Human Molecular Genetics, 2001, Vol. 10, No. 24 2803-2811
© 2001 Oxford University Press
Developmental expression of the fragile X-related 1 proteins in mouse testis: association with microtubule elements
1Unité de Recherche en Génétique Humaine et Moléculaire and 2Unité d’Endocrinologie de la Reproduction, Hôpital St. François d’Assise du CHUQ, 10, rue de l’Espinay, Québec G1L 3L5, Canada and 3Faculté de Médecine, Université Laval, Québec, Canada
Received August 1, 2001; Revised and Accepted September 11, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fragile X mental retardation 1 protein (FMRP) is the archetype of a class of cytoplasmic mRNA-binding proteins that includes the fragile X-related 1 and 2 proteins (FXR1P and FXR2P). Whereas absence of FMRP is the cause of fragile X syndrome, it is not known if FXR1P and FXR2P are associated with any pathology. It is also still elusive whether these homologous proteins can partially compensate for the absence of FMRP in the case of the fragile X syndrome. FXR1 is widely expressed in mammals and its expression pattern is complex since several mRNA variants and protein isoforms are detected. In mouse, we observed that the highest level of FXR1 is found in the adult testis. This tissue is an exception, since all known FXR1P isoforms, some of which have been considered as tissue specific, are detected in it. In young animals, changes in mRNA-spliced variants and their corresponding protein isoforms occur during spermatogenesis. Using biochemical, immunohistochemical and electron microscopic techniques, we show that FXR1P is associated with microtubule elements. Since the cytoskeletal framework is implicated in cellular plasticity as well as in mRNA transport, we propose new possibilities for the function(s) of the FXR proteins.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fragile X-related 1 (FXR1) is a member of a small gene family that includes fragile X mental retardation 1 (FMR1) and fragile X-related 2 (FXR2; reviewed in 1–3). FMR1 was the first to be discovered and absence of its expression was found to be associated with fragile X syndrome (4). Whereas FMR1 is located on human chromosome X at q27.3 (5), FXR1 and FXR2 are autosomal genes mapping at 3q28 and 17p13.1, respectively (6,7). These two genes are not associated with any known pathology or defect. The proteins coded by these genes are structurally very similar: they share high amino acid homologies in clustered regions and contain two KH boxes and an RGG domain which are motifs known to interact with RNA (7–9). Also, the three proteins contain nuclear localization and export signals (NLS and NES, respectively) (10), and while predominantly localized in the cytoplasm, they accumulate within the nucleus when cells are treated with Leptomycine B, a drug that prevents nuclear export mediated by the export receptor Exportin1 (11). The three members of this RNA-binding protein family have been shown to associate with messenger RiboNucleoParticules (mRNP) derived from heavy sedimenting polyribosomes, suggesting that these proteins play a role in the transport and/or translation of mRNA (12–14). Indeed, recent results from Laggerbauer et al. (15) and Li et al. (16) point to the possibility that FMRP may act as a negative regulator of translation, at least in an in vitro system.
FXR1P is widely distributed in human and mouse tissues (14,17,18), and its expression pattern is complex. Using antibodies to different domains of the protein, we have described six distinct isoforms of 70, 74, 78, 80, 82 and 84 kDa (19). In agreement with these observations, Kirkpatrick et al. (20) have observed seven mRNA variants resulting from alternative splicing of the FXR1 primary transcript. Some of these variants differ by three to six nucleotides, making it impossible to distinguish by SDS–PAGE the corresponding proteins that vary only by one or two amino acids. Whereas the 70–80 kDa proteins have been detected in all tissues, albeit at different levels, in heart and skeletal muscles these isoforms are replaced by super-long proteins of 82 and 84 kDa (14,19). These shifts in apparent molecular weights are observed during in vitro differentiation of myocysts and transition of myoblast to myotubes.
During a study on the expression of the different FXR1 mRNA variants in the developing mouse, we observed that transcripts containing exon 15 were detected in RNA preparations from testis of adult mice. This exon is an 81 bp insert that encodes 27 amino acids and has been isolated from muscle (19) as well as from heart RNA (20). Since these mRNA variants were reported to be muscle specific, this observation prompted us to investigate whether different FXR1P isoforms and their corresponding mRNA variants could be expressed during spermatogenesis. Here we report changes occurring in the distribution of FXR1 mRNA variants and their corresponding protein isoforms during spermatogenesis. We also show that FXR1P is localized to mature spermatocytes where it is found to be associated with microtubules elements.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Characterization of FXR1 mRNA variants and protein isoforms in testis
Previous comparative analyses of FXR1 expression have shown that high levels of mRNA were detected in muscle; however, these analyses were conducted on RNA obtained from a restricted number of human tissues (6). Other studies using a larger number of tissues from mouse showed that indeed high levels of FXR1 mRNA were found in muscle, but that the highest amounts were observed in testis (20,21). In the present study, we used poly(A)+ mRNA preparations from mouse liver, muscle and testis and determined FXR1 mRNA levels using a full-length FXR1 cDNA probe (9). Northern blot analyses showed that low levels of FXR1 mRNA were detected in liver preparations, whereas the highest signals were observed in testis (Fig. 1A). In addition, in liver and muscle preparations, two distinct RNA species of 3.2 and 2.4 kb of relative equivalent intensities were observed as previously reported (6,12,20,21). In contrast, the predominant signal detected in testis was at 2.4 kb whereas trace amounts of the 3.2 kb could be revealed only after prolonged exposure of the films. We have determined that both 3.2 and 2.4 kb species contain different combinations of RNA variants (+/– exons 14–17) necessary to synthesize all the FXR1P isoforms. The only difference between these two RNA species resides in the presence of a long 3'-UTR that was cloned, sequenced and shown to be composed of 613 nt (manuscript in preparation).
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To determine whether transcripts containing exon 15 were present in RNA extracted from testis, membranes carrying 2 µg of size fractionated poly(A)+ mRNA from liver, muscle and testis were probed with the 81 bp stretch. The results showed that no hybridization signals could be detected in liver RNA. On the other hand, hybridization signals were observed for both muscle 3.2 and 2.4 kb mRNA whereas only a single band at 2.4 kb was present in testis RNA (Fig. 1A) indicating that mRNA variants containing exon 15 were also present in testis RNA. To confirm these results, we next determined the distribution of the different FXR1 isoforms present in the same tissues by immunoblot analyses using different antibodies. mAb3FX specific to an epitope present in all isoforms confirmed that whereas P70, P74, P78 and P80 were detected in liver preparations, only the super-long P82 and P84 were present in muscle as previously shown (19). In addition to the four standard proteins of 70–80 kDa, testis also contains P82 and P84 (Fig. 1B) in agreement with the results obtained at the RNA level (Fig. 1A). These results indicate that when compared to the other tissues previously studied (14), testis makes exception in expressing all FXR1P isoforms.
Modulation of FXR1 expression during testis maturation
Our initial comparative protein analyses of FXR1P isoforms in testis between suckling new born (10 days after birth) and adult mice showed subtle differences in FXR1P isoforms distribution. These results prompted us to analyze in detail FXR1 gene expression during testis maturation. Protein extracts were prepared from testis 6, 8, 10, 12, 14, 16, 18 and 20 days after birth and the distribution of the different isoforms were analyzed by immunoblotting using different antibodies to FXR1P. These periods of development were chosen following Bellvé et al.’s (22) chart describing the different cell populations at equilibrium during the first wave of spermatogenesis.
Time-course analyses with mAb3FX showed that FXR1P70, FXR1P74, FXR1P78 and FXR1P80 were all present in testis regardless of the developmental stage; however, their levels increased steadily after day 14 to reach a plateau by day 20 (data not shown). A clearer picture was obtained using serum 830, since this antibody reacts with a less complex pattern of isoforms (Fig. 2A). Increased P78 and P80 levels were clearly detected by day 14, and this was confirmed after reaction with antibody 27–15 (Fig. 2A). As internal control, we used the ribosomal protein L7 whose levels remained relatively constant from day 6 to adulthood.
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To determine whether the appearance of the super-long FXR1P82 and FXR1P84 isoforms corresponded to the expression of newly spliced mRNA variants, total RNA was prepared from testis at the same stages used for protein analyses. Using primers lying in exons 14 and 17, semi quantitative RT–PCR analyses were performed using only 15 amplification cycles. The results of such analyses confirmed that a fragment of 417 bp containing exon 15 became clearly detectable by day 14, whereas trace amounts were present at day 12 (Fig. 2B). Other amplicons of 249 and 336 bp corresponding to the mRNA specific for P70 and P78 isoforms were also detected and their levels increased by day 14 in agreement with the results obtained at the protein level. Each DNA fragment was then sequenced and the results confirmed the presence or absence of exon 15 in the different variants. As an internal control, PCR amplification of testis RNA using FMR1-specific primers resulted in a 714 bp amplicon, the level of which remained constant throughout the time-course analysis (Fig. 2B).
Immunolocalization of FXR1P in testis of young and adult mice
To localize the distribution of FXR1P in the heterogeneous cell population present in testis, we performed immunohistological staining of this organ at different stages of mouse development. A low level of staining was observed in testis sections from 6-day-old young animals. At this age, primitive type A spermatogonia that account for 
16% of total cells in the semineferous epithelium, are attached to the basement membrane (22). In cross-sections, FXR1P staining was restricted to the primitive spermatogonia (Fig. 3). At day 14, an increase in staining was observed predominantly towards the center area containing primary spermatocytes at the early pachytene stage. Finally, in 20-day-old and adult testes, positive cytoplasmic signals were observed in almost all cells ranging from pachytene to round spermatids. Surprisingly, in testis from adult mice, strong staining was also present in the center of the lumens where spread bundles of flagella from spermatids ready to be released (stage VIII, step 16 of the maturation cycle; 23) were detected. This staining was related to the asynchronous epithelium cycle of the seminiferous tubules, and different levels of positive reactions in the lumen were observed. Also, no staining was observed in lumens devoid of mature spermatozoa (Fig. 3). Such a distribution of FXR1P might have escaped detection in previous studies (17), perhaps due to the restricted number of tubules and maturing sperm cells observed. A more convincing image was obtained at higher magnification, showing the staining in the principal piece of the sperm tails (Fig. 3).
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In an attempt to determine which cell type present in testis expressed P82 and P84, cells at different meiotic and mitotic stages were separated by fluorescence-activated cell sorting (FACS) according to their DNA content (24). Spermatogonia, leptotene+zygotene, pachytene cells, secondary spermatocytes and spermatids were isolated and protein extracts were analyzed. P78 and P80, as well as P70 and P74 (data not shown), were detected in all cell populations. Whereas only these four isoforms were present in spermatogonia and primary spermatocytes at their early stages in the prophase of meiosis I, cells at later stages (pachytene) and those undergoing meiotic reduction, expressed in addition the super-long P82 and P84 isoforms (Fig. 4).
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To determine whether in testis FXR1Ps are also associated with mRNP as seen in cells grown in culture as well as in brain (12,19,25), cytoplasmic extracts were analyzed by sedimentation through sucrose density gradients and each collected fraction was subjected to immunoblot analysis using antibody 830. Whereas FXR1P co-fractionated with polyribosomes in the presence of MgCl2, further evidence that it was associated with mRNPs was obtained after dissociation of the polyribosomes with EDTA, yielding slower sedimenting structures (Fig. 5A and B).
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FXR1Ps are present in sperm flagella and are associated with microtubules
The results presented above strongly suggested that FXR1Ps are present in the flagella. To further identify the isoforms present in these structures, mature spermatozoa obtained from the epididymis of adult mice were collected and concentrated by sedimentation after two cycles of washing with phosphate buffered saline (PBS). Purified spermatozoa were either directly lysed in SDS-buffer sample or sonicated to separate the heads from the tails following centrifugation on Percoll pads. The distribution of the outer dense fiber protein Odf84 (Odf2), a protein marker of the tail (26) indicated that tails were recovered above the Percoll interface. Immunoblot analyses for P82 and P84 clearly showed that these isoforms are present in the flagellum (Fig. 6). Using different antibodies to the FXR family members, the presence of FXR2P was also detected but not that of FMRP (data not shown).
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In further analyses, we concentrated on FXR1P82–FXR1P84. Immunofluorescence studies on fixed spermatozoa with sera 27–17 (or 27–15), both specific to P82 and P84, showed a regular but punctuated staining along the flagellum including the middle and the principal pieces (Fig. 7B). Furthermore, the immunogold labeling provided much greater resolution since gold deposits were localized along the microtubules of the axoneme (Fig. 7C). This was also demonstrated by pre-embedding immunolabeling of P82 and P84 after treatment of the spermatozoa with non-ionic detergents which induced the release of bundles of microtubule structures (Fig. 7D).
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Finally, we used a biochemical method to isolate polymerized microtubules and their associated proteins that was first devised for brain microtubules (27). Testis extracts were subjected to two cycles of temperature-dependent polymerization and depolymerization in the presence of 1 mM GTP, and an enriched fraction of microtubules was obtained (Fig. 8). Immunoblot analyses with anti-tubulin mAbE7 showed indeed an enrichment of tubulin. Reaction of the same blot with antiserum 27–17 (and 27–15), showed the presence of P82 and P84, indicating that these proteins can be purified along with microtubular structures. Further analyses are required to determine the stochiometry of FXR1P and tubulin subunits in microtubules.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fragile X-related 1 protein belongs to the FXR family of RNA-binding proteins that are constituents of mRNPs. Due to complex alternative splicing of the FXR1 primary transcript, several tissue-specific mRNA variants have been reported (20).
In this study, we present evidence that the highest expression of FXR1 in mouse is found in testis. We also show that this organ differs from the other tissues tested in expressing all known FXR1P isoforms. Since FMRP has not been detected in maturing germ cells in human and mouse (17,28), it is possible that FXR1P replaces FMRP for an as yet unknown specific function in these cells. It is worth mentioning that Coy et al. (6) have observed a pattern of expression of FXR1 mRNA in post-meiotic sperm cells by in situ hybridization analyses. However, the cDNA probe used by these authors did not allow discrimination between the different mRNA variants, since no alternative splicing of FXR1 transcript was known at that time.
FXR1P82 and FXR1P84, as well as their corresponding transcripts, are detected in 14-day-old mice testes at a time when the bulk of transcription occurs, while germ cells are at the pachytene stage of the first prophase of meiosis. Although some of the RNA is translated at that stage and de novo protein synthesis is required during meiosis, other repressed mRNA are stocked until germ cells become haploid and differentiate into early elongated spermatids (29). It is difficult a priori to conceive that FXR1P, an RNA-binding protein known to shuttle between the nucleus and the cytoplasm in cells maintained in culture (11,14,30), can be found in a structure such as the flagellum of spermatozoa. However, the case of FXR1P seems not to be unique since it is reminiscent of the spermatid perinuclear ribonucleic acid-binding protein (Spnr). This RNA-binding protein is associated with the manchette, a spermatid-specific microtubular array, and is thought to be involved in the transport or translation of selected mRNAs (31,32). Spnr was shown to move along the manchette and was detected further onto the flagellar axoneme during spermatids maturation (31). Our observation that FXR1P isoforms are present in the sperm tail leads to more questions than answers. Whereas it is possible that FXR1P plays a role in differentiation of spermatozoa as part of mRNPs that are required during spermiogenesis, its presence within the axoneme is puzzling. Since gene transcription in the haploid genome of the spermatids ceases as cells are still developing and differentiating, translation is dependent on mRNAs that are possibly stored on the cytoskeletal structure. While these mRNAs are translated, FXR1P might remain associated with the microtubule arrays in the manchette, and once this is done, it ends up within the elongating axoneme as mature spermatozoa. Therefore, it is possible that the presence of FXR1P in the axoneme is a ‘relic’, due to an exceptional stability and affinity to microtubules.
The presence of FXR1P in myocontractile bands in striated muscle (14,19) and in sperm flagella (this report), in addition to their ability to co-purify with microtubules, strongly suggests that these proteins might be implicated in structures involved in movement/motility that are energy dependent. Transfer and active transport of mRNPs and proteins are also energy dependent (33–35). Interestingly, Schenck et al. (36) recently reported the identification of two novel FXR interactors. These proteins, CYFIP1 and CYFIP2 (for Cytoplasmic FMRP Interacting Proteins), have been found to interact with the small GTPase Rac1 (37), which in turn plays a role in the dynamic reorganization of the actin cytoskeleton (38). It has also been proposed that GTPases Rac and Rho play roles in the maintenance of neuron dendritic spines and branches (39). Indeed, FXR1P contains the sequence 456-GGPRGGKS-463 that is an ATP/GTP consensus binding-site motif, supporting the notion that it might interact with complexes involved in energy transfer and/or consumption. Work is in progress to determine whether this domain is functional in FXR1P.
Interactions of RNA-binding proteins with microtubules have been documented among others, for the Drosophila and mammalian Staufen (40–43), murine protamine RNA-binding protein (PRBP, Prm-1) (44), and TB-RBP/translin (45–48). Such associations have been suggested to be required for the long distance travel of RNA in oocytes as well as in neurons (49,50). This could also be the case for FMRP that has been detected in the synaptosomes at distant locations from the neuron body (51). Based on our observations that FXR1P (this report), as well as FMRP and FXR2P (M.-E.Huot and E.W.Khandjian, unpublished data), co-purify with polymerized tubulin, we suggest that the FXR protein members, as RNA transporters or ‘chaperons’, are associated with the cytoskeletal framework. Since these proteins do not harbor any canonical tubulin-binding domain (TBD), as is the case for Stauffen (42), we propose that the FXR proteins interact with yet unknown partners that in turn are microtubule-associated proteins.
FXR1 is widely expressed in mouse. Due to the multiple encoded protein isoforms, it is not known which specific proteins might be required to control different functions. Also, it is not known whether mutations or gene defects could induce any abnormalities such as neurological, spermatogenical or others as seen in mice deficient for Spnr (52) or in male infertility (53).
In a more general context, it is accepted that the three members of the FXR RNA-binding protein family might interact with each other in the same mRNP complexes (7,36,54,55). However, the possibility that FXR1P and FXR2P could partially compensate for the absence of FMRP in the fragile X syndrome is still an open question. In the case of the absence of FMRP, the transport process of these mRNP complexes, regardless of their mRNA content, to distal locations in neurons, might be less efficient leading to reduced and/or incomplete local stocking of mRNAs. Since synaptosomes, which are far distant from the neuronal cell soma, contain FMR1 mRNA that is rapidly translated into FMRP in response to neurotransmitters stimulation (51), its absence might induce alteration in local protein synthesis required for the remodeling of synaptic connectivity (56). This might induce abnormal spine maturation (57,58) leading to higher cognitive dysfunctions. As a working hypothesis and based on the fact that the functional RNA-binding hierarchy of the three FXR members is still unknown, we speculate that the long-distance translocation of mRNPs in neurons requires the presence of all FXR members for optimal transport and translation of mRNAs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Animals
Male CD-1 mice at different ages were obtained from Charles River Canada. Animals were killed by asphyxia in a CO2 chamber in agreement with the recommendations of the Canadian Council on Animal Care.
Protein studies
Immunoblot analyses. Organs were removed from animals and processed according to our standard protocols and protein extracts were prepared for SDS–PAGE as described (59). Immunoblot analyses were performed using mAb3FX and antisera 830, 27–17 and 27–15 to FXR1P at 1:2000, 1:25 000, 1:5000 and 1:2500 dilutions, respectively. The specificity of these antibodies was reported elsewhere (14,19). Other antibodies and sera used were: anti-ß-tubulin mAbE7 (obtained from the Developmental Studies Hybridoma Bank University of Iowa), anti-Odf2 against the outer dense fiber protein (from F.A.van der Hoorn) (26), mAb1C3 against FMRP (28), mAb42A against FXR2P (from G.Dreyfuss) (7), and anti-L7 ribosomal protein (from A.Ziemiecki) (60). Detection of bound antibodies was performed with HRP-coupled secondary antibodies followed by ECL reaction as described (61).
Purification of microtubules. Microtubules and associated proteins were purified by two cycles of temperature-dependent assembly and disassembly in the presence of GTP as described (27) with the exception that testis of adult mice were used instead of brain. After two cycles of polymerization/depolymerization, microtubule extracts were denatured in SDS-sample buffer and used for immunoblot analyses after SDS–PAGE. Protein concentration was determined using the Bradford method after TCA precipitation.
Polyribosome analyses. Testes from adult CD-1 mice were rapidly removed from animals and finely minced in PBS buffer. The fragments were homogeneized in a solution containing 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 100 mM NaF, 10 ng/ml aprotinin, 1 mM phenylmethyl-sulfonylfluoride, 1 mM DTT, 10 U/ml RNasin (Pharmacia) and 1% IGEPAL and subjected to sucrose density ultracentrifugation as described (61). Each collected fraction was denatured with a 3-fold concentrated SDS-lysis buffer and subjected to immunoblot analysis using the Gibco BRL Convertible Filtration Manifold System.
RNA studies
Northern blot analysis. Total RNA was extracted using the Trizol reagents according to the manufacturer’s protocol (Gibco). Poly(A)+ RNA from different mouse tissues was purchased from Clontech. Northern blot analyses were performed as described (63) with the exception that RNA was transferred to nylon membranes by vacuum blotting. 32P-labeled probes were prepared by random-priming and used at 106 c.p.m./ml of hybridization solution.
Reverse transcription and PCR. RT was performed on 0.2 µg of total RNA prepared from testis using reverse primers (see below). For FXR1 mRNA variants, one fifth of the resulting reaction was used to perform a PCR with the following oligonucleotides: 5'-CGTCGTAGGCGGTCTCGTAG-3' (forward) and 5'-ACCATTCAGGACTGCTGCTT-3' (reverse). For FMR1 we used the primers: 5'-GCCTTGCTGTTGGTGGTTAGC-3' (forward) and 5'-CACAACTCCTGACTTGTCCACGAT-3' (reverse). PCR was performed initially by denaturation at 95°C for 5 min, followed by 15 cycles of denaturation at 95°C for 30 s, annealing at 66°C for 30 s, extension at 72°C for 1 min and a final extension step at 72°C for 5 min. Amplified DNA fragments were fractionated on 1.5% agarose gels, stained with ethidium bromide, eluted and sequenced to validate the RNA variants.
Germ cell isolation
Germ cells at different differentiation stages (spermatogonia, spermatocytes and round spermatids) were obtained from adult (90 days) CD-1 mice. The preparation and isolation of the different cells was based on the DNA content of the different germ cell populations using FACS according to Mays-Hoopes et al. (24).
Spermatozoa studies
Protein studies. An incision was made on the cauda epididymis of CD-1 adult mice (90 days old) and low pressure was applied with forceps on the epididymis to expel spermatozoa. The cells were resuspended in PBS for 30 min at 37°C, then washed twice by centrifugation at room temperature (20 min, 150 g). The final pellet was resuspended in HBS (10 mM HEPES pH 7.2, 150 mM NaCl).
To determine whether FXR1P was present in the flagellum, washed spermatozoa were resuspended in HBS supplemented with protease inhibitors (500 µM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A) and sonicated for 5 s on ice (64). Heads and flagella fragments were then separated by a 20 min centrifugation (700 g) at 4°C through a 90% Percoll layer in HBS. Flagella fragments were recovered at the surface of the Percoll layer whereas the heads were found in the pellet. The flagella fraction was centrifuged once more on a Percoll pad to ensure minimal contamination of the tails by the heads. The flagella were next washed in HBS and the proteins extracted in the presence of SDS-sample buffer.
Immunocytochemistry and indirect immunofluorescence
Testis were rapidly removed from animals and directly fixed in a freshly prepared 4% paraformaldehyde solution in PBS for 18 h at 4°C. After dehydration in ethanol series, tissues were embedded in paraffin. Five micron sections were processed for deparaffinization and for microwave oven treatments. Endogenous peroxidase was inhibited by a 60 min treatment with 0.5% H2O2 solution in PBS and the sections were next incubated for 18 h at 4°C with antiserum 830 (1:2000). Bound Ig was reacted with biotinylated secondary antibodies followed by a streptavidin-peroxidase conjugate and stained with the AEC chromogen (Histomouse-SP Kit; Zymed). The slides were counterstained with hematoxylin and mounted in GVA Mount (Zymed). Light microscopy was viewed with a Nikon TE300 microscope connected to a CoolSnap camera (RS Photometrics) using 10x, 40x and 60x objectives. Images were transferred to the Adobe PhotoShop program.
Washed spermatozoa were resuspended in PBS and placed on a poly-L-lysine coated coverslip, fixed for 15 min in 4% paraformaldehyde in PBS, permeabilized for 10 min in 0.2% Triton X-100 in PBS, and rinsed with PBS. Non-specific sites were blocked with PBS supplemented with 1% bovine serum albumin (BSA). Samples were then incubated for 1 h at 37°C with the different antisera diluted in PBS supplemented with 0.1% BSA, rinsed with PBS, and stained with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG antibodies. The antisera used were 830, 27–17 and 27–15, diluted 1:200, 1:100 and 1:50, respectively. Rabbit IgG (10 µg/ml) was used as a negative control. Samples were mounted in PBS–glycerol containing 0.1% diazabicyclo[2,2,2]octane as anti-bleaching agent. Fluorescent and light microscopy was viewed with a Nikon TE300 microscope equipped for epifluorescence and phase-contrast microscopy, using a 100x oil immersion objective.
Electron microscopy studies
Purified spermatozoa, concentrated after low-speed centrifugation, were fixed in 4% paraformaldehyde solution in PBS for 18 h at 4°C and then embedded in LR-White resin (London Resin Co.). Ultrathin sections (70–90 nm) on formvar-coated gold grids were preblocked in PBS containing 2% normal rabbit serum and 0.1% Tween 20 and reacted for 2 h with the primary antibodies. Sections were then incubated with secondary antibodies conjugated to 15 nm gold particles (BBI). Sections were stained with uranylacetate and lead nitrate and examined in a JEOL 1200EX electron microscope at 80 kV. For pre-embedding studies, purified spermatozoa in PBS were permeabilized twice with 0.2% Triton, for 20 min at room temperature, washed twice and reacted with the primary antibodies and subsequently reacted with the gold-labeled secondary antibodies. After extensive washing, the material was fixed in 4% paraformaldehyde solution in PBS for 18 h at 4°C, followed by osmium impregnation in Na-Cacodylate buffer and embedding in PolyBed 812 epoxy (Polyscience). Sections were counterstained with uranylacetate and lead nitrate before examination.
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ACKNOWLEDGEMENTS |
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We thank Jacquetta Trasler, Frans van der Hoorns, Jean-Marc Matter and Robert Bauchwitz for helpful discussions, Yves Labelle and Paul H.Naccache for critical reading of the manuscript and Dominique Heitz for her help in the RNA studies. Thanks are also due to Helène Chamberland and Aristide Pusterla from the Electron Microscopy Core Facilities for their precious help, and Sylvie Giroux for her initial observation of flagella staining. We are grateful to Barbara Bardoni, Jean-Lou Mandel, Gideon Dreyfuss, Frans van der Hoorns and Andrew Ziemiecki for providing antibodies. This work was supported mainly by the Natural Sciences and Engineering Research Council of Canada and in part by the Fragile X Research Foundation of Canada. M.E.H. holds a scholarship from the Canadian Institutes of Health Research and R.M. is a recipient of a postdoctoral fellowship from the Fragile X Research Foundation of Canada/Canadian Institutes of Health Research Partnership Challenge Fund program. P.L. is a Scholar of the Fonds de la Recherche en Santé du Québec.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 418 525 4402; Fax: +1 418 525 4195; Email: edward.khandjian@crsfa.ulaval.ca
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REFERENCES |
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