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Gamete Biology |
a INSERM U.567 CNRS-UMR 8104, Institut Cochin, Departement d'Hematologie, Maternité de Port-Royal, Université Rene Descartes, 75014 Paris, France
b GERM-INSERM U. 435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, Bretagne, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, meiosis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human and mouse PHTF1 mRNAs were detected in all the tissues analyzed, but their levels were remarkably high in the testis [1]. Spermatogenesis is a complex differentiation program leading to development of highly specialized haploid male gametes from primordial diploid cells. This differentiation program starts with the formation of spermatogonia from germ line stem cells followed by mitotic divisions to produce primary spermatocytes. Primary spermatocytes then undergo meiotic division to give rise eventually to haploid spermatid cells. Spermatids undergo extensive morphological changes, a phenomenon known as spermiogenesis, to produce spermatozoa. The essential morphological events consistently described during spermiogenesis are the condensation of the nucleus, the construction of the acrosome and flagellum, and the elimination of the residual cytoplasm [3]. These changes, which lead to profound modifications of preexistent structures or accompany the formation of new organelles, have been clearly characterized at the electron microscopy level, but their biochemistry still remains largely obscure.
The endoplasmic reticulum undergoes noticeable transformations during spermiogenesis and becomes closely associated with several components of the cell as they are formed or being transformed [3, 4]. These de novo structures include a network of tubular cisternae located along the convex surface of the Golgi apparatus, a fenestrated sleeve in association with the manchete, another closely associated to the flagellum, an extensive and conspicuous network located close to the plasma membrane, and a radial body. [3, 5].
The Golgi apparatus also undergoes singular transformations, the best known of which are related to the acrosomic system [6]. During the formation of the acrosome, the Golgi changes its orientation and adopts a cis-trans configuration facing the nucleus. This new configuration puts the trans-Golgi in close proximity to the nucleus and could permit direct vesicular flow from the Golgi to the acrosomal vesicle. [7, 8]. When the addition of material to the acrosomic system terminates, the Golgi apparatus migrates toward the opposite pole of the cell. Most of the Golgi stacks are seen in the residual body eliminated by the mature spermatid [6], but some remnants are still present in the cytoplasmic droplet of spermatozoa during epididymal maturation [7].
We report here the first attempt to investigate phtf1 expression. The protein identifies specific ER domains in meiotic and postmeiotic germinal cells. Its localization is intimately linked to the changes undergone by the Golgi apparatus and the endoplasmic reticulum. Phtf1 could help us to understand the process of germ cell maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals used in these studies were maintained and killed according to the guidelines for the approval for animal experimentation. Sertoli cells and peritubular cells were isolated from 20-day-old Sprague Dawley rats as described previously with degrees of purity of 98% and >96%, respectively, [9, 10]. The isolation of Leydig cells and of testicular resident macrophages from 90-day-old rats was carried out during the same preparation as described previously [11], and the purity was >98% and >94%, respectively. Spermatogonia (purity 
94%) were prepared from 9-day-old Sprague Dawley rats using a protocol described by Bellve et al. [5]. Germ cells with a purity of greater than 90% were prepared from adult rat testes as described before [12] except that enzymatic dissociation of cells was replaced by a mechanical dispersion. Pachytene spermatocytes, round spermatids, and cytoplasmic fragments of elongated spermatids plus residual bodies (CES/RB) were prepared by centrifugal elutriation with a purity >90%.
RNA Analyses
For reverse transcription-polymerase chain reaction (RT-PCR) analyses, 5 µg of RNA were reverse transcribed as described in Cudicini et al. [13] and the reaction mixture brought to 100 µl. Each PCR was performed using 1 µl as follows: 94°C for 30 sec, 54°C (phtf1) or 57°C (actin) for 30 sec, and 72°C for 2 min and 35 cycles. The rat phtf1 oligonucleotides are reported on legends of figures, and the actin oligonucleotides were previously described [14]. Amplified products (289 base pairs [bp] for phtf1 and 1020-bp products for actin) were analyzed on agarose gels.
Plasmids and Transient Transfection of Cell Lines
A NotI-ScaI fragment comprising the entire coding sequence from the mouse phtf1 cDNA (EMBL/GENBANK AJ133721 and AJ242864) was cloned between the NotI-EcoR V sites of the pCDNA3-1 expression vector (Invitrogen). To generate the N-terminal myc-tagged protein (myc-phtf1), we mutated the first amino acid position of the protein from G
C (ATG corresponds to the first Met) to G
C, obtaining an EcoRV site. The six myc tags (BamHI-EcoRI blunted fragment from the pCS3-MT vector) were inserted at this site. The C-terminal tagged protein (phtf1-myc) was obtained by deleting the phtf1 sequence from the plasmid myc-phtf1 with EcoRI (by blunting this site, we created a stop codon after the 6-myc tag). The entire phtf1 sequence was then inserted at the ClaI site in the 5' sequence of the tag sequence.
Cell lines were maintained in Dulbecco modified minimal essential medium supplemented with 10% fetal calf serum. NIH3T3 and COS7 cells were transfected with 1 µg of plasmid per 10 mm well by using the Fugene reagent as recommended by suppliers (Roche, France).
Coupled Transcription-Translation and In Vitro Membrane Association
Coupled transcription-translation was performed in rabbit reticulate lysate (Promega) by using the plasmid phtf1-myc, with or without canine microsomal membranes (Promega). For membrane experiences, after translation, volume was adjusted to 750 µl with TBS (NaCl 150 mM, Tris 10 mM, pH 7.4), and microsomes were pelleted by centrifugation for 30 min at 150 000 x g in a TL80.4 rotor. For carbonate extraction, pellets were suspended in ice-cold 0.1 M sodium carbonate, pH 11 [15]. For Triton X-114 extraction [16], membranes were solubilized in 1% Triton X-114 for 60 min at 4°C, phase separation was induced at 37°C, and centrifugation was done for 5 min at 13 000 rpm in a microfuge. TBS and carbonate pellets were washed three times and Triton phase five times. All the corresponding supernatant fractions were pooled and acetone precipitated.
Subcellular Fractionation of Germ Cells
The different fractions of cells (spermatogonia, spermatocytes, round spermatids, and CES/RB) were prepared as described by Goodwin [17]. Briefly, cells were suspended at a cell density of 10–30 x 106 in 1 ml of buffer A containing 20 mM HEPES, pH 7.5, 1 mM EDTA, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride (25 µg/ml), aprotinin (1 µg/ml), and leupeptin (10 µg/ml), and 10 µM E-64. The cell suspension was homogenized in a glass Dounce homogenizer and then centrifuged at 2000 x g to pellet the nuclei. The postnuclear supernatant was further centrifuged at 105 000 x g to pellet the microsomal membranes while the supernatant was collected for the cytosolic fraction. The whole cells and pellets of microsomal membranes were suspended in buffer A (1 ml). Fifty micrograms of total protein was then mixed in 2x Laemly buffer, boiled, and loaded in an 8% SDS-PAGE gel.
Antibodies
The region between amino acid residues 351 and 454 of the predicted mouse phtf1 protein was inserted into a pRSET-derived (Invitrogen) his-tagged vector. The corresponding recombinant protein was expressed in bacteria and was affinity purified (Xpress system protein expression pRSET; Invitrogen) to raise a polyclonal antiserum, mp71, in rabbit (Eurogentec).
Dr. Bunick (University of Pennsylvania) generously donated the monoclonal 1C9 anti-calmegin antibody [18]. Antibodies against ß-cop, TGN 38, 
-adaptin, and ß-adaptin were a gift of Dr. Benichou (Institut Cochin, Paris, France) and were purchased from Affinity Bioreagents. Antibodies against p58 were purchased from Sigma (France).
Western Blot Analyses
Protein extracts were separated on 8% acrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham). Membranes were incubated in 10 mM Tris-Hcl, pH 7.4; 150 mM NaCl, 20% Tween-20 (TBST), and 2% BSA at room temperature for 2 h. The rabbit polyclonal mp71 antiserum was diluted 1/500 and peroxidase conjugated donkey anti-rabbit IgG (or the donkey anti-mouse) (Promega) diluted 1/2500 in TBST-BSA. The commercial monoclonal antibody 9E10 (Roche) was diluted 1/2500 and peroxidase conjugated donkey anti-mouse IgG (Promega) diluted 1/2500 in TBST-BSA. Membranes were subjected to chemiluminescent detection using an enhanced chemiluminescence Western blotting detection kit (Amersham). Blocking mp71 antiserum was achieved using 5 µg of the purified mouse phtf1 corresponding to amino acids 351–454 protein. Bound antibodies were subsequently removed using 10 µl Ni-NTA magnetic agarose beads (Qiagen, France).
Immunohistochemistry
For the immunohistochemical analysis, paraffin sections of Bouin-fixed rat testis were mounted on glass slides. Sections were deparaffinized, rehydrated, and antigens unmasked by using a pressure cooker (2 min in 0.01 M sodium citrate buffer, pH 6, at high pressure and 15 min of cooling under tap water). Sections were incubated for 60 min with a 2% blocking solution goat serum, 1% BSA in PBS, followed by incubation with mp71 at 1:100 dilution for 60 min. For peroxidase staining, we used an anti-rabbit avidin and biotinylated horseradish peroxidase (ABC) staining kit (Vector Laboratories, Santa Cruz, CA) and diamino benzidine (DAB) (Sigma).
For immunofluorescence analysis, testicular sections were processed as previously described except that the secondary antibody was replaced by commercially anti-rabbit tetramethyl rhodamine isothiocyanate (TRITC)-conjugated or anti-mouse fluorescein isothiocyanate (FITC)-conjugated. Anti-ß-cop was used at 1:50, anti-TGN 38 and - or ß-adaptin were used at 1:100 dilution, and anti-p-58 was used at 1:200. Confocal images were acquired with a MRC-1000 confocal microscope (Bio-Rad) and processed in Adobe Photoshop.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blot studies have previously shown that phtf1 mRNA is abundantly present in testicular tissues [1]. We extended our analysis to identify the cells expressing phtf1. Because of the possibility of obtaining large and pure populations of testicular cells from rat, we performed these experiments on this model. The high phtf1 conservation (98% human-mouse) [1, 2] permitted generating a partial rat phtf1 cDNA (AJ 437403) and designing rat-specific primers. This phtf1 fragment represents the rat ortholog, as it harbors 97% sequence homology and 100% amino acid identity. The seminiferous epithelium of mammalian testis undergoes extensive postnatal development in a characterized sequence of germ differentiation that continues cyclically throughout adulthood. The first wave of spermatogenesis in the rat involves the appearance of pachytene spermatocytes at 15–18 days postpartum (dpp), of haploid spermatids at about 22 dpp, and the first release of mature spermatozoa around 45 dpp; the rat is considered to be adult after about 70 days. The onset of gene expression during this period can be used to predict the pattern of type-specific germ cell expression [19, 20]. RT-PCR analysis detected a specific 289 bp in RNA from all testis samples, but a marked rise in its expression occurred about 20 days after birth (Fig. 1A) that correlated with the onset of meiosis. The presence of a faint band at 9 days old indicated that there was some level of expression in other testicular cells.
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To identify exactly which testicular cells were expressing phtf1, RNA from myoid peritubular cells, Leydig cells, Sertoli cells, testicular macrophages, spermatogonia, spermatocytes, and spermatids were subjected to RT-PCR. Using this technique in three independent experiments, the specific phtf1 band appeared more abundant (at least 20-fold greater when quantified by a PhosphorImager) in pachytene spermatocytes and round spermatids than in the somatic cell populations (Fig. 1B). No signal was visible in the spermatogonia (Fig. 1B). These findings suggest that phtf1 gene expression is associated with germinal cell maturation. The presence of a very faint signal in the somatic cells probably explains the low levels of phtf1 mRNA detected in 9-day-old rats (Fig. 1A).
Phtf1 Is an Integral Membrane Protein Present from Meiotic Spermatocytes to the End of Spermiogenesis
To extend our RNA analysis at the protein level, we raised a rabbit polyclonal antiserum against a specific phtf1 region (see Materials and Methods). The specificity of the phtf1 antibody, mp71, was established by Western blot analysis of the total proteins extracted from COS cells transfected with a myc-mouse phtf1 expression vector. As expected, the 9E10 anti-myc monoclonal antibody revealed an 89-kDa immunoreactive band in protein extracts (Fig. 2A). The same band was observed in extracts from transfected cells when using the mp71 antibody (Fig. 2A). The 89-kDa band was specific because it was blocked when the mp71 antibody was preincubated with the purified protein preparation used as immunogen. Because the region used to raise the polyclonal antibody was highly conserved (96% amino acids identity between mouse and human), we used it to reveal phtf1 in rat.
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In order to correlate the RT-PCR data with protein expression, we prepared extracts from somatic cells (peritubular and Sertoli cells), germ cells (spermatogonia, spermatocytes, and spermatids), and CES/RB and analyzed them by Western blot with the mp71 antiserum. No immunoreactive band was present in Sertoli or peritubular cells (Fig. 2B). In germ cells, the 83-kDa phtf1 band was observed in pachytene spermatocytes, and it was dramatically increased (>70-fold) in spermatids. It was still present at very high levels in CES/RB (Fig. 2B). Two further lower bands (70 and 51 kDa) were detected in CES/RB and could correspond to protein degradation.
Cell fractionation was then used to assess the cellular compartmentalization of phtf1 in germ cells. Spermatogonia, spermatocytes, early spermatids, and CES/RB were fractionated by Dounce homogenization and, after separation of the nuclei and unbroken cells, centrifuged to obtain a high-speed pellet consisting mainly of membranes (CM, microsomal fraction) and a cytosolic fraction (CY). Most of the phtf1 was found in the microsomal fraction (Fig. 2B), indicating that it was associated with a cell membrane. As the predicted primary sequence did not contain a signal peptide, we wanted to find out whether phtf1 was simply associated with the membrane or was actually incorporated into it. Rabbit reticulocyte coupled transcription/translation was performed with a plasmid coding for a fusion mouse phtf1-myc in the presence of canine microsomal membranes. Microsomes were then isolated by centrifuging, and phtf1 was found in the pellet, indicating that it was associated with the lipid membrane fraction (Fig. 2C). When the membrane fraction was extracted using alkaline carbonate buffer (0.1 M, pH 11), phtf1 remained in the pellet (Fig. 2C). When microsomes were solubilized by using the detergent Triton X-114, phtf1 partitioned with the detergent phase (Fig. 2C). Taken together, these findings indicate phtf1 is an integral membrane protein.
Developmental Stage-Specific Expression of Phtf1
To identify the stage at which the cells express phtf1, immunohistochemical studies of adult rat testicular sections were performed using mp71 antibody and horseradish peroxidase. Typical sections after immunocytochemical staining of phtf1 with the mp71 antibody are shown in Figure 3. Somatic cells (peritubular, Leydig, and Sertoli cells) did not exhibit any detectable signal, whereas some germ cell categories were strongly stained in every seminiferous tubule (Fig. 3b). No reactivity was ever found in the spermatogonia. Specific immunolabeling was first detected in primary spermatocytes as a very faint signal close to the nuclei at all stages where this germ cell category is present (Fig. 3, c to f, and the enlargement in the inner figures). Such labeled vesicles were also observed in step 1–2 spermatids (Fig. 3c). The labeling progressively extended to the cytoplasm of early spermatids (Fig. 3d) and then gained in intensity (Fig. 3e). It was intense from step 4 spermatid to the end of spermiogenesis (Fig. 3, b and d). Staining was finally associated with the CES/RB (Fig. 3b). The specificity of this labeling was ascertained by using blocked serum (Fig. 3a). Figure 3g summarizes phtf1 expression in germ cells during the development cycle of the rat seminiferous epithelium.
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Phtf1 Localizes at the Endoplasmic Reticulum and the Peripheral cis Face of the Golgi
To assess the subcellular localization of phtf1, an expression vector containing myc-tagged phtf1 was transfected into NIH 3T3 cells. The protein appeared as a fine reticular network extending throughout the cytoplasm (Fig. 4a), and as it was reminiscent of that displayed by endoplasmic reticulum (ER) markers, we checked for possible colocalization with the ER resident chaperone calnexin. Both proteins displayed the same localization (Fig. 4, a to c), indicating that phtf1 was synthesized in the ER.
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Because the mp71 antibody was not suitable for electron microscopy studies, we performed double-labeling experiments in rat testis sections using antibodies recognizing phtf1 and the testis ER chaperone calmegin [18]. Figure 4, d to m, shows the results obtained after confocal microscopy examination. Phtf1 labeling exactly matched that of calmegin in step 3–8 spermatids (Fig. 4, d to f) and early elongating spermatids (Fig. 4, g to i). However, the presence of phtf1 was not always restricted to the regions where calmegin was located. Phtf1 described a horseshoe-like region at one pole of the nucleus in pachytene spermatocytes (Fig. 4f, inner figure) and step 1–2 spermatids (Fig. 4m, inner figure), whereas calmegin covered the entire cell. Distribution of both proteins was the same during steps 3–9 of spermiogenesis, but calmegin appeared to be more concentrated in the region neighboring the nuclear membrane. By step 13–14, both calmegin and phtf1 intensively stained the late spermatid cytoplasmic lobe (Fig. 4, g to i). Calmegin-positive structures, putatively corresponding to condensing ER regions in proximity to the radial body [18, 21], also stained intensely for phtf1 (arrowheads in Fig. 4, g to j). Nevertheless, phtf1 evidenced a network throughout the cytoplasm that persisted in the cytoplasmic lobe of step 15–19 spermatids, whereas calmegin was no longer observed after step 14 (Fig. 4, k to m).
The position and structure of the perinuclear labeling observed in pachytene spermatocytes was suspected of corresponding to the Golgi apparatus. In order to check this, testis sections were double labeled with antibodies against TGN38 and against phtf1 (Fig. 5, a to f). Phtf1 did indeed predict the location of the Golgi apparatus, but it was evident that phtf1 and Golgi markers were identifying different structures. The mp71 antibody labeled structures situated between the nucleus and TGN of pachytene spermatocytes from stage III to XII (Fig. 5f, inside top). During the following stages and up to the end of the Golgi phase, the TGN38 labeling was superimposed over the phtf1 labeling. This pointed out the profound reorganizing process that culminated by the inversion of the position of the organelle. At this time, phtf1 occupied the cis elements position (Fig. 5f, inside bottom). The cytoplasmic lobes of the elongating spermatids did not exhibit any significant phtf1-TGN38 overlaps (Fig. 5c, inside top).
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To reveal a situation consistent with the cis-Golgi saccules, we stained the entire Golgi apparatus with antibodies against p58, a soluble protein that links the cytoskeleton to Golgi membranes (Fig. 5, g to l). Spermatids strongly expressing phtf1 showed significant overlapping with both phtf1 and p58 (Fig. 5i, inside right). Nevertheless, high magnification and thinner sections (0.15 µm) revealed that phtf1 labeling of pachytene spermatocytes and step 1–3 spermatids could be distinct from p58-positive structures. Phtf1 was mainly located at the cis side and sometimes seemed to be going through the bracket described by the Golgi marker (see arrowheads in Fig. 5l, inside).
To ascertain if phtf1 location corresponded to vesicles in transit between the Golgi elements, we probed rat testis slides with mp71 and anti-ß-COP antibodies. As described previously by Moreno et al. [8] and Hermo et al. [7], ß-COP vesicles were abundant between the Golgi apparatus and the expected acrosome region, and phtf1 labeling did not show any correspondence with this signal (data not shown). We addressed the possibility that phtf1 labeling in pachytene spermatocytes and Golgi phase spermatids could identify elements in transit between the Golgi system and the plasma membrane. Clathrin-coated vesicles are the most well-known carrier vesicles connecting both membranes. Clathrin associates with the membranes through aptamer complexes. AP1 complex is associated with Golgi-endosome clathrin-coated vesicles, and AP2 with endosome-plasma membrane [22]. We used an anti-ß-adaptin antibody to recognize both complexes. Aptamers labeling became stronger with meiosis progression, suggesting changes in transport activity. No significant overlapping between ß-adaptin and phtf1 was observed (Fig. 6, a to i). Phtf1 was generally observed as a cage-like structure surrounding aptamers (see Discussion). ß-Adaptin labeling reinforces the idea that phtf1 resides in a restricted ER or ER-Golgi membrane.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The findings of a previous homology analysis had suggested that phtf1 could belong to the homeobox gene family [1]. However, on the basis of the presence of 6–8 stretches of hydrophobic amino acid residues, phtf1 could also be a transmembrane protein [1]. We demonstrate here that phtf1 indeed has some of the characteristic properties of a membrane protein. First, most of it fractionated with the high-speed membrane pellet subcellular fraction of germinal cells, and in vitro-translated protein was translocated into the microsomal fraction when translation was performed in the presence of canine microsomal membranes. Furthermore, when microsomes were washed with an alkaline carbonate buffer, the protein remained in the pellet, indicating that phtf1 was not associated with a membrane component but rather inserted into the lipid bilayer. Finally, Triton X-114 extraction demonstrated that phtf1 partitioned as a hydrophobic integral membrane protein. Because phtf1 contains 6–8 stretches of hydrophobic amino acid residues, it could be a multipass transmembrane protein (class III). Immunostaining of phtf1-transfected cells was consistent with this conclusion, as the protein could be synthesized in the endoplasmic reticulum (see below).
In adult rat testicular sections, antibodies revealed that phtf1 first appeared in early rat pachytene spermatocytes in which the protein was restricted to a juxtanuclear region. By step 2–3 of spermiogenesis, the concentration of the protein had risen considerably and the reactivity extended to the entire cytoplasm. Phtf1 colocalized with the ER chaperone calmegin in step 3–8 spermatids and early elongating spermatids. However, we noticed also phtf1 in or in close proximity to the Golgi apparatus in meiotic and early spermatids. By using conjoint labeling of phtf1 with the Golgi-associated protein p58 and the TGN-resident protein TGN38, we deduced that phtf1 was linked to the cis face but was not part of the cis saccules of Golgi. Phtf1 was clearly not transported between Golgi saccules or between the Golgi and plasma membrane because its labeling was not related to ß-COP or aptamer-coated vesicles. Furthermore, phtf1 sometimes looked as if it was going through the bracket described by the Golgi complex. The cisternae of the ER have been shown to be closely applied to the convex outer surface of the Golgi, where they were found to be separated from the stacks of saccules by a region called the peripheral region [4]. This structural continuity between ER and Golgi was observed only on the cis face of the Golgi apparatus, but peripheral tubules have been shown crossing the gaps between the stacks of Golgi saccules in noncompact zones [4, 7]. As phtf1 labeling is reminiscent of these structures, we suggest that it may identify the peripheral Golgi region (Fig. 7A).
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Phtf1 in combination with several markers revealed the transformations undergone by the Golgi complex, changing from the typical flat-shaped organelle through the spherical form of meiotic spermatocytes to the final flattened but inverted Golgi of the cap-phase spermatids. It was proposed that the increased size of the Golgi apparatus of pachytene spermatocytes could be predominantly due to heightened secretory activity and/or to a mechanism of partitioning the Golgi complex [27]. We observed a remarkably high level of chlatrin AP1-AP2 transport in pachytene cells that argued in favor of enhanced secretory activity. Interestingly, the transporters were always found in the circular region delimited by phtf1. These findings suggest that the chlatrin vesicles could be engaged in a new traffic route. These transport changes in pachytene cells, together with the previous evidence of acrosin synthesis in spermatocytes [28], raise the possibility of a meiotic proacrosomal vesicle origin. A schematic representation of the relationship between phtf1 and the rough changes suffered by the Golgi apparatus is presented in Figure 7B. An increase in the concentration of phtf1 was concomitant with its extension over the entire ER, perfectly matching the calmegin labeling. This colocalization decreased during the acrosomal phase, when calmegin was concentrated in a specific region, probably around the radial body [29]. Both proteins strongly stained this region, but whereas calmegin had vanished by step 15–16, phtf1 continued to reveal a network in the cytoplasmic lobe of step 15–19 spermatids. Because protein synthesis has stopped by step 13–14 of spermiogenesis, chaperoning would no longer be necessary and the usual rough-ER apparatus would disappear. Phtf1 could identify a different ER domain or be addressed to another membranous organelle, as we have already described in the ER-Golgi region. Electron microscopy has shown that most of the ER has disappeared by step 15 [3, 4], but little information has emerged about the smooth ER or about the lamellar organelles present in the cytoplasmic lobe. Phtf1 would provide a new marker for following many of these obscure transformations undergone by the endoplasmic reticulum.
Phtf1 possesses an evolutionarily-conserved structure and also displays a conserved pattern of expression even in Drosophila (data not shown), which strongly suggests that this protein may play an important role in the differentiation of germinal cells. Its expression accompanies many still unknown transformations undergone by the Golgi apparatus and the endoplasmic reticulum. Thus, phtf1 would probably be of interest in investigating the origin, nature, and function of many of the de novo issued organelles during the transformation of the spermatic cell.
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ACKNOWLEDGMENTS |
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- and ß-adaptin antibodies. Special thanks are addressed to Florence Heutte for her assistance with the confocal microscopy. We acknowledge helpful discussions with P.H. Roméo. |
FOOTNOTES |
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2 Correspondence: Natacha Raich, INSERM U 567-UMR 8104, 123 Boulevard de Port-Royal, 75014 Paris, France. FAX: 33 0 1 43 25 11 67; e-mail: raich{at}infobiogen.fr
Received: 25 July 2002.
First decision: 20 August 2002.
Accepted: 3 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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