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J. Biol. Chem., Vol. 277, Issue 8, 6311-6317, February 22, 2002

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Isolation and Properties of Gas8, a Growth Arrest-specific Gene Regulated during Male Gametogenesis to Produce a Protein Associated with the Sperm Motility Apparatus^*

Shauh-Der Yeh^§¶¶¶, Ying-Jiun Chen^¶, Annie C. Y. Chang^**, Rabindranath Ray, Bin-Ru She, Wen-Sen Lee^§, Han-Sun Chiang^¶¶§, Stanley N. Cohen^**, and Sue Lin-Chao^§

From the  Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, ^§ Graduate Institute of Medical Sciences, and ^¶¶ Department of Urology, Taipei Medical University, Taipei 110, Taiwan,  Institute of Biochemistry, National Taiwan University School of Medicine, and ^** Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5120

Received for publication, July 23, 2001, and in revised form, December 17, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth arrest-specific (Gas) genes are expressed during serum starvation or contact inhibition of cells grown in culture. Here we report the isolation and characterization of Gas8, a novel gene identified on the basis of its growth arrest-specific expression in murine fibroblasts. We show that production of Gas8 mRNA and protein occurs in adult mice predominantly in the testes, where expression is regulated during postmeiotic development of male gametocytes. Whereas a low level of Gas8 mRNA was detected by Northern blotting in testes of murine male neonates and young adolescents, Gas8 mRNA increased rapidly postmeiotically. In adult males, both Gas8 mRNA and protein reached steady state levels in testes that were 10-fold higher than in other tissues. Immunohistochemical analyses showed that Gas8 protein accumulates in gametocytes as they approach the lumen of seminiferous tubules and is localized to the cytoplasm of round spermatids, the tails of elongating spermatids, and mature spermatid tail bundles protruding into the lumen; in epididymal spermatozoa Gas8 protein was present in the flagella. However, premeiotic murine gametocytes lacked detectable Gas8 protein, as did seminiferous tubules in biopsy specimens from seven human males having cytological evidence of non-obstructive azoospermia secondary to Sertoli cell-only syndrome. Our findings, which associate Gas8 production developmentally with the later stages of spermatogenesis and spatially with the sperm motility apparatus, collectively suggest that this growth arrest-specific gene product may have a role in sperm motility. This postulated role for Gas8 is supported by our observation that highly localized production of Gas8 protein occurs also in the cilia of epithelial cells lining pulmonary bronchi and fallopian tubes and by the flagellar association of a Trypanosoma brucei ortholog of Gas8.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth arrest-specific (Gas) genes are structurally and functionally diverse genetic loci that are expressed preferentially in cultured cells that enter a quiescent state, commonly as a consequence of serum deprivation or growth to confluence. The cDNA sequences of these Gas genes show no similarity. Their activities have been found to range from the control of nerve cell differentiation to the regulation of microfilament organization, apoptosis, cell proliferation, and cell cycling (1-12). We previously reported the use of retroviral based gene search vectors to identify two murine chromosomal loci (354-6 and 354-7) whose expression in fibroblasts is activated during growth arrest (13). The locus in 354-7 encodes Gas7, which is expressed prominently during terminal differentiation of cultured Purkinje neurons and affects neurite outgrowth (14). Here we report the isolation and properties of the gene present at the 354-6 locus, which we previously named Gas8 (GenBank^TM accession number U19859 and Ref. 40, deposited in 1995). A human ortholog of unknown function was designated as a Gas gene on the basis of its sequence similarity to Gas8 and assigned the name of GAS11 (15).

In the production of male gametocytes, diploid spermatogonia or stem cells proliferate, undergo meiosis, and differentiate in the testis into haploid spermatozoa containing flagella required for normal fertile function. This process involves complex regulation of at least in part the post-transcriptional level of the expression of a variety of testis-specific genes (16). We show here that Gas8 is predominantly a testicular protein, whose expression in mice is developmentally regulated during puberty and spermatogenesis and in humans is absent in infertile males who lack the ability to generate gametes. The localization of Gas8 in the motility apparatus of postmeiotic gametocytes and mature spermatozoa, together with the detection of Gas8 also in cilia at the apical surfaces of epithelial cells lining the pulmonary bronchi and fallopian tubes suggests that the Gas8 protein may have a role in the functioning of motile cellular appendages.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Sequencing of Gas8 cDNA from Serum-starved NIH3T3 Cells-- A lacZ-containing fragment of genomic DNA from the 354-6 cell, in which a Moloney murine leukemia virus (MoMuLV)¹ lac provirus was integrated downstream of the Gas8 genomic DNA (13), was introduced by cloning and subcloning into the pSP72NOT plasmid (Promega), yielding pGas354-6. A series of nested deletions of this fragment was sequenced as described (14). 5' and 3' rapid amplification of cDNA ends cloning was also described in Ju et al. (14).

Sequencing of Drosophila Gas8 cDNA-- Using a TBLASTN data base search, we detected one Drosophila melanogaster EST (LD14709; 721-bp mRNA) that has extensive sequence homology to mouse Gas8. We obtained the cDNA clone containing this EST from Genome Systems Inc. and sequenced it as described (14). The GenBank^TM accession number for the determined sequence is AF468957.

Northern Blotting-- Different tissues, e.g. lung, liver, kidney, heart, testis, cerebellum, cerebrum, adnexa (ovary and fallopian tube), spleen, and skeletal muscle of albino mouse (ICR strain; adult male/female mice) more than 12 weeks old, were collected, immediately frozen in liquid nitrogen, and stored at 70 °C until use. For the collection of developing testes, the albino mice were housed in a cage in a 1:5 male to female ratio. The presence of a vaginal plug was the positive indication of mating and was considered as day 1 of gestation. Neonatal testes were collected from day 0 (samples were collected by 3 h after birth) to postnatal day 30 and immediately frozen in liquid nitrogen and stored at 70 °C until use. Total RNA was isolated from different tissues following standard protocol (17). Twenty µg of total RNA from different tissues was fractionated on a 1% agarose-formaldehyde gel as described (18). RNAs were transferred onto Zeta-probe membranes (Bio-Rad) and then cross-linked by UV by following the vendor's protocol. Membranes were hybridized with random primed [-³²P]dCTP-labeled Gas8 cDNA probe. Equal loading was monitored by ethidium bromide staining of 18 and 28 S rRNAs or by hybridization with -actin cDNA probes.

Construction of pET15b-DH4-Gas8 and Preparation of Gas8 Antigen and Anti-Gas8 Antibodies-- The end-filled AflIII DNA fragment containing Gas8 cDNA derived from pGas354-6 was inserted in the SmaI site of pET15bDH4 (a modified version of pET15b) to produce a His-tagged Gas8 protein expressed from a T7 bacterial phage promoter. The His-tagged Gas8 protein was overexpressed in Escherichia coli BL21(DE3)pLysS (Novagen, WI). His-tagged Gas8 used for antiserum preparation was purified from the supernatant fraction by separating the proteins by SDS-PAGE. The band corresponding to Gas8 was excised and homogenized in PBS and stored at 20 °C until use for immunizing the rabbit by subcutaneous injection as described (19).

Immunoprecipitation and Immunoblotting-- Various tissues from adult mice were homogenized by a polytron (PT3100, Kinematica) in buffer containing 1 mM EDTA, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM dithiothreitol, and protease inhibitors. After centrifugation, a sample of supernatant containing 1 mg of protein was used for each immunoprecipitation. The extract was first incubated with 25 µl of Sephadex G-100 at 4° for 2 h. The supernatant was centrifuged and then incubated with 9.7 µl of Sephadex G-100 and 0.3 µl of Sepharose CL-4B conjugated with the anti-Gas8 antiserum or the preimmune serum at 4 °C overnight. The beads were washed three times with the extraction buffer before resuspension in SDS-PAGE loading buffer and Western blot analysis (20, 21).

Mouse sperm cells were collected from cauda epididymis of adult mice (ICR strain, 3 months old). Protein lysate was made by homogenizing the sperm cells in RIPA buffer using a polytron homogenizer at 8,000 rpm for 10-20 s. The lysate was centrifuged at 10,000 rpm for 15 min at 4 °C. Protein concentration was estimated by using Bradford reagent (Bio-Rad). Protein samples were resolved on SDS-PAGE and detected by immunoblotting as described (20, 21). Positive immunoreaction was detected by an enhanced chemiluminescence system (Pierce) according to the manufacturer's instructions.

Immunohistochemistry and Immunofluorescence-- Perfusion-fixed, adult (3 months) ICR mouse testis was dehydrated and then embedded in paraffin. Tissue sections 4-5 µm in thickness were collected for immunohistochemical study. The sections were rehydrated and then balanced in PBS buffer. After blocking by 10% normal goat serum in PBS, they were incubated with anti-Gas8 antiserum (1:300 dilution) in PBS containing 0.4% Triton X-100. The antibody-antigen complex was detected by an avidin-biotin-peroxidase complex method and then developed in diaminobenzidine (DAB)-hydrogen peroxide (22). The nuclei were counterstained with either Mayer's hematoxylin or methyl green as indicated.

Mouse ciliated respiratory epithelial cells were brushed from adult mouse pulmonary bronchi. Mouse epididymal sperm were collected surgically from adult ICR mice. The sperm or epithelial cells were smeared on glass slides and then were fixed in ice-cold acetone for 20 min. After washing in PBS for 30 min, they were blocked with 5% non-fat milk in PBS for 30 min and incubated with a 1:300 dilution of anti-Gas8 antiserum or preimmune serum for 1 h. The antibody-antigen complex was detected with fluorescein-conjugated swine anti-rabbit IgG antibody (Dako, Glostrup, Denmark). The excess of antibodies was washed out with PBS, and samples were examined by confocal microscopy LSM510 (Zeiss, Jena, Germany).

Analysis of Gas8 Protein Expression in Human Testes-- Human testicular tissues were biopsied from infertile patients or excised from patients with prostate cancer for androgen ablation therapy at Taipei Medical University Hospital. The tissues were prepared and stained using the En-Vision Plus system (Dako) and the manufacturer's protocol. The primary antibody (anti-Gas8 antiserum) was diluted (1:300) in PBS. After being stained with En-Vision Plus reagent, slides were developed in DAB-hydrogen peroxide. For comparison, adjacent sections were stained with hematoxylin and eosin according to a standard protocol.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of Gas8 Genomic DNA and Transcripts-- The NIH3T3-derived cell line 354-6 (13) contains a single chromosomal insertion of a MoMuLV retroviral construct carrying the E. coli lacZ gene. The lacZ reporter gene in this insert is expressed from a chromosomally located promoter that is activated upon cell growth arrest. Cloning of the DNA segment 5' to lacZ yielded a 8.7-kb plasmid DNA insert whose restriction map (Fig. 1A) corresponded to the chromosomal DNA region containing the retroviral construct (13). Sequence analysis showed that the distal 2 bp of the native long terminal repeat sequence of the retrovirus (Fig. 1A, indicated as aa on the coding strand) had been deleted at the chromosome/provirus junction, as is characteristic of retroviral integration. The analysis showed also that retroviral insertion had occurred near a polypurine/polypyrimidine (Pu/Py) sequence (23), which typically is seen in S1 hypersensitive regions located at 5' to transcribed genes.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   A, map and structure of genomic DNA in the region of integration of MoMuLVlac in cell line 354-6. The scale at the right indicates DNA length in base pairs for each part of the figure. The solid horizontal lines represent introns. Exon I is indicated by the shaded box. Restriction endonuclease cleavage sites are indicated, as are the long terminal repeat (LTR) and lacZ segments (open boxes) of the retrovirus construct. Enlargement of the region containing the provirus integration site is shown in the lower part of the figure. The nucleotide sequences shown by bold letters indicate the chromosomal sequence, whereas the lowercase letters indicate the MoMuLVlacZ/chromosomal DNA junction. The letters A, B, and C above the lacZ box mark the three splice acceptor sites on the retroviral construct as shown in B. The location of the polypurine/polypyrimidine (Pu/Py) is indicated. Exon I is spliced to lacZ via splice acceptor site B to produce fusion cDNA. B, sequences at the junction of the fused cDNA derived from cell line 354-6. The sequences at the three splice acceptor sites 5' to the lacZ reporter gene of the retroviral vector are indicated. The translation initiation codon ATG used for synthesis of -galactosidase is underlined in the fusion cDNA diagram. The boxed areas indicate the lacZ segment of the fusion cDNA, as determined from the cDNA sequence.

Eight independent cDNAs derived from lacZ-containing fusion transcripts contained sequences of a gene that corresponded to the genomic DNA segment we had identified. This gene was designated as Gas8. Further analysis indicated that the retrovirus had inserted into the first exon of Gas8 and that an ATG codon in-frame with lacZ was generated at the splice junction of the chromosomal and retrovirus components of the fusion transcript (Fig. 1B). These findings are consistent with the earlier observation that 354-6 cells synthesize a LacZ fusion protein that is approximately the same size as native LacZ (13).

Analysis of Gas8 cDNAs derived from native 3T3 cells showed two different species. These were identical through nucleotide 1126, where the sequences diverged, implying alternative splicing of Gas8 transcripts near the 3' end. The open reading frames (ORFs) of 334 and 489 amino acids encoded by the two transcripts specify putative proteins that have calculated molecular masses of 40 and 57.9 kDa, respectively. The longer Gas8 transcript (GenBank^TM accession number U19859) contains the hexanucleotide GAGGAC corresponding to the Kozak consensus sequence (24) immediately 5' to the predicted start site of the translation. Following the translational open reading frame (ORF) in the transcript is an AUUUA sequence, which has been implicated in determining mRNA stability (25). A polyadenylation signal (26), AAUGAAA, is located 20 nucleotides upstream from the poly(A) tract present at the 3' end of this transcript.

Gas8 Is Expressed Predominantly in the Testes, and Its Expression Is Developmentally Regulated-- We used Northern blot analysis of total RNA isolated from various tissues of adult mice to investigate possible tissue-specific differences in the expression of Gas8. These experiments identified a 1.8-kb Gas8-specific transcript made predominantly in the testes along with much smaller amounts of two larger transcripts detected by the same probe (Fig. 2A). Limited amounts of the 1.8-kb transcript (approximately one-tenth the steady-state concentration in testes) were detected also in the kidney, cerebrum, and female adnexa (ovary and fallopian tube) (Fig. 2A). Consistent with our Northern blot results, immunoprecipitation and Western blot analyses using rabbit polyclonal antibodies raised against the bacterially expressed Gas8 protein showed prominent accumulation of Gas8 protein, which approximated a 60-65-kDa position, in adult mouse testes and also a small amount of Gas8 in female adnexa (Fig. 2B). The size of the protein detected in both tissues is slightly larger than the predicted product of the ORF encoded by the Gas8 transcript we isolated.

View larger version (76K): [in this window] [in a new window]   Fig. 2.   Gas8 mRNA and protein are predominantly expressed in adult mouse testis. A, 20 µg of total RNA from various tissues were used for Northern blot analysis using [-32P]dCTP-labeled probe synthesized on full-length Gas8 cDNA by random priming. The left panel shows ethidium bromide-stained 28 and 18 S rRNAs on the preblotted gel. The right panel is the autoradiogram of the same gel showing the relative abundance of Gas8 mRNA in various tissues as indicated. Significant degradation of RNA preparations from the spleen and lung was observed. B, proteins extracted in radioimmune precipitation buffer (750 µg/lane) from various adult mouse tissues as indicated were used for immunoprecipitation by preimmune or antiserum conjugated to Sepharose CL-4B beads. The immunoprecipitated products were examined by Western blot analysis using anti-Gas8 antiserum. I, samples immunoprecipitated by anti-Gas8 antibody; P, samples immunoprecipitated by preimmune antibody. The bands specifically detected in female adnexa and testes by anti-Gas8 antibody are indicated by dots located to their left. The Gas8 protein detected by our polyclonal antibody migrates at a position corresponding to a molecular mass of 60 to 65 kDa, which was slightly larger than the mass deduced from the predicted Gas8 amino acid sequence. The major band migrating between 50 and 55 kDa represents the IgG heavy chain as indicated. The protein detected from muscle tissues was performed along with a testis tissue sample as shown on the last two wells.

Northern blot analysis of testis mRNA isolated from animals of various ages showed that Gas8 transcription is developmentally regulated during pubertal maturation (Fig. 3). Only a small amount of Gas8 transcripts was detected in neonates and young adolescents (through day 15) when the germ cells in the first wave of spermatogenesis reach the early pachytene stage. However, Gas8 mRNA increased dramatically by day 20, in which germ cells in the majority of tubules reach late pachytene stage, and early spermatids are found in a small number of tubules (27) and reached a plateau at day 30, when mature spermatozoa are produced. Transcription of Gas8 then continued at a high level throughout adulthood (Fig. 3).

View larger version (88K): [in this window] [in a new window]   Fig. 3.   The expression of Gas8 mRNA is regulated in developing testis. Twenty µg of total RNA isolated from different stages, i.e. postnatal day 0/1, 5, 10, 15, 20, 25, 30, and adult as indicated, of developing testis were used for Northern blot analysis using random primed [-32P]dCTP-labeled Gas8 cDNA probe. The lower panel shows comparable loading of RNA in different lanes by probing the same blot with a specific mouse -actin probe.

Association of Gas8 Protein Production with Later Stages of Spermatogenesis and Specifically with Production of the Sperm Motility Apparatus-- Immunohistochemical analyses to localize the site(s) of Gas8 protein in adult testes showed that Gas8 accumulated in gametocytes as they matured and approached the lumen of seminiferous tubules (brown precipitates in Fig. 4A versus controls in Fig. 4B). In particular, Gas8 was present in round spermatids, in the tails of elongating spermatids, and in mature spermatids present in the lumen of seminiferous tubules, all of which are cells in the postmeiotic stages of spermatogenesis (e.g. spermiogenesis). In contrast, no Gas8 protein was detected in gametocytes at early stages of spermatogenesis. Although a small amount of immunoreative material was found also in hormone-producing Leydig cells, which like mature spermatozoa are in a terminally differentiated state (28), this signal was not reversed by preabsorption of antibody with Gas8 protein, suggesting that it is nonspecific.

View larger version (97K): [in this window] [in a new window]   Fig. 4.   Gas8 protein in mouse testes sections predominantly detected in gametocytes. Cross-sections of perfusion-fixed adult mouse testis were subjected to immunoperoxidase staining by the avidin-biotin-peroxidase complex method followed by DAB-hydrogen peroxide detection as described under "Experimental Procedures." The brown colored precipitate indicates the positive reaction by anti-Gas8 antibodies. The nuclei were counterstained with Mayer's hematoxylin. A, Gas8 protein was detected at the tails, which projected into the lumen of the seminiferous tubules, of spermatids and in the cytoplasm of the Leydig cells, which surrounded the small vessels in the testis. B, the cross-sectional view of seminiferous tubule stained by anti-Gas8 antibody preabsorbed with purified Gas8 protein isolated from E. coli showing the absence of the signal detected in the sperm tails but not Leydig cells in A. The scale bar indicates 50 µm.

In the adult mouse testis, the seminiferous epithelium undergoes cyclic changes during spermatogenesis, and sections showing seminiferous tubules at the various stages of spermatogenesis contain collections of gametocytes developing in synchrony (29). The presence of Gas8 protein in seminiferous tubules was strikingly correlated by immunohistochemical analysis with the stage of spermatogenesis and, specifically, with the transition from round to elongated spermatids (spermiogenesis). Whereas the Gas8 protein was not detected in gametocytes in the premeiotic stages of spermatogenesis, sections of seminiferous tubules containing gametes at different stages of postmeiotic spermatogenesis (29) showed markedly different staining patterns by anti-Gas8 antibody. Gas8 was detected specifically in tubules in which short tails of elongating spermatids are embedded in the cytoplasm of Sertoli cells (Fig. 5, A and D), tubules in which elongating spermatids have moved to the apical region of Sertoli cells (Fig. 5, B and E), and tubules in which tail bundles of mature spermatids still embedded in Sertoli cells protrude into the lumen (Fig. 5, C and F). The staining of gametocytes in these tubules by anti-Gas8 antibody confirmed that Gas8 protein accumulation is associated with later stages of spermatogenesis and further suggested a possible association of Gas8 with tail maturation in postmeiotic gametocytes.

View larger version (88K): [in this window] [in a new window]   Fig. 5.   Association of Gas8 protein production with later stages of spermatogenesis. Seminiferous epithelium showing cyclic changes during spermatogenesis were examined by immunohistochemical methods as in Fig. 3, except that methyl green rather than Mayer's hematoxylin was used as nuclear stain. A and D, section of seminiferous tubules showing regions at stages IX to XII. The short tails of elongating spermatids, which were embedded in the cytoplasm of Sertoli cells, were positively stained by anti-Gas8 antibody. B and E, section of seminiferous tubules at stages I to VI. The cytoplasm of round spermatids and the tails of elongating spermatids, which have moved to the apical region of Sertoli cells, are stained with anti-Gas8 antibody. C and F, longitudinal section of seminiferous tubules at stages VII to VIII. The longer tails of elongated spermatids, which form bundles in the lumen, are strongly stained with anti-Gas8 antiserum. The size markers indicate 30 µm in A, B, and C and 15 µm in D, E, and F.

Consistent with the above findings, immunofluorescent staining of epididymal sperm indicated that Gas8 protein is present also in the flagella of mature spermatozoa (Fig. 6A, top panel); control experiments using preimmune serum showed only nonspecific staining of proteins in the sperm head (Fig. 6B, top panel).

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Detection and localization of Gas8 protein in flagella of mouse epididymal sperm and at the apical portion of bronchial or fallopian epithelium in mouse lung and fallopian tube, respectively. Top panel, confocal microscopy of mature sperm collected from the cauda epididymis of adult mouse shows specific localization of Gas8 protein in the sperm flagella. Epididymal sperm stained with preimmune serum (B) instead of anti-Gas8 antibody (A) as negative control. Sperm samples were prepared as described under "Experimental Procedures." The scale bar indicates 10 µm in length. Middle panel, immunolocalization of Gas8 protein shows Gas8 protein localized at the apical portion of bronchial epithelium (A) and fallopian epithelium (C). B and D are adjacent sections of A and C, respectively, which were stained with anti-Gas8 antibody preabsorbed with recombinant Gas8 showing decreased signals of Gas8 specifically at the apical regions. E, higher magnification view of lung section stained with anti-Gas8 antiserum showing brown precipitate was located at the apical portion of ciliated epithelial cells. The scale bars represent 100 µm (in A and B), 50 µm (in C and D), and 10 µm (in E), respectively. Tissue sections with 4-5 µm in thickness were collected and prepared for immunohisto- chemical study as described under "Experimental Procedures." Immunohistochemical localizations of Gas8 in mouse lung and fallopian tube were performed as in Fig. 5, except that methyl green rather than Mayer's hematoxylin was used as the nuclear stain. Bottom panel, mouse ciliated respiratory epithelial cells were brushed from adult mouse pulmonary bronchi and were stained with anti-Gas8 antiserum (A) or preimmune serum (B) using the same method described for Fig. 5. The arrows indicate the region of cilia. The scale bar represents 10 µm. Confocal images were recorded by Carl Zeiss confocal microscope LSM510.

The association of Gas8 expression with later stages of sperm cell differentiation and our finding that Gas8 protein is present in both tails of elongating spermatids and flagella of mature epididymal spermatozoa raised the prospect that Gas8 may have a role in the action of motile cellular appendages of male gametocytes. This notion was supported by the finding that the Gas8 protein also accumulated selectively in the cilia of epithelial cells lining both bronchi (shown as brown precipitates in Fig. 6, middle panels A and E; as arrowheads in bottom panel A) and fallopian tubes (brown precipitates in Fig. 6, middle panel C; see also controls in Fig. 6, middle panels B and D and bottom panel B).

Absent Gas8 Expression in Testis of Infertile Human Males Lacking Gametocytes-- Consistent with the selective occurrence of Gas8 protein in mouse gametocytes, testes biopsies from seven Sertoli cell-only syndrome patients, which lack gametocytes (30) (Fig. 7C), showed no detectable Gas8 staining in seminiferous tubules (Fig. 7F). In contrast, immunohistochemical staining of biopsied testes from fertile human males or from five adult males with obstructive azoospermia all showed Gas8 protein in the cytoplasm of round spermatids, the tails of elongating spermatids, and testicular spermatozoa (Fig. 7, D and E).

View larger version (149K): [in this window] [in a new window]   Fig. 7.   Gas8 expression in human testes from infertile males. Tissue sections from human testes biopsies taken from two males being evaluated for infertility (B and C) is compared here with the tissue from the testis of a fertile male whose testis was resected for the treatment of prostate cancer (A). The sections from infertile males show seminiferous tubules in the testes of patients diagnosed as having Sertoli cell-only syndrome (C) or obstructive azoospermia (B). Negative controls for immunohistochemical staining used preimmune serum at the same dilution as primary antibody (G-I). A-C, photomicrographs of human testis sections stained with hematoxylin and eosin. D-F, immunohistochemical staining of human testis sections using anti-Gas8 serum as primary antibody. Immunohistochemical staining and negative controls were adjacent sections of the same tissue block for each patient. The brown precipitate indicates immunoreactive material except for I showing a nonspecific staining. The scale bar indicates 30 µm.

The Gas8 Protein Sequence Is Highly Conserved in Flagellated Protozoa, Drosophila, and Humans-- Recently, a human gene of unknown function was given a Gas gene designation on the basis of its similarity to the GenBank^TM sequence we deposited for Gas8 and was named GAS11 (15). The T lymphocyte triggering factor of Trypanosoma brucei (GenBank^TM accession number AF012853) (31) shows 59% amino acid similarity and 35% identity with Gas8 (and with GAS11) along the entire sequence of both proteins. Using mouse Gas8 DNA sequences as a probe, we performed a TBLASTN data base search and identified a 721-nucleotide EST (clone LD14709) that has significant sequence alignment to the mouse gene in a cDNA library prepared from D. melanogaster embryo mRNA. This EST encodes an open reading frame whose amino acid residues have 61% similarity and 43% identity to the mouse Gas8 protein and its human ortholog, GAS11 (Fig. 8). Amino acid residues and motifs that are conserved between the mouse and Drosophila Gas8 proteins are distributed over the entire length of these polypeptides. The Drosophila ortholog of Gas8 (GenBank^TM AE003427) is located on the X chromosome.

View larger version (115K): [in this window] [in a new window]   Fig. 8.   The predicted amino acid composition of Gas8 is highly conserved in organisms ranging among flagellated protozoa, Drosophila, and humans. Alignments of amino acids predicted to be encoded by the murine Gas8 gene, the T. brucei, the T lymphocyte triggering factor (TLTF), the D. melanogaster LD14709 EST (GenBankTM number AF468957), and the human GAS11 gene. Shaded areas indicate amino acids in common at specific positions of the proteins.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gas8, a gene whose product is preferentially induced in growth-arrested murine fibroblasts growing in culture is shown here to be expressed in vivo in testes during postmeiotic development of male germ cells. Our finding that Gas8 is produced in the later stages of differentiation of spermatozoa is reminiscent of results obtained for another developmentally regulated Gas gene we have studied, Gas7, which is expressed predominantly during terminal differentiation of Purkinje neurons (14).

During the premeiotic phase of spermatogenesis, diploid spermatogonia and their progeny divide mitotically to generate large numbers of germ cells. Later, the first and the second divisions of meiosis yield haploid gametocytes; it is in this phase (i.e. spermiogenesis) that biochemical and morphological differentiation take place to produce mature spermatozoa (29). This entire process begins at puberty, which is also when Gas8 transcription starts to rise. Gas8 mRNA is present prominently by postnatal day 20 and continues to increase through day 30, reaching a plateau that persists through adulthood.

From 17 to 19 days, the majority of seminiferous tubules contain late pachytene spermatocytes and by 22 to 24 days, early (round) spermatids are detected in many tubules (32). Despite the accumulation of Gas8 transcripts during this period, as shown by Northern blotting, only a small amount of Gas8 protein was detected in round spermatids by immunocytochemical analysis, suggesting that translation of Gas8 mRNA is delayed until later in spermatogenesis. Consistent with this interpretation, despite the presence or a low level of Gas8 mRNA from birth (Fig. 3), no Gas8 protein was detected until after testes maturation. Similar transcription and translation patterns have been observed for other genes regulated during gametogenesis (16). As transcription is known to cease in germ cells during midspermiogenesis and protein production is regulated during spermiogenesis by post-transcriptional mechanisms (16, 33), the postmeiotic developmental regulation of Gas8 production we have observed seems likely to occur at the level of protein synthesis.

Localized expression of Gas8 was also observed in the cilia of epithelial cells lining mouse bronchi and fallopian tubes, although the steady state levels of Gas8 mRNA and protein in these structures were far lower than in adult testes. The cilia of bronchi and fallopian tubes are structurally similar to sperm cell flagella and have a similar mechanism of movement (34, 35).

Analysis of the amino acid composition of the Gas8 protein revealed that it is rich in hydrophilic amino acids. It contains 20% positively charged residues (Asp + Glu = 90/489) and 20% negatively charged residues (Arg + Lys = 90/489). There is no typical signal peptide sequence in the N terminus; however, a putative nuclear localization signal (¹⁶⁵KKMKMLRDELDLRRKTE) was observed toward the center of the protein, raising the prospect that Gas8 may be transiently translocated into the nucleus during some stage of spermatogenesis. The Gas8 protein sequence and particularly domains extending from amino acids 47 through 76 and 198 through 260 is remarkably conserved in multiple organisms (Fig. 8). Recent evidence that a trypanosomal protein T lymphocyte triggering factor localizes near the flagellar pocket (36) and contains sequence similarity to Gas8 (31) is consistent with the notion that Gas8 and related proteins may carry out a motility function expressed in organisms ranging from protozoa to humans. However, other functions that Gas8 may have cannot be excluded because Gas8 protein is present in round spermatids, which lack flagella.

GAS11, the human counterpart of Gas8, has 87 and 96% identity at the nucleotide and amino acid sequence level, respectively, with the murine gene (Fig. 8). The human sequence maps to chromosome 16q24.3, a locus frequently associated with loss of heterozygosity in breast, prostate, hepatocellular, and ovarian carcinomas and for this reason was initially suspected of being a tumor suppressor gene (15). Anti-mouse Gas8 antibodies detected GAS8/GAS11 in the flagella of human sperm (data not shown) and in the testes of both fertile human males and males having obstructive azoospermia. However that GAS8/GAS11 protein is absent from the testes of infertile human males with Sertoli cell-only syndrome, which is associated with an absence of gametocytes in seminiferous tubules (30), is consistent with evidence that Gas8 expression in mouse testes occurs specifically in gametocytes. Given the localization of Gas8 in motile cellular appendages of both sperm and bronchial epithelial cells, it is of some interest that a clinical association between human male infertility due to reduced sperm motility and respiratory disease due to immotile cilia of bronchial epithelial cells has been observed (37). Similarly, ciliary dyskinesis in the human respiratory tract has been associated with female infertility due to dysfunction of cilia in the oviduct (38).

Enhanced expression of at least 10 separate Gas genes has been found in cultured growth-arrested NIH3T3 cells by using cDNA subtraction (39) and retrovirus-based gene-trapping vectors (13, 14, 40). Like Gas8, Gas7 is expressed predominantly in terminally differentiated cells (14). Like Gas8, Gas6, a ligand for Tyr-3 receptor tyrosine kinase families (41-43), is expressed predominantly in testes; however, unlike Gas8, the Gas6 protein is localized primarily to Sertoli and Leydig cells. Recent work has shown that knockout mice lacking Gas6 as well as other multireceptor tyrosine kinases are viable, but infertile, and show disordered spermatogenesis and reduced numbers of mature epididymal sperm (44).

	ACKNOWLEDGEMENTS

We thank Marco Conti and Margaret T. Fuller for helpful advice and suggestions.

	FOOTNOTES

^* These studies were supported by an intramural fund and a Program Project Grant from Academia Sinica, by the grant of Frontier Sciences from the National Science Council (NSC) of Taiwan (to S. L. C.), and by Grants NSC84-0412/2331-B001-094-Y and HG00325 from the NSC and National Institutes of Health, respectively (to S. N. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF468957.

^¶ These authors contributed equally to this work.

This study fulfilled in part the requirements for the Ph.D. thesis, Graduate Institute of Medical Sciences, Taipei Medical University, Taiwan.

To whom correspondence and reprint and material requests should be addressed. Tel.: 886-2-2789-9218; Fax: 886-2-27826085; E-mail: mbsue@ccvax.sinica.edu.tw.

Published, JBC Papers in Press, January 7, 2002, DOI 10.1074/jbc.M106941200

	ABBREVIATIONS

The abbreviations used are: MoMuLV, Moloney murine leukemia virus; EST, expressed sequence tag; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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