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Spats 1 (Srsp1) is differentially expressed during testis development of the rat
Gene Expression Patterns 10 (2010) 1–8
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Spats 1 (Srsp1) is differentially expressed during testis development of the rat Carlos A. Capoano a, Rodolfo Wettstein a, Alejandra Kun b,c, Adriana Geisinger a,c,* a
Department of Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay Department of Proteínas y Ácidos Nucleicos, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay c Sección Bioquímica, Facultad de Ciencias, Montevideo, Uruguay b
a r t i c l e
i n f o
Article history: Received 17 July 2009 Received in revised form 19 November 2009 Accepted 24 November 2009 Available online 3 December 2009 To the late Dr. Rodolfo Wettstein Keywords: Testis development Spermatogenesis Meiosis Spermatocytes Gonocytes Peritubular myoid cells Serine stretch Spats1 Srsp1
a b s t r a c t Spats1 encodes the first reported testis-specific protein containing a long serine stretch. Besides, it bears a probable bipartite nuclear localization signal. Here, we describe the expression pattern of Spats1 in rat along embryonic and postnatal testis development by immunoblots and confocal immunohistochemistry. Spats1 is first expressed in the embryo at 17.5 days post-coitum, coinciding with the time when gonocytes acquire a quiescent state. At this time expression is detected in peritubular myoid cells and gonocytes. Spats1 attains maximum levels during meiosis of the first spermatogenic wave, mainly in pachytene spermatocytes, while a lower signal is also observed in spermatogonia, Sertoli cells and myoid cells. Protein levels dramatically decay afterwards, with minimum expression in adult individuals, where no signal was detected in elongating spermatids or spermatozoa. Spats1 is mostly cytoplasmic, although in pachytene spermatocytes it mapped to nuclei as well. Alkaline phosphatase treatment showed that this protein would be highly phosphorylated. Moreover, we show that the protein is highly conserved along metazoan evolution. Our results suggest a role in the initiation of the first spermatogenic wave, and in the establishment or progression of the first male meiotic division. Ó 2009 Elsevier B.V. All rights reserved.
1. Results and discussion Testis is a relatively unique tissue because it is the site of a developmental process that continues through the entire life. Unfortunately, the developmental and differentiation programs that lead to the production of mammalian male germ cells are still poorly understood at the molecular level due to some testis intrinsic difficulties, mainly the great tissue heterogeneity. Spats1 (‘‘spermatogenesis associated, serine-rich 1”; also called Srsp1) was previously identified in our laboratory with the aid of the differential display method (e.g. Geisinger et al., 1996, 2005), as a differentially expressed gene during spermatogenesis of the rat (Rattus norvegicus). Northern blot analyses against RNAs from ten adult different tissues showed that Spats1 mRNA is testis-specific (Geisinger et al., 2002). In this work we approached for the first time the characterization of the expression pattern of Spats1 along testis development, as a way to contribute to the elucidation of its biological function. To obtain information on the expression pattern of Spats1 at the
* Corresponding author. Address: Departamento de Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), CP 11600, Avda. Italia 3318, Montevideo, Uruguay. Tel.: +5982 4872605; fax: +5982 4875548. E-mail address: [email protected] (A. Geisinger). 1567-133X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2009.11.006
protein level, we have cloned and expressed a cDNA region coding for 234 amino acids of the putative protein. This region included the serine stretch and the presumptive nuclear localization signal (NLS) (Fig. 1). Polyclonal antibodies were generated in rabbit, and employed in confocal immunohistochemistry and immunoblotting experiments. 1.1. Tissue specificity of Spats1 expression Immunohistochemical methods using fluorescence confocal microscopy with anti-Spats1 antibody detected a strong signal on sections of 21 day-old rat testes (see Fig. 2A). A weaker signal was also detectable in testes from other ages (18 dpc–40 dpp, see below), while no signal was observed in sections from ovary (Fig. 2B), liver, or kidney (not shown). Besides, no signal was visible in control experiments either omitting the incubation with antiSpats1 antibody (not shown) or treated with rabbit pre-immune serum (Fig. 2C), thus indicating that the anti-Spats1 serum is target-specific. Western-blot experiments revealed the presence of the protein in lysates from testes of different ages, but not in lysates from liver or brain (see Fig. 4A). These results, together with previous Northern blot assays showing the presence of Spats1 mRNA in adult rat testis but its absence in epididymis, ovary, brain, heart, lung, liver,
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Figure 1. Predicted full amino acidic sequence of Spats1 protein of the rat. The identifiable domains are a long serine stretch and a probable bipartite NLS. The region in bold characters was expressed as a GST-fusion protein in pGEX-5x-3 and used for immunization. The serine stretch (18 serines interrupted by a threonine and a proline) and putative NLS are underlined with continuous and discontinuous lines, respectively.
Figure 2. Immunostaining and confocal microscopy on cryosections from rat testis (A) and ovary (B). (C) Negative control on testis sections with the pre-immune serum. Spats1 signal is shown in green. The same sections were co-stained with the DNA-specific fluorochrome TOPRO-3 (red signal). Bar: 100 lm.
intestine, spleen and kidney (Geisinger et al., 2002) allow us to assert that Spats1 is a testis-specific protein. Moreover, this work is the first evidence on the existence of a protein product for the gene Spats1. This is not trivial, considering that testis is subjected to unusually high levels of post-transcriptional repression, with some mRNAs expressed at high levels but partially repressed or inactivated, and generating little or no protein at all (Kleene, 2003).
ing that gonocytes are not synchronized within each developmental stage, but rather there is an overlap of cells expressing different genes within the same seminiferous cord section (Culty, 2009, and references therein). At 20 dpc (i.e. the day before birth), the number of gonocytes expressing Spats1 seems to be higher (Fig. 3A, panels c, c00 ). 1.3. Differential expression of Spats1 along postnatal testis development
1.2. Spats1 expression during embryonic development To determine when Spats1 was first expressed, gonad sections of male and female embryos starting at 13 days post-coitum (dpc; i.e. when gonocytes become resident in the forming gonad [reviewed by Culty (2009)]) were immunostained with anti-Spats1 antibody and analysed by confocal microscopy. A signal was first detected in embryo testes at 17.5–18 dpc (Fig. 3A, frame b), but not in ovaries from the same age (not shown), while no signal was found before 17 dpc neither in female nor in male gonads (Fig 3A, frame a). Interestingly, Spats1 appearance coincides with the time when gonocytes change from a mitotic to a quiescent stage in rat. In fact, although germ cell stages between primordial germ cells and the spermatogonial stem cells are often indiscriminately called gonocytes, these cells represent several successive developmental stages: a fetal mitotic phase that takes place when the germ cell becomes resident in the gonad (dpc 13–18 in rat), a postnatal mitotic phase (3 to 4–5 days post-partum [dpp] in rat), and a quiescent phase in between. These stages express different levels and combinations of genes (Culty, 2009). On the other hand, Spats1 signal is also coincident with the moment when peritubular myoid cells first appear delineating the periphery of the nascent seminiferous cords (dpc 17) (Culty, 2009), and a signal surrounding the seminiferous cords was detected starting at 17.5 dpc (Fig. 3A, panel b). Co-immunostaining for myosin indicated that the peritubular signal corresponded to myoid cells (Fig. 3A, panels c–c000 ). Besides, a positive signal was observed in the cytoplasm of a gonocyte subpopulation at 17.5– 18 dpc, mostly in those cells nearer to the basement membrane (Fig. 3A, panel b). This would be in agreement with reports show-
At birth, when only gonocytes still in the quiescent phase and Sertoli cells are present inside the seminiferous cords, the highest Spats1 expression was detected in gonocytes (Fig. 3A, panel d). When Spats1 expression was studied along the first spermatogenic wave, starting at 12 dpp (i.e. before the onset of the first meiotic division) up to 40 dpp, when spermatozoa are already present, a signal was detected before the beginning of meiosis (Fig. 3B, frame a). The signal intensity was maintained during early meiotic prophase (17–18 dpp), when the predominant cell types are leptotene and zygotene spermatocytes (Fig. 3B, frame b), and increased to attain its maximum by day 21 (medium meiotic prophase), coinciding with the appearance of pachytene spermatocytes (Malkov et al., 1998) (Fig. 3B, frame c). Later, at postmeiotic stages (i.e. 27 dpp), the signal was clearly weaker (Fig. 3B, frame d). It still decreased until the adult stage (40 dpp), when only a faint signal was observed (Fig. 3B, panel e). The decrease in protein signal as the first spermatogenic wave progresses is more evident in Fig. 3B, panels a0 –e0 . These results were further supported by Western-blot experiments, which detected the protein in lysates from testes of 17.5 dpc to 21 dpp individuals, but not from 40 dpp. A weaker signal was observed at postnatal day 27 that was intermediate between 21 dpp and adult testes (see Fig. 4A). Among all the stages and cell types that expressed Spats1, the strongest signal was found in spermatocytes at 21 dpp (Fig. 3C, frames a and b), which was confirmed by co-immunostaining for Spats1 and Sycp3, a marker for the lateral elements of synaptonemal complexes (Lammers et al., 1994) (Fig. 3C, frames b–b000 ). Besides, the other cell types present in the testis at 21 dpp, namely spermatogonia, supporting Sertoli and myoid cells (Fig. 3C, panels c–c0 00 ) would also express relatively high levels of Spats1 (see Fig. 3C).
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Figure 3. Expression of Spats1 along testis development analyzed by confocal immunohistochemistry. (A) Expression analysis from embryonic stages until birth. Frame a: 15.5–16 dpc (E16); b: 17.5–18 dpc (E17.5); c: 20 dpc (E20); and d: 0 dpp (P0). In frames a, b and d testes cryosections were stained with anti-Spats1 antibody (green) and TOPRO-3 (red). Frames c–c0 0 0 show co-immunostaining of Spats1 (green) and myosin (MYH) to label peritubular myoid cells (red). The cell nuclei were counterstained with DAPI (blue). Arrowheads point at gonocytes (G). The bars in a and b correspond to 100 lm, and in c–c0 0 0 and d to 25 lm. (B) Expression of Spats1 along the first spermatogenic wave. Vibratome transversal sections of seminiferous tubules from 12 (P12), 18 (P18), 21 (P21), 27 (P27) and 40 (P40) dpp rats were incubated with anti-Spats1 antibody (green) and TOPRO-3 (red) (frames a–e). Frames a0 –e0 show Spats1 staining alone for better visualization of variations in signal intensity. Bar: 100 lm. (C) Detailed expression analysis of Spats1 on transversal cryosections of seminiferous tubules from 21 day-old-specimens. The picture on frame a was stained with anti-Spats1 antibody (green), and nuclei were counterstained with TOPRO-3 (red). Frames b–b0 0 show co-immunostaining for Spats1 (red in this case) and Sycp3 (green) as a marker for synaptonemal complexes. b0 -b0 0 0 correspond to a section from frame b. Frames c–c0 0 are co-stainings for Spats1 (green) and myosin (MYH, red). c0 0 0 and d0 0 0 show nuclei counterstained with DAPI. The arrowheads in frame a, point at spermatocytes (Sc), Sertoli (S) and peritubular myoid cells (M). (D) and (E) Detailed expression analysis on testis sections of 27 and 40 dpp individuals, respectively, stained with anti-Spats1 antibody (green) and TOPRO-3 (red). Arrowheads point at spermatocytes (Sc), round spermatids (Rs), and spermatozoa (Sz). All the bars in C, D and E correspond to 25 lm.
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Figure 4. Immunoblot with anti-Spats1 antibody. (A) Protein lysates of testis from rats of 17.5–18 dpc (TE17.5), 0 dpp (T0), 12 dpp (T12), 18 dpp (T18), 21 dpp (T21), 27 dpp (T27) and 40 dpp (T40) were loaded with protein lysates from liver (L) and brain (B) from 40 days-old rats. (B) Testicular cell suspensions from 21 day-old rats were treated with alkaline phosphatase (T21/AP) and loaded into polyacrylamide gels. As a control, the same amount of cells was treated in the same way but in the absence of alkaline phosphatase (T21). A0 and B0 : Coomasie blue stained gels are shown as loading controls.
As the first spermatogenic wave progresses, a much lower signal was observed in round spermatids (Fig. 3D), while no signal was found in elongating spermatids or spermatozoa (Fig. 3E). This is in agreement with the lower overall expression levels observed at 27 and especially at 40 dpp, since the proportion of meiotic cells progressively decreases (Bellvé et al., 1977; Malkov et al., 1998). The high protein expression levels in spermatocytes, with notably lower levels in round spermatids, is in agreement with previously reported Northern blot results that showed a stronger signal in meiotic prophase cells (Geisinger et al., 2002), and indicates that Spats1 is one of a high number of genes whose expression is increased in meiocytes (Shima et al., 2004). Altogether, these results suggest that although important developmental delays in the translation of a large number of mRNAs (such as those coding for protamine 1 and 2 [Prm1 and Prm2]) is a relevant aspect of gene expression in spermatogenic cells (Kleene, 2003), this is not the case for Spats1 mRNA, whose translation time would match transcription. 1.4. Intracellular Spats1 distribution The labelling pattern of anti-Spats1 antibody was observed as a disperse signal in the cytoplasm of all the cell types in which the protein was detected (see Fig. 3A frames b and d, 3C frame a, 3D and E). However, pachytene spermatocytes from 21 day-old testes also showed intranuclear labelling (Fig. 3C, frames a and b, central part of the tubules). The nuclear signal, which was confirmed by performing confocal Z stacks, was detected in all the inner Z-axis planes (not shown), but was never found colocalizing with DNA. As the protein sequence exhibits a putative bipartite NLS, we propose that the NLS would be functional to address Spats1 to the nucleus in spermatocytes at 21 dpp. It is worth mentioning that although we have found this intranuclear labelling in spermatocytes, we cannot rule out the possibility that a small amount of the protein could be located in the nucleus of other cell types but remain undetected due to low expression levels. In fact, some intranuclear signal is apparent in 20 dpc gonocytes (Fig. 3A, frame c). 1.5. Spats1 phosphorylation As can be seen in Fig. 4A, Western-blot experiments detected two bands in the testes lysates of 17.5 dpc, 0, 12, 18 and 21 dpp, while in 27 day-old individuals the signal was weaker, mainly
allowing visualization of the lower mobility band. The faster migration band had an apparent molecular mass of 32 kDa; this is coincidental with the in silico predicted molecular mass for Spats1 (Geisinger et al., 2002). The lower migration band had an apparent molecular mass of 40 kDa, suggesting a post translational modification. To investigate if this lower mobility band could be due to phosphorylation, we treated 21 day-old testicular cell suspensions with alkaline phosphatase followed by Western-blot analysis. Treatment with phosphatase resulted in complete disappearance of the 40 kDa band (Fig. 4B), thus indicating that phosphorylation was responsible for the observed shift in the electrophoretic mobility of Spats1. Two previously studied proteins, nucleolar protein Nopp140 (Meier and Blobel, 1992) and dehydrin Rab17 from plants (Plana et al., 1991), which exhibit serine stretches and NLS (Jensen et al., 1998), have shown to be phosphorylated in their serine stretches, in an ‘‘all or none” fashion. As a consequence, only two bands were observed in immunoblots, with no detectable intermediate phosphorylation forms (Plana et al., 1991; Meier and Blobel, 1992), which coincides with our observations regarding Spats1. Moreover, taking the length of the serine stretches of those two proteins and the molecular masses of their phosphorylated forms (Plana et al., 1991; Meier and Blobel, 1992) into consideration, the size of the Spats1 lower migration band matches its expected molecular mass if all the residues in the serine stretch were phosphorylated. As a consequence, we suggest that phosphorylation would be in the serine stretch and that the protein may exist either in a completely phosphorylated or dephosphorylated state. 1.6. Spats1 evolutionary conservation In a previous work (Geisinger et al., 2002) we had shown similarity of the predicted Spats1 protein sequence with conceptually translated cDNA clones from mouse, human, and the urochordate Ciona intestinalis. Interestingly, the C. intestinalis sequence was derived from the testis of this primitive chordate. We have now extended our homology findings to a high number of mammals including different eutherian orders, metatherians (Monodelphis domestica) and prototherians (Ornithorhyncus anatinus) (Fig. 5A). Besides Spats1 being a single copy gene, a unique transcript was found in all the analyzed species. Although three presumptive isoforms are annotated in the GenBank for Pan troglodytes and Macaca mulatta, we have noticed that the three GenBank entrances correspond to exactly the same transcript. The presence of Spats1 homologs in primitive chordates was further confirmed by finding a matching sequence in a conceptually translated mRNA from testis of the cephalochordate Branchiostoma floridae (GenBank accession No. XM_002214124). Moreover, we have found Spats1 homologous sequences in some lower invertebrate species, namely the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) and the cnidarians Acropora millepora and Nematostella vectensis (Fig. 5A). For the case of S. purpuratus, the sequence matched the conceptual translation of a clone from a testis cDNA library (GenBank accession No. EC431083.1). When the predicted Spats1 sequences from invertebrates were compared to the O. anatinus putative protein, eliminating most of the mammalian homologs, the similarity between the invertebrate sequences was remarkable (Fig. 5B). Due to the unusually rapid evolution rates of reproductive proteins (reviewed in Swanson and Vacquier, 2002), they are frequently poorly conserved even between closely related mammalian species (e.g. Lin et al., 2006). Therefore, the high Spats1 conservation supports the idea that this protein could have an important, conserved role in testis development and meiotic onset. Although no identifiable domains were found in the C-terminal region, this
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Figure 5. Alignment of Spats1 amino acidic sequence from different species. Alignment was performed with ClustalW2 and Bioedit software programs. (A) The protein sequence from Rattus norvegicus was aligned with matching sequences from eutherian (Mus musculus, Homo sapiens, Pan troglodytes, Macaca mulatta, Equus caballus, Canis familiaris, Sus scrofa, Bos taurus), metatherian (Monodelphis domestica), and prototherian (Ornithorhyncus anatinus) mammals, primitive chordates (Branchiostoma floridae and Ciona intestinalis), cnidarians (Acropora millepora and Nematostella vectensis), and the echinoderm Strongylocentrotus purpuratus. Identical amino acids are highlighted in black and similar amino acids in grey. Serine-rich regions are enclosed within a red frame. A second serine-rich region in some species is included within a blue frame. Putative NLSs are indicated in green. Some putative proline-directed kinase sites and conserved putative CK2 sites are underlined in red and blue respectively. (B) Same alignment as in A, but eliminating all the mammalian sequences except O. anatinus to show Spats1 conservation among invertebrates.
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is where the highest conservation was found, suggesting that this region may represent a functional domain. 1.7. Conclusions The results presented here suggest that Spats1 of the rat would exhibit a dynamic expression pattern, being expressed and distributed in a stage-specific, cell-specific and spatial-specific way along testis development. Its expression would start at the time when gonocytes enter the embryonic quiescent phase, accumulating in myoid cells and gonocytes. During the following days expression would increase in gonocytes. Spats1 expression augments as the testis progresses through the first spermatogenic wave, reaching its maximum in spermatocytes I mainly at the time when these cells arrive at the pachytene stage. At that time, the protein is also detected in spermatogonia, Sertoli and peritubular myoid cells. Protein levels start to decay afterwards, indicating that Spats1 could not be longer necessary after the first meiotic division is established or, alternatively, that its decrease would be required for spermiogenesis to progress. Interestingly, this is the first reported testis-specific protein bearing a phosphorylatable serine stretch. The Spats1 testis-specific expression, high protein levels, widespread distribution and conspicuous timely-spatial-stage-specific expression pattern, together with the high sequence conservation, prompt us to think that its role, whatever it is, should be very important for the organization of seminiferous tubules and to the onset and/or progression of meiosis during the first spermatogenic wave. Further investigations will be required to understand the role of this protein as well as the significance of phosphorylation for its function. It will be interesting to determine which protein kinase is responsible for the serine stretch phosphorylation of Spats1. We are currently exploring the physiological role of Spats1 in relation to testis development and meiosis by obtaining Spats1 knockout mice. 2. Experimental procedures 2.1. Animals and tissues Male Sprague–Dawley rats (Rattus novergicus) of different ages (0, 12, 18, 21, 27 and 40-dpp) were obtained from the animal facility at the Instituto de Investigaciones Biológicas Clemente Estable (IIBCE, Montevideo, Uruguay). Animals were killed by cervical dislocation (juvenile individuals) or by administration of an overdose of sodium pentobarbital (adults), following the recommendations of the Uruguayan National Commission of Animal Experimentation (CHEA). Then the different tissues were excised, and the tunica albuginea was removed from testes. Pregnant rats were sacrificed in the same way, and embryos were dissected in ice-cold PBS. Heads, torsos and viscera overlying the urogenital systems were removed and gonads were dissected free from the mesonephros. Tissues for immunoblotting were immediately frozen in liquid nitrogen and kept at 80 °C for further processing. Tissues destined to immunohistochemistry were fixed and processed as described later. Testicular cell suspensions for alkaline phosphatase treatment were prepared essentially as instructed (Meistrich, 1977). For polyclonal antibody production a healthy, parasite- and disease-free New Zealand white adult male rabbit was purchased from the breeding facility at the Faculty of Medicine (Universidad de la República, Montevideo). All the experimental protocol was in accordance with the requirements of the CHEA. 2.2. cDNA cloning PCR primers (forward: 50 -CCTTAAGGATCCGAGATGCCCAGCACA GT-30 ; reverse: 50 -CATGAACTCGAGCCTTCTCCCAGAAAGGCAGA-30 )
were designed to allow the amplification and sub-cloning of a 702 bp fragment from the coding region of a Spats1 cDNA clone contained in a pBluescript SK (Stratagene, La Jolla, CA, USA) vector (Geisinger et al., 2002). Italicized sequences correspond to BamHI and XhoI restriction sites added to the 50 ends of forward and reverse primers, respectively, to facilitate cloning. The primers also had additional overhangs besides the restriction sites. Amplification was done from 10 pg of plasmid DNA in a standard PCR mix, using a two-step PCR reaction (94 °C 2 min, 5 cycles of 94 °C 30 s, 57 °C 30 s, 72 °C 30 s, and 30 cycles of 94 °C 30 s, 60 °C 30 s, 72 °C 30 s; final extension: 72 °C 5 min). PCR products were eluted from agarose gels with the Concert Rapid Gel Extraction System™ (Life Technologies, Rockville, MD, USA), digested with BamHI and XhoI, directionally cloned into BamHI/XhoI-digested pGEX-5x-3 GST-fusion vector (Amersham/GE, Little Chalfont, UK) and transformed into Escherichia coli BL21 STAR™ (Invitrogene, Carlsbad, CA, USA) competent cells. Clones were subsequently sequenced according to standard protocols. 2.3. Protein expression and purification Selected clones were grown in LB containing 50 mg/L ampicillin at 37 °C to DO600 = 0.5–0.6. Gene expression was induced with 0.3 mM isopropyl-b-D-thiogalactoside (IPTG) for 3 h at 30 °C. Cells were harvested, resuspended in B-PER™ protein extraction reagent (Pierce, Rockford, IL, USA) containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA; Product number P 2714) 0.5 mg/g of culture, and GST-fusion protein was purified with the B-PER GST Spin Purification Kit™ (Pierce), according to the instructions of the manufacturer. The identity of the purified protein was confirmed by means of a Western-blot with an anti-GST antibody and reconfirmed by trypsine digestion and MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) analysis (not shown). Eluted protein was acetone-precipitated and resuspended in SDS-sample buffer. One-dimensional SDS–PAGE was performed on 12% polyacrylamide gels (Laemmli, 1970), and the gels were stained with Coomasie blue. Bands were excised from the gel, frozen at -80 °C, subsequently crushed with a Teflon pestle (Sigma) and suspended in PBS. 2.4. Antibody production Prior to immunization, a 2–3 mL of test bleed was taken from the rabbit to provide a source of pre-immune antiserum. 300 lg of the protein/polyacrylamide in PBS was mixed with an equal volume of complete Freund’s adjuvant, inoculated subcutaneously at eight points into the back of the animal and allowed to react for 20 days. Subsequently, three boosters were performed every 10 days. Immunogens for the boosters were prepared the same way as for the initial injection but with incomplete Freund’s adjuvant, and were applied by intramuscular injection into the leg. Final bleeding was taken at day 50. The obtained polyclonal antibody recognized the GST-Spats1 fusion protein in E. coli protein lysates on Western-blot experiments (not shown). The specificity of the antibody could be verified as the signal was blocked by adding the eluted protein prior to incubation with the samples (not shown). 2.5. SDS–PAGE and Western-blot Frozen tissues or cell suspensions were resuspended in SDSsample buffer and boiled for 5 min at 95 °C. SDS–PAGE was carried out on 12% polyacrylamide gels. 15 mg of protein lysate or 4.5 106 cells were loaded per lane. Protein gels were stained with
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Coomasie blue or transferred to nitrocellulose membranes as described by Matsudaira, 1987. For protein transfers, membranes were blocked for 2 h at room temperature (RT) in TBST buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20 v/v) containing 5% (w/v) non-fat milk. Membranes were washed 3 times for 10 min with TBST and incubated for 2 h on a rotary platform with anti-Spats1 antibody diluted 1:1000 in blocking buffer. Two washes of 10 min with TBST were performed before incubating for 1 h with goat anti-rabbit IgG coupled to peroxidase (Pierce) diluted 1:30,000 in blocking solution, and membranes were washed again with TBST. Bound secondary antibodies were detected using Supersignal West Pico Chemiluminescent Substrate Kit™ (Pierce).
co-staining, goat anti-rabbit Alexa 488 was applied first, followed by goat anti-mouse Alexa 633 (for MYH, Invitrogen, dilution 1/ 2000). For Scp3-Spats1 co-staining, slides were first blocked with 0.1% BSA, 50 mM glycine, 5% normal donkey serum (v/v) in buffer PHEM prior to incubation with donkey anti-goat 488 (for Scp3, Santa Cruz, sc-2024, dilution 1/100). Slides were then re-blocked with the blocking buffer containing 5% normal goat serum (v/v), and then incubated with goat anti-rabbit 633 antibody. In all coimmunostaining experiments the sections were co-stained with the DNA intercalant DAPI (Santa Cruz).
2.6. Sample processing for immunohistochemistry
4.5 106 cells from a 21 dpp rat testis suspension was resuspended in NEB-3 buffer (New England Biolabs, Ipswich, MA, USA) with protease inhibitor cocktail (Sigma P 2714, 6 ll of a 10X solution) and incubated with 30 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs) in a final volume of 50 ll for 1 h at 37 °C. CIP was inactivated by heating at 80 °C for 15 min. A control reaction was treated in identical conditions but omitting CIP. The proteins were acetone-precipitated, resuspended in SDSsample buffer and subjected to SDS–PAGE and immunoblot analysis with anti-Spats1 antibody.
The testicles were cut into small pieces and fixed by immersion in 4% paraformaldehyde (PFA) in buffer PHEM (25 mM Hepes, 10 mM EGTA, 2 mM MgCl2, 60 mM PIPES, pH 7.4) for 1 h at RT. Embryonic gonads were fixed in the same way. Then, the samples were washed in buffer PHEM for 2 h with gentle stirring, changing solution every 10 min. Sections were obtained by two methods: vibratome sections (floating immunostaining) and cryosections (immunostaining onto slides). 40–100 lm vibratome thick sections (MA752 vibratome, Campden Instruments, Loughborough, UK) were obtained after immersing the seminiferous tubules into a support medium (35% BSA w/v, 5% gelatine w/v, in buffer PHEM), and polymerized by the addition of 20 ll/mL of 25% v/v glutaraldehyde per ml of solution. Seminiferous tubules destined to cryosection were previously cryoprotected with sucrose in buffer PHEM, increasing its concentration from 15% to 30% (w/v), and then embedded into increasing cryo-embedding medium (Jung, Nussloch, Germany) from 25 to 100% (v/v). 10–15 lm cryosections were obtained with a Leica CM1510-1 cryostat (Wetzlar, Germany). Liver, kidney and ovary from adult animals were similarly processed and immunostained as controls.
2.8. Alkaline phosphatase treatment
Acknowledgements This work was supported by CSIC, Uruguay (Grant I+D C19 to A.G.) and a postgraduate fellowship of PEDECIBA/DICYT/ANII to C.A.C. The authors would also like to thank Ricardo Benavente (University of Würzburg) and Rosana Rodríguez-Casuriaga for helpful comments, Jessica Urbanavicius and Héctor Rodríguez for providing the rats, Monica Brauer and Analía Ricceri for assistance with the cryostat, and Marcelo Fernández (animal facility, IIBCE) for technical assistance with the immunization protocol.
2.7. Immunostaining and confocal microscopy
Appendix A. Supplementary material
Sections were permeabilized by a single incubation with 0.1% Tween 20 (v/v) in buffer PHEM for 1 h at RT. Subsequently, the free aldehyde groups were blocked by incubating in 1% (w/v) natriumborhydrid (Sigma) in PHEM for 30 min at RT (Bunea and Zarnescu, 2001), followed by three washes of 5 min with buffer PHEM. Non-specific antibody-binding sites were blocked afterwards for 30 min at RT with blocking buffer (0.1% BSA, 50 mM glycine, 5% normal goat serum v/v in buffer PHEM). Then, the sections were incubated for 2 h at RT with the specific antibody (polyclonal anti-Spats1, dilution 1/400) in blocking buffer. Incubation with anti-antibody (goat anti-rabbit Alexa 488, Invitrogen, dilution 1/ 2000) was performed for 1 h at RT, and DNA was labelled with a monomeric cyanine nucleic acid stain (TOPRO-3, Invitrogen, dilution 1/4000, 5 min). Sections were washed three times for 5 min with buffer PHEM between each incubation step. Finally, the slides were mounted with antifade mounting medium (Immunogold, Invitrogen), and stored at 4 °C protected from light until observation under a confocal microscope Olympus FV300 (Olympus, Melville, NY, USA). Negative controls were performed in parallel under identical conditions, but either omitting the primary antibody (not shown) or replacing it with pre-immune serum. For co-immunostaining experiments, the slides were sequentially incubated (with intermediate washes), first with monoclonal anti-MYH(G4) (Santa Cruz, sc-6956) or polyclonal anti Scp3(K-13) (Santa Cruz, sc-33875), and then with anti-Spats1 antibody, for 2 h at RT each. The secondary anti-antibodies were also sequentially incubated (with intermediate washes). In the case of MYH-Spats1
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.gep.2009.11.006. References Bellvé, A.R., Cavicchia, J.C., Milletfe, C.F., O´Brien, D.A., Bhatnagar, Y.M., Dym, M., 1977. Spermatogenic cells of the prepubral mouse. Isolation and morphological characterization. J. Cell Biol. 74, 68–85. Bunea, M., Zarnescu, O., 2001. New current aspects on the immunohistochemical techniques. Roumanian Biotechnol. Lett. 6, 177–207. Culty, M., 2009. Gonocytes, the forgotten cells of the germ cell lineage. Birth Defects Res. (C) 87, 1–26. Geisinger, A., Alsheimer, M., Baier, A., Benavente, R., Wettstein, R., 2005. The mammalian gene pecanex 1 is differentially expressed during spermatogenesis. Biochim. Biophys. Acta 1728, 34–43. Geisinger, A., Dos Santos, A., Benavente, R., Wettstein, R., 2002. Identification and characterization of Srsp1, a rat gene differentially expressed during spermatogenesis and coding for a serine stretch-containing protein. Cytogenet. Genome Res. 98, 249–254. Geisinger, A., Wettstein, R., Benavente, R., 1996. Stage-specific gene expression during rat spermatogenesis: application of the mRNA differential display method. Int. J. Dev. Biol. 40, 385–388. Jensen, A.B., Goday, A., Figueras, M., Jessop, A.C., Pages, M., 1998. Phosphorylation mediates the nuclear targeting of the maize Rab17 protein. Plant J. 13, 691–697. Kleene, K.C., 2003. Patterns, mechanisms, and functions of translation regulation in mammalian spermatogenic cells. Cytogenet. Genome Res. 103, 217–224. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lammers, J.H.M., Offenberg, H.H., van Aalderen, M., Vink, A.C.G., Dietrich, A.J.J., Heyting, C., 1994. The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyteregulated genes. Mol. Cell Biol. 14, 1137–1146. Lin, Y.H., Lin, Y.M., Teng, Y.N., Hsieh, T.Y.T., Lin, Y.S., Kuo, P.L., 2006. Identification of ten novel genes involved in human spermatogenesis by microarray analysis of testicular tissue. Fertil. Steril. 86, 1650–1658.
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Malkov, M., Fisher, Y., Don, J., 1998. Developmental schedule of the postnatal rat testis determined by flow cytometry. Biol. Reprod. 59, 84–92. Matsudaira, P., 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262, 10035–10038. Meier, U.T., Blobel, G., 1992. Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell 70, 127–138. Meistrich, M.L., 1977. Separation of spermatogenic cells and nuclei from rodent testes. Methods Cell Biol. 15, 15–44.
Plana, M., Itarte, E., Eritjas, R., Goday, A., Pages, M., Martinez, M.C., 1991. Phosphorylation of maize Rab-17 protein by casein kinase 2. J. Biol. Chem. 266, 22510–22514. Shima, J.E., McLean, D.J., McCarrey, J.R., Griswold, M.D., 2004. The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis. Biol. Reprod. 71, 319–330. Swanson, W.J., Vacquier, V.D., 2002. The rapid evolution of reproductive proteins. Nat. Genet. 3, 137–144.
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Spats 1 (Srsp1) is differentially expressed during testis development of the rat Carlos A. Capoano a, Rodolfo Wettstein a, Alejandra Kun b,c, Adriana Geisinger a,c,* a
Department of Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay Department of Proteínas y Ácidos Nucleicos, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay c Sección Bioquímica, Facultad de Ciencias, Montevideo, Uruguay b
a r t i c l e
i n f o
Article history: Received 17 July 2009 Received in revised form 19 November 2009 Accepted 24 November 2009 Available online 3 December 2009 To the late Dr. Rodolfo Wettstein Keywords: Testis development Spermatogenesis Meiosis Spermatocytes Gonocytes Peritubular myoid cells Serine stretch Spats1 Srsp1
a b s t r a c t Spats1 encodes the first reported testis-specific protein containing a long serine stretch. Besides, it bears a probable bipartite nuclear localization signal. Here, we describe the expression pattern of Spats1 in rat along embryonic and postnatal testis development by immunoblots and confocal immunohistochemistry. Spats1 is first expressed in the embryo at 17.5 days post-coitum, coinciding with the time when gonocytes acquire a quiescent state. At this time expression is detected in peritubular myoid cells and gonocytes. Spats1 attains maximum levels during meiosis of the first spermatogenic wave, mainly in pachytene spermatocytes, while a lower signal is also observed in spermatogonia, Sertoli cells and myoid cells. Protein levels dramatically decay afterwards, with minimum expression in adult individuals, where no signal was detected in elongating spermatids or spermatozoa. Spats1 is mostly cytoplasmic, although in pachytene spermatocytes it mapped to nuclei as well. Alkaline phosphatase treatment showed that this protein would be highly phosphorylated. Moreover, we show that the protein is highly conserved along metazoan evolution. Our results suggest a role in the initiation of the first spermatogenic wave, and in the establishment or progression of the first male meiotic division. Ó 2009 Elsevier B.V. All rights reserved.
1. Results and discussion Testis is a relatively unique tissue because it is the site of a developmental process that continues through the entire life. Unfortunately, the developmental and differentiation programs that lead to the production of mammalian male germ cells are still poorly understood at the molecular level due to some testis intrinsic difficulties, mainly the great tissue heterogeneity. Spats1 (‘‘spermatogenesis associated, serine-rich 1”; also called Srsp1) was previously identified in our laboratory with the aid of the differential display method (e.g. Geisinger et al., 1996, 2005), as a differentially expressed gene during spermatogenesis of the rat (Rattus norvegicus). Northern blot analyses against RNAs from ten adult different tissues showed that Spats1 mRNA is testis-specific (Geisinger et al., 2002). In this work we approached for the first time the characterization of the expression pattern of Spats1 along testis development, as a way to contribute to the elucidation of its biological function. To obtain information on the expression pattern of Spats1 at the
* Corresponding author. Address: Departamento de Biología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), CP 11600, Avda. Italia 3318, Montevideo, Uruguay. Tel.: +5982 4872605; fax: +5982 4875548. E-mail address: [email protected] (A. Geisinger). 1567-133X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2009.11.006
protein level, we have cloned and expressed a cDNA region coding for 234 amino acids of the putative protein. This region included the serine stretch and the presumptive nuclear localization signal (NLS) (Fig. 1). Polyclonal antibodies were generated in rabbit, and employed in confocal immunohistochemistry and immunoblotting experiments. 1.1. Tissue specificity of Spats1 expression Immunohistochemical methods using fluorescence confocal microscopy with anti-Spats1 antibody detected a strong signal on sections of 21 day-old rat testes (see Fig. 2A). A weaker signal was also detectable in testes from other ages (18 dpc–40 dpp, see below), while no signal was observed in sections from ovary (Fig. 2B), liver, or kidney (not shown). Besides, no signal was visible in control experiments either omitting the incubation with antiSpats1 antibody (not shown) or treated with rabbit pre-immune serum (Fig. 2C), thus indicating that the anti-Spats1 serum is target-specific. Western-blot experiments revealed the presence of the protein in lysates from testes of different ages, but not in lysates from liver or brain (see Fig. 4A). These results, together with previous Northern blot assays showing the presence of Spats1 mRNA in adult rat testis but its absence in epididymis, ovary, brain, heart, lung, liver,
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Figure 1. Predicted full amino acidic sequence of Spats1 protein of the rat. The identifiable domains are a long serine stretch and a probable bipartite NLS. The region in bold characters was expressed as a GST-fusion protein in pGEX-5x-3 and used for immunization. The serine stretch (18 serines interrupted by a threonine and a proline) and putative NLS are underlined with continuous and discontinuous lines, respectively.
Figure 2. Immunostaining and confocal microscopy on cryosections from rat testis (A) and ovary (B). (C) Negative control on testis sections with the pre-immune serum. Spats1 signal is shown in green. The same sections were co-stained with the DNA-specific fluorochrome TOPRO-3 (red signal). Bar: 100 lm.
intestine, spleen and kidney (Geisinger et al., 2002) allow us to assert that Spats1 is a testis-specific protein. Moreover, this work is the first evidence on the existence of a protein product for the gene Spats1. This is not trivial, considering that testis is subjected to unusually high levels of post-transcriptional repression, with some mRNAs expressed at high levels but partially repressed or inactivated, and generating little or no protein at all (Kleene, 2003).
ing that gonocytes are not synchronized within each developmental stage, but rather there is an overlap of cells expressing different genes within the same seminiferous cord section (Culty, 2009, and references therein). At 20 dpc (i.e. the day before birth), the number of gonocytes expressing Spats1 seems to be higher (Fig. 3A, panels c, c00 ). 1.3. Differential expression of Spats1 along postnatal testis development
1.2. Spats1 expression during embryonic development To determine when Spats1 was first expressed, gonad sections of male and female embryos starting at 13 days post-coitum (dpc; i.e. when gonocytes become resident in the forming gonad [reviewed by Culty (2009)]) were immunostained with anti-Spats1 antibody and analysed by confocal microscopy. A signal was first detected in embryo testes at 17.5–18 dpc (Fig. 3A, frame b), but not in ovaries from the same age (not shown), while no signal was found before 17 dpc neither in female nor in male gonads (Fig 3A, frame a). Interestingly, Spats1 appearance coincides with the time when gonocytes change from a mitotic to a quiescent stage in rat. In fact, although germ cell stages between primordial germ cells and the spermatogonial stem cells are often indiscriminately called gonocytes, these cells represent several successive developmental stages: a fetal mitotic phase that takes place when the germ cell becomes resident in the gonad (dpc 13–18 in rat), a postnatal mitotic phase (3 to 4–5 days post-partum [dpp] in rat), and a quiescent phase in between. These stages express different levels and combinations of genes (Culty, 2009). On the other hand, Spats1 signal is also coincident with the moment when peritubular myoid cells first appear delineating the periphery of the nascent seminiferous cords (dpc 17) (Culty, 2009), and a signal surrounding the seminiferous cords was detected starting at 17.5 dpc (Fig. 3A, panel b). Co-immunostaining for myosin indicated that the peritubular signal corresponded to myoid cells (Fig. 3A, panels c–c000 ). Besides, a positive signal was observed in the cytoplasm of a gonocyte subpopulation at 17.5– 18 dpc, mostly in those cells nearer to the basement membrane (Fig. 3A, panel b). This would be in agreement with reports show-
At birth, when only gonocytes still in the quiescent phase and Sertoli cells are present inside the seminiferous cords, the highest Spats1 expression was detected in gonocytes (Fig. 3A, panel d). When Spats1 expression was studied along the first spermatogenic wave, starting at 12 dpp (i.e. before the onset of the first meiotic division) up to 40 dpp, when spermatozoa are already present, a signal was detected before the beginning of meiosis (Fig. 3B, frame a). The signal intensity was maintained during early meiotic prophase (17–18 dpp), when the predominant cell types are leptotene and zygotene spermatocytes (Fig. 3B, frame b), and increased to attain its maximum by day 21 (medium meiotic prophase), coinciding with the appearance of pachytene spermatocytes (Malkov et al., 1998) (Fig. 3B, frame c). Later, at postmeiotic stages (i.e. 27 dpp), the signal was clearly weaker (Fig. 3B, frame d). It still decreased until the adult stage (40 dpp), when only a faint signal was observed (Fig. 3B, panel e). The decrease in protein signal as the first spermatogenic wave progresses is more evident in Fig. 3B, panels a0 –e0 . These results were further supported by Western-blot experiments, which detected the protein in lysates from testes of 17.5 dpc to 21 dpp individuals, but not from 40 dpp. A weaker signal was observed at postnatal day 27 that was intermediate between 21 dpp and adult testes (see Fig. 4A). Among all the stages and cell types that expressed Spats1, the strongest signal was found in spermatocytes at 21 dpp (Fig. 3C, frames a and b), which was confirmed by co-immunostaining for Spats1 and Sycp3, a marker for the lateral elements of synaptonemal complexes (Lammers et al., 1994) (Fig. 3C, frames b–b000 ). Besides, the other cell types present in the testis at 21 dpp, namely spermatogonia, supporting Sertoli and myoid cells (Fig. 3C, panels c–c0 00 ) would also express relatively high levels of Spats1 (see Fig. 3C).
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Figure 3. Expression of Spats1 along testis development analyzed by confocal immunohistochemistry. (A) Expression analysis from embryonic stages until birth. Frame a: 15.5–16 dpc (E16); b: 17.5–18 dpc (E17.5); c: 20 dpc (E20); and d: 0 dpp (P0). In frames a, b and d testes cryosections were stained with anti-Spats1 antibody (green) and TOPRO-3 (red). Frames c–c0 0 0 show co-immunostaining of Spats1 (green) and myosin (MYH) to label peritubular myoid cells (red). The cell nuclei were counterstained with DAPI (blue). Arrowheads point at gonocytes (G). The bars in a and b correspond to 100 lm, and in c–c0 0 0 and d to 25 lm. (B) Expression of Spats1 along the first spermatogenic wave. Vibratome transversal sections of seminiferous tubules from 12 (P12), 18 (P18), 21 (P21), 27 (P27) and 40 (P40) dpp rats were incubated with anti-Spats1 antibody (green) and TOPRO-3 (red) (frames a–e). Frames a0 –e0 show Spats1 staining alone for better visualization of variations in signal intensity. Bar: 100 lm. (C) Detailed expression analysis of Spats1 on transversal cryosections of seminiferous tubules from 21 day-old-specimens. The picture on frame a was stained with anti-Spats1 antibody (green), and nuclei were counterstained with TOPRO-3 (red). Frames b–b0 0 show co-immunostaining for Spats1 (red in this case) and Sycp3 (green) as a marker for synaptonemal complexes. b0 -b0 0 0 correspond to a section from frame b. Frames c–c0 0 are co-stainings for Spats1 (green) and myosin (MYH, red). c0 0 0 and d0 0 0 show nuclei counterstained with DAPI. The arrowheads in frame a, point at spermatocytes (Sc), Sertoli (S) and peritubular myoid cells (M). (D) and (E) Detailed expression analysis on testis sections of 27 and 40 dpp individuals, respectively, stained with anti-Spats1 antibody (green) and TOPRO-3 (red). Arrowheads point at spermatocytes (Sc), round spermatids (Rs), and spermatozoa (Sz). All the bars in C, D and E correspond to 25 lm.
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Figure 4. Immunoblot with anti-Spats1 antibody. (A) Protein lysates of testis from rats of 17.5–18 dpc (TE17.5), 0 dpp (T0), 12 dpp (T12), 18 dpp (T18), 21 dpp (T21), 27 dpp (T27) and 40 dpp (T40) were loaded with protein lysates from liver (L) and brain (B) from 40 days-old rats. (B) Testicular cell suspensions from 21 day-old rats were treated with alkaline phosphatase (T21/AP) and loaded into polyacrylamide gels. As a control, the same amount of cells was treated in the same way but in the absence of alkaline phosphatase (T21). A0 and B0 : Coomasie blue stained gels are shown as loading controls.
As the first spermatogenic wave progresses, a much lower signal was observed in round spermatids (Fig. 3D), while no signal was found in elongating spermatids or spermatozoa (Fig. 3E). This is in agreement with the lower overall expression levels observed at 27 and especially at 40 dpp, since the proportion of meiotic cells progressively decreases (Bellvé et al., 1977; Malkov et al., 1998). The high protein expression levels in spermatocytes, with notably lower levels in round spermatids, is in agreement with previously reported Northern blot results that showed a stronger signal in meiotic prophase cells (Geisinger et al., 2002), and indicates that Spats1 is one of a high number of genes whose expression is increased in meiocytes (Shima et al., 2004). Altogether, these results suggest that although important developmental delays in the translation of a large number of mRNAs (such as those coding for protamine 1 and 2 [Prm1 and Prm2]) is a relevant aspect of gene expression in spermatogenic cells (Kleene, 2003), this is not the case for Spats1 mRNA, whose translation time would match transcription. 1.4. Intracellular Spats1 distribution The labelling pattern of anti-Spats1 antibody was observed as a disperse signal in the cytoplasm of all the cell types in which the protein was detected (see Fig. 3A frames b and d, 3C frame a, 3D and E). However, pachytene spermatocytes from 21 day-old testes also showed intranuclear labelling (Fig. 3C, frames a and b, central part of the tubules). The nuclear signal, which was confirmed by performing confocal Z stacks, was detected in all the inner Z-axis planes (not shown), but was never found colocalizing with DNA. As the protein sequence exhibits a putative bipartite NLS, we propose that the NLS would be functional to address Spats1 to the nucleus in spermatocytes at 21 dpp. It is worth mentioning that although we have found this intranuclear labelling in spermatocytes, we cannot rule out the possibility that a small amount of the protein could be located in the nucleus of other cell types but remain undetected due to low expression levels. In fact, some intranuclear signal is apparent in 20 dpc gonocytes (Fig. 3A, frame c). 1.5. Spats1 phosphorylation As can be seen in Fig. 4A, Western-blot experiments detected two bands in the testes lysates of 17.5 dpc, 0, 12, 18 and 21 dpp, while in 27 day-old individuals the signal was weaker, mainly
allowing visualization of the lower mobility band. The faster migration band had an apparent molecular mass of 32 kDa; this is coincidental with the in silico predicted molecular mass for Spats1 (Geisinger et al., 2002). The lower migration band had an apparent molecular mass of 40 kDa, suggesting a post translational modification. To investigate if this lower mobility band could be due to phosphorylation, we treated 21 day-old testicular cell suspensions with alkaline phosphatase followed by Western-blot analysis. Treatment with phosphatase resulted in complete disappearance of the 40 kDa band (Fig. 4B), thus indicating that phosphorylation was responsible for the observed shift in the electrophoretic mobility of Spats1. Two previously studied proteins, nucleolar protein Nopp140 (Meier and Blobel, 1992) and dehydrin Rab17 from plants (Plana et al., 1991), which exhibit serine stretches and NLS (Jensen et al., 1998), have shown to be phosphorylated in their serine stretches, in an ‘‘all or none” fashion. As a consequence, only two bands were observed in immunoblots, with no detectable intermediate phosphorylation forms (Plana et al., 1991; Meier and Blobel, 1992), which coincides with our observations regarding Spats1. Moreover, taking the length of the serine stretches of those two proteins and the molecular masses of their phosphorylated forms (Plana et al., 1991; Meier and Blobel, 1992) into consideration, the size of the Spats1 lower migration band matches its expected molecular mass if all the residues in the serine stretch were phosphorylated. As a consequence, we suggest that phosphorylation would be in the serine stretch and that the protein may exist either in a completely phosphorylated or dephosphorylated state. 1.6. Spats1 evolutionary conservation In a previous work (Geisinger et al., 2002) we had shown similarity of the predicted Spats1 protein sequence with conceptually translated cDNA clones from mouse, human, and the urochordate Ciona intestinalis. Interestingly, the C. intestinalis sequence was derived from the testis of this primitive chordate. We have now extended our homology findings to a high number of mammals including different eutherian orders, metatherians (Monodelphis domestica) and prototherians (Ornithorhyncus anatinus) (Fig. 5A). Besides Spats1 being a single copy gene, a unique transcript was found in all the analyzed species. Although three presumptive isoforms are annotated in the GenBank for Pan troglodytes and Macaca mulatta, we have noticed that the three GenBank entrances correspond to exactly the same transcript. The presence of Spats1 homologs in primitive chordates was further confirmed by finding a matching sequence in a conceptually translated mRNA from testis of the cephalochordate Branchiostoma floridae (GenBank accession No. XM_002214124). Moreover, we have found Spats1 homologous sequences in some lower invertebrate species, namely the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) and the cnidarians Acropora millepora and Nematostella vectensis (Fig. 5A). For the case of S. purpuratus, the sequence matched the conceptual translation of a clone from a testis cDNA library (GenBank accession No. EC431083.1). When the predicted Spats1 sequences from invertebrates were compared to the O. anatinus putative protein, eliminating most of the mammalian homologs, the similarity between the invertebrate sequences was remarkable (Fig. 5B). Due to the unusually rapid evolution rates of reproductive proteins (reviewed in Swanson and Vacquier, 2002), they are frequently poorly conserved even between closely related mammalian species (e.g. Lin et al., 2006). Therefore, the high Spats1 conservation supports the idea that this protein could have an important, conserved role in testis development and meiotic onset. Although no identifiable domains were found in the C-terminal region, this
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Figure 5. Alignment of Spats1 amino acidic sequence from different species. Alignment was performed with ClustalW2 and Bioedit software programs. (A) The protein sequence from Rattus norvegicus was aligned with matching sequences from eutherian (Mus musculus, Homo sapiens, Pan troglodytes, Macaca mulatta, Equus caballus, Canis familiaris, Sus scrofa, Bos taurus), metatherian (Monodelphis domestica), and prototherian (Ornithorhyncus anatinus) mammals, primitive chordates (Branchiostoma floridae and Ciona intestinalis), cnidarians (Acropora millepora and Nematostella vectensis), and the echinoderm Strongylocentrotus purpuratus. Identical amino acids are highlighted in black and similar amino acids in grey. Serine-rich regions are enclosed within a red frame. A second serine-rich region in some species is included within a blue frame. Putative NLSs are indicated in green. Some putative proline-directed kinase sites and conserved putative CK2 sites are underlined in red and blue respectively. (B) Same alignment as in A, but eliminating all the mammalian sequences except O. anatinus to show Spats1 conservation among invertebrates.
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is where the highest conservation was found, suggesting that this region may represent a functional domain. 1.7. Conclusions The results presented here suggest that Spats1 of the rat would exhibit a dynamic expression pattern, being expressed and distributed in a stage-specific, cell-specific and spatial-specific way along testis development. Its expression would start at the time when gonocytes enter the embryonic quiescent phase, accumulating in myoid cells and gonocytes. During the following days expression would increase in gonocytes. Spats1 expression augments as the testis progresses through the first spermatogenic wave, reaching its maximum in spermatocytes I mainly at the time when these cells arrive at the pachytene stage. At that time, the protein is also detected in spermatogonia, Sertoli and peritubular myoid cells. Protein levels start to decay afterwards, indicating that Spats1 could not be longer necessary after the first meiotic division is established or, alternatively, that its decrease would be required for spermiogenesis to progress. Interestingly, this is the first reported testis-specific protein bearing a phosphorylatable serine stretch. The Spats1 testis-specific expression, high protein levels, widespread distribution and conspicuous timely-spatial-stage-specific expression pattern, together with the high sequence conservation, prompt us to think that its role, whatever it is, should be very important for the organization of seminiferous tubules and to the onset and/or progression of meiosis during the first spermatogenic wave. Further investigations will be required to understand the role of this protein as well as the significance of phosphorylation for its function. It will be interesting to determine which protein kinase is responsible for the serine stretch phosphorylation of Spats1. We are currently exploring the physiological role of Spats1 in relation to testis development and meiosis by obtaining Spats1 knockout mice. 2. Experimental procedures 2.1. Animals and tissues Male Sprague–Dawley rats (Rattus novergicus) of different ages (0, 12, 18, 21, 27 and 40-dpp) were obtained from the animal facility at the Instituto de Investigaciones Biológicas Clemente Estable (IIBCE, Montevideo, Uruguay). Animals were killed by cervical dislocation (juvenile individuals) or by administration of an overdose of sodium pentobarbital (adults), following the recommendations of the Uruguayan National Commission of Animal Experimentation (CHEA). Then the different tissues were excised, and the tunica albuginea was removed from testes. Pregnant rats were sacrificed in the same way, and embryos were dissected in ice-cold PBS. Heads, torsos and viscera overlying the urogenital systems were removed and gonads were dissected free from the mesonephros. Tissues for immunoblotting were immediately frozen in liquid nitrogen and kept at 80 °C for further processing. Tissues destined to immunohistochemistry were fixed and processed as described later. Testicular cell suspensions for alkaline phosphatase treatment were prepared essentially as instructed (Meistrich, 1977). For polyclonal antibody production a healthy, parasite- and disease-free New Zealand white adult male rabbit was purchased from the breeding facility at the Faculty of Medicine (Universidad de la República, Montevideo). All the experimental protocol was in accordance with the requirements of the CHEA. 2.2. cDNA cloning PCR primers (forward: 50 -CCTTAAGGATCCGAGATGCCCAGCACA GT-30 ; reverse: 50 -CATGAACTCGAGCCTTCTCCCAGAAAGGCAGA-30 )
were designed to allow the amplification and sub-cloning of a 702 bp fragment from the coding region of a Spats1 cDNA clone contained in a pBluescript SK (Stratagene, La Jolla, CA, USA) vector (Geisinger et al., 2002). Italicized sequences correspond to BamHI and XhoI restriction sites added to the 50 ends of forward and reverse primers, respectively, to facilitate cloning. The primers also had additional overhangs besides the restriction sites. Amplification was done from 10 pg of plasmid DNA in a standard PCR mix, using a two-step PCR reaction (94 °C 2 min, 5 cycles of 94 °C 30 s, 57 °C 30 s, 72 °C 30 s, and 30 cycles of 94 °C 30 s, 60 °C 30 s, 72 °C 30 s; final extension: 72 °C 5 min). PCR products were eluted from agarose gels with the Concert Rapid Gel Extraction System™ (Life Technologies, Rockville, MD, USA), digested with BamHI and XhoI, directionally cloned into BamHI/XhoI-digested pGEX-5x-3 GST-fusion vector (Amersham/GE, Little Chalfont, UK) and transformed into Escherichia coli BL21 STAR™ (Invitrogene, Carlsbad, CA, USA) competent cells. Clones were subsequently sequenced according to standard protocols. 2.3. Protein expression and purification Selected clones were grown in LB containing 50 mg/L ampicillin at 37 °C to DO600 = 0.5–0.6. Gene expression was induced with 0.3 mM isopropyl-b-D-thiogalactoside (IPTG) for 3 h at 30 °C. Cells were harvested, resuspended in B-PER™ protein extraction reagent (Pierce, Rockford, IL, USA) containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA; Product number P 2714) 0.5 mg/g of culture, and GST-fusion protein was purified with the B-PER GST Spin Purification Kit™ (Pierce), according to the instructions of the manufacturer. The identity of the purified protein was confirmed by means of a Western-blot with an anti-GST antibody and reconfirmed by trypsine digestion and MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) analysis (not shown). Eluted protein was acetone-precipitated and resuspended in SDS-sample buffer. One-dimensional SDS–PAGE was performed on 12% polyacrylamide gels (Laemmli, 1970), and the gels were stained with Coomasie blue. Bands were excised from the gel, frozen at -80 °C, subsequently crushed with a Teflon pestle (Sigma) and suspended in PBS. 2.4. Antibody production Prior to immunization, a 2–3 mL of test bleed was taken from the rabbit to provide a source of pre-immune antiserum. 300 lg of the protein/polyacrylamide in PBS was mixed with an equal volume of complete Freund’s adjuvant, inoculated subcutaneously at eight points into the back of the animal and allowed to react for 20 days. Subsequently, three boosters were performed every 10 days. Immunogens for the boosters were prepared the same way as for the initial injection but with incomplete Freund’s adjuvant, and were applied by intramuscular injection into the leg. Final bleeding was taken at day 50. The obtained polyclonal antibody recognized the GST-Spats1 fusion protein in E. coli protein lysates on Western-blot experiments (not shown). The specificity of the antibody could be verified as the signal was blocked by adding the eluted protein prior to incubation with the samples (not shown). 2.5. SDS–PAGE and Western-blot Frozen tissues or cell suspensions were resuspended in SDSsample buffer and boiled for 5 min at 95 °C. SDS–PAGE was carried out on 12% polyacrylamide gels. 15 mg of protein lysate or 4.5 106 cells were loaded per lane. Protein gels were stained with
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Coomasie blue or transferred to nitrocellulose membranes as described by Matsudaira, 1987. For protein transfers, membranes were blocked for 2 h at room temperature (RT) in TBST buffer (10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20 v/v) containing 5% (w/v) non-fat milk. Membranes were washed 3 times for 10 min with TBST and incubated for 2 h on a rotary platform with anti-Spats1 antibody diluted 1:1000 in blocking buffer. Two washes of 10 min with TBST were performed before incubating for 1 h with goat anti-rabbit IgG coupled to peroxidase (Pierce) diluted 1:30,000 in blocking solution, and membranes were washed again with TBST. Bound secondary antibodies were detected using Supersignal West Pico Chemiluminescent Substrate Kit™ (Pierce).
co-staining, goat anti-rabbit Alexa 488 was applied first, followed by goat anti-mouse Alexa 633 (for MYH, Invitrogen, dilution 1/ 2000). For Scp3-Spats1 co-staining, slides were first blocked with 0.1% BSA, 50 mM glycine, 5% normal donkey serum (v/v) in buffer PHEM prior to incubation with donkey anti-goat 488 (for Scp3, Santa Cruz, sc-2024, dilution 1/100). Slides were then re-blocked with the blocking buffer containing 5% normal goat serum (v/v), and then incubated with goat anti-rabbit 633 antibody. In all coimmunostaining experiments the sections were co-stained with the DNA intercalant DAPI (Santa Cruz).
2.6. Sample processing for immunohistochemistry
4.5 106 cells from a 21 dpp rat testis suspension was resuspended in NEB-3 buffer (New England Biolabs, Ipswich, MA, USA) with protease inhibitor cocktail (Sigma P 2714, 6 ll of a 10X solution) and incubated with 30 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs) in a final volume of 50 ll for 1 h at 37 °C. CIP was inactivated by heating at 80 °C for 15 min. A control reaction was treated in identical conditions but omitting CIP. The proteins were acetone-precipitated, resuspended in SDSsample buffer and subjected to SDS–PAGE and immunoblot analysis with anti-Spats1 antibody.
The testicles were cut into small pieces and fixed by immersion in 4% paraformaldehyde (PFA) in buffer PHEM (25 mM Hepes, 10 mM EGTA, 2 mM MgCl2, 60 mM PIPES, pH 7.4) for 1 h at RT. Embryonic gonads were fixed in the same way. Then, the samples were washed in buffer PHEM for 2 h with gentle stirring, changing solution every 10 min. Sections were obtained by two methods: vibratome sections (floating immunostaining) and cryosections (immunostaining onto slides). 40–100 lm vibratome thick sections (MA752 vibratome, Campden Instruments, Loughborough, UK) were obtained after immersing the seminiferous tubules into a support medium (35% BSA w/v, 5% gelatine w/v, in buffer PHEM), and polymerized by the addition of 20 ll/mL of 25% v/v glutaraldehyde per ml of solution. Seminiferous tubules destined to cryosection were previously cryoprotected with sucrose in buffer PHEM, increasing its concentration from 15% to 30% (w/v), and then embedded into increasing cryo-embedding medium (Jung, Nussloch, Germany) from 25 to 100% (v/v). 10–15 lm cryosections were obtained with a Leica CM1510-1 cryostat (Wetzlar, Germany). Liver, kidney and ovary from adult animals were similarly processed and immunostained as controls.
2.8. Alkaline phosphatase treatment
Acknowledgements This work was supported by CSIC, Uruguay (Grant I+D C19 to A.G.) and a postgraduate fellowship of PEDECIBA/DICYT/ANII to C.A.C. The authors would also like to thank Ricardo Benavente (University of Würzburg) and Rosana Rodríguez-Casuriaga for helpful comments, Jessica Urbanavicius and Héctor Rodríguez for providing the rats, Monica Brauer and Analía Ricceri for assistance with the cryostat, and Marcelo Fernández (animal facility, IIBCE) for technical assistance with the immunization protocol.
2.7. Immunostaining and confocal microscopy
Appendix A. Supplementary material
Sections were permeabilized by a single incubation with 0.1% Tween 20 (v/v) in buffer PHEM for 1 h at RT. Subsequently, the free aldehyde groups were blocked by incubating in 1% (w/v) natriumborhydrid (Sigma) in PHEM for 30 min at RT (Bunea and Zarnescu, 2001), followed by three washes of 5 min with buffer PHEM. Non-specific antibody-binding sites were blocked afterwards for 30 min at RT with blocking buffer (0.1% BSA, 50 mM glycine, 5% normal goat serum v/v in buffer PHEM). Then, the sections were incubated for 2 h at RT with the specific antibody (polyclonal anti-Spats1, dilution 1/400) in blocking buffer. Incubation with anti-antibody (goat anti-rabbit Alexa 488, Invitrogen, dilution 1/ 2000) was performed for 1 h at RT, and DNA was labelled with a monomeric cyanine nucleic acid stain (TOPRO-3, Invitrogen, dilution 1/4000, 5 min). Sections were washed three times for 5 min with buffer PHEM between each incubation step. Finally, the slides were mounted with antifade mounting medium (Immunogold, Invitrogen), and stored at 4 °C protected from light until observation under a confocal microscope Olympus FV300 (Olympus, Melville, NY, USA). Negative controls were performed in parallel under identical conditions, but either omitting the primary antibody (not shown) or replacing it with pre-immune serum. For co-immunostaining experiments, the slides were sequentially incubated (with intermediate washes), first with monoclonal anti-MYH(G4) (Santa Cruz, sc-6956) or polyclonal anti Scp3(K-13) (Santa Cruz, sc-33875), and then with anti-Spats1 antibody, for 2 h at RT each. The secondary anti-antibodies were also sequentially incubated (with intermediate washes). In the case of MYH-Spats1
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