animals depends on transcriptional defects.
The reduction in MSJ-1 levels could be part of a general alteration in testicular metabolism. Therefore, we characterized wobbler testis by investigating the expression of some genes involved in the regulation of testicular activity and assaying intratesticular androgen content. We evaluated levels of several transcripts involved in testicular metabolism by semiquantitative RT-PCR: the ER and AR, steroid hormone receptors which are essential for testis activity; the nuclear orphan receptor dax-1, which interacts with steroidogenic factor 1 to modulate male sexual differentiation and testis development [19, 31]; and the proto-oncogene c-fos, which is involved in premeiotic stage progression in mammals [32]. Only ER transcripts showed strongly reduced levels in the wobbler mouse testis. Additionally, we evaluated intratesticular androgen levels by RIA. Indeed, circulating and intratesticular profiles have been shown to be mismatched in several species [33, 34]. We observed significant differences between +/+ and wr/wr mice, the latter showing a several fold lower androgen content. This result indicates that the normal value previously detected in the plasma of wobbler mice has lead to misleading conclusions [12] with respect to the steroidal environment of the wobbler testis. Since we show that some testicular markers, AR included, are normally expressed in wobbler mice, the difference in androgen levels between testes of wild-type and wobbler mice could be due to defects in the androgen-binding protein system or to failures of intratesticular feedback mechanisms. However, the significance of low androgen levels in wobbler mice needs to be further investigated in light of our results from in vitro incubation showing that testosterone is not involved in the regulation of MSJ-1 protein level in either mice or frogs.
Thus, the very scanty expression of MSJ-1 in postmeiotic stages of wr/wr mouse testis strongly suggests that this protein is involved in acrosome formation. MSJ-1 could affect a cytoskeletal factor, modulating acrosome vesicle movement and position. For example, in the rat, filamentous actin and the relative actin-capping proteins concentrate in the developing subacrosomal space in round spermatids and are responsible for acrosome position and shape [35]. Alternatively, MSJ-1 might be involved in the fusion of acrosomic vesicle themselves. Indeed, DnaJ-like proteins modulate synaptic neurotransmitter release [4] and coated vesicle traffic, and during placenta formation, mammalian relative of dnaJ is important in chorioallantoic fusion at embryonic Day 8.5 [36]. Alterations have also been noted in the wobbler mouse sperm tail; in particular, the sperm appear to have average-length tails, but 70% of the sperm in the vas deferens have ultrastructural defects in the flagellum [13].
In conclusion, MSJ-1 in both mammals and lower vertebrates shows a similar pattern of expression. The use of different animal models belonging to different classes of vertebrates distant in the evolutionary tree (amphibians first appeared 400 million yr ago) indicates that MSJ-1 must exert a highly conserved basic function during spermiogenesis. In this view, MSJ-1 seems to be involved in mechanisms related to acrosome formation as is suggested by our results in wr/wr mice, which lack a true acrosome.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from MURST "ex40% Geremia" and 60%, the Association Française contre les Myopathies (M.P.J., S.B.) and was carried out within the CNR "Target Project on Biotechnology." 
2 Correspondence: Riccardo Pierantoni, Dipartimento di Medicina Sperimentale, Sez. "F.BOTTAZZI," II Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy. FAX: 19 39 081 5667536/7500; riccardo.pierantoni{at}unina2.it
Accepted: November 28, 2001.
Received: June 25, 2001.
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REFERENCES |
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) expression by degenerative motoneurons of the murine mutant wobbler: a neuronal signal for astrogliosis?. J Neurosci 1994; 14:4206-4216[Abstract]This article has been cited by other articles:
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  G. Berruti and E. Martegani The Deubiquitinating Enzyme mUBPy Interacts with the Sperm-Specific Molecular Chaperone MSJ-1: The Relation with the Proteasome, Acrosome, and Centrosome in Mouse Male Germ Cells Biol Reprod, January 1, 2005; 72(1): 14 - 21. [Abstract] [Full Text] [PDF]
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