Glyceraldehyde 3-Phosphate Dehydrogenase-S Protein Distribution ...

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Numbers indicate steps of spermatids nearest each tubule lumen. a) x250, b) X125 (reproduced at ... cated (Tissue Tearor; Biospec Products, Inc., Racine, WI).
BIOLOGY OF REPRODUCTION 58, 834-841 (1998)

Glyceraldehyde 3-Phosphate Dehydrogenase-S Protein Distribution during Mouse Spermatogenesis' Donna 0. Bunch,2 ,3 Jeffrey E. Welch, 4 Patricia L. Magyar, 5 E.M. Eddy, 3 and Deborah A. O'BrienS Gamete Biology Section,3 Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 United States Environmental Protection Agency, 4 Research Triangle Park, North Carolina 27711 The Laboratories for Reproductive Biology,5 Departments of Pediatrics and Cell Biology & Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599 ABSTRACT The spermatogenic cell-specific isoform of glyceraldehyde 3phosphate dehydrogenase (GAPD-S) may regulate glycolysis and energy production required for sperm motility. Although the steady-state level of Gapd-s mRNA ismaximal at step 9 of mouse spermatogenesis, GAPD-S protein was not detected by immunohistochemistry until steps 12-13. This result suggests that Gapd-s is translationally regulated. Western blot analysis of isolated germ cells confirmed that GAPD-S is not detected in pachytene spermatocytes or round spermatids. A major immunoreactive protein migrating with a molecular weight (Mr) of 69 200 was observed in condensing spermatids and cauda sperm. Additional minor proteins that migrated at M, 55 200, 32 500, and 27 500 were detected in sperm. The molecular weight of GAPD-S ishigher than the predicted molecular weight of 47 445, apparently due to a proline-rich 105-amino acid domain at the N-terminus. Recombinant GAPD-S protein lacking the proline-rich region migrated at Mr 38 250, comparably to somatic GAPD, which also lacks the proline-rich domain. Indirect immunofluorescence demonstrated that GAPD-S isrestricted to the principal piece in the sperm flagellum. Western blot analysis indicated that GAPD-S is tightly associated with the fibrous sheath of the flagellum, consistent with a potential role in regulating sperm motility. INTRODUCTION Although glycolysis is a highly conserved process for energy production, several enzymes in this pathway have spermatogenic cell-specific isozymes [1]. These glycolytic enzymes having a protein isoform or mRNA found only in spermatogenic cells include hexokinase, phosphoglucoisomerase, aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPD-S), phosphoglycerate kinase, enolase, and lactate dehydrogenase. A mouse gene has been identified for a unique glyceraldehyde 3-phosphate dehydrogenase (Gapd-s) that is expressed only in spermatids [2-4]. Before GAPD-S was identified, it had been suggested that GAPD is a key regulator of glycolysis in spermatogenic cells and the target of compounds that suppress sperm glycolysis and fertility [5, 6]. Lactate is the preferred energy substrate for round spermatids in the rat [7-10], mouse [11], hamster [12], and, presumably, other mammals. Incubation of isolated round Accepted October 31, 1997. Received September 9, 1997. 'This work was supported in part by the Andrew W. Mellon Foundation and NIH Grants U54HD35041 (The Laboratories for Reproductive Biology) and CA16086 (UNC Lineberger Comprehensive Cancer Center). 2Correspondence: Donna O'Dell Bunch, P.O. Box 12233 MD C4-04, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. FAX: (919) 541-3800; e-mail: [email protected]

spermatids in the presence of glucose without other energy substrates soon results in a depletion of ATP and cell death [10, 13, 14]. The accumulation of fructose 1,6-diphosphate and trioses following addition of glucose [10, 13, 14] indicates that an enzyme downstream of phosphofructokinase is inhibited. Metabolic intermediates downstream of GAPD do not accumulate, providing evidence that GAPD is inhibited, thereby limiting glycolysis in round spermatids [13-15]. In contrast to round spermatids, sperm have a high glycolytic capacity [16] and are able to utilize glucose effectively. In the mouse, glucose is required for hyperactivated motility of sperm [17, 18] and for fertilization in vitro [19]. Glycolysis apparently serves as the major source of energy production during fertilization, since neither pyruvate nor lactate can substitute for glucose as an energy substrate [19] and since inhibitors of oxidative phosphorylation do not affect fertilization in vitro [17]. Glucose is also required for hyperactivated motility of sperm from other species including humans [20], guinea pigs [21], and macaques [22]. The absence of glucose significantly decreases human fertilization rates in vitro [23, 24]. In the absence of hyperactivated motility, sperm fail to penetrate the zona pellucida in both humans [20] and mice [25]. GAPD-S appears to be the target of several reproductive toxicants that adversely affect male fertility, including atchlorohydrin, epichlorohydrin, 6-chloro-6-deoxyglucose, and ornidazole. These compounds are converted to the common metabolite (S)-3-chlorolactaldehyde, a competitive substrate inhibitor of the GAPD isoform in sperm that reduces fertility in several mammalian species [6, 26-31]. Infertility mediated by (S)-3-chlorolactaldehyde is associated with reduced motility and reduced enzymatic activity in sperm, at concentrations that do not inhibit activity of the GAPD isoform in somatic tissues [32]. The mouse Gapd-s gene encodes a protein of 438 amino acids, whereas the Gapd gene expressed in somatic cells encodes a protein of 333 amino acids [33]. The additional 105 amino acids of GAPD-S are on the N-terminus; 49.5% of these residues are proline. The overall homology between GAPD-S and somatic GAPD is 71%. The cysteine required for substrate binding is conserved, as are seven of eight amino acids that form the NAD + binding pocket [2]. Northern blot analysis and in situ hybridization studies have shown that Gapd-s mRNA is present only in round and condensing spermatids, first appearing during the cap phase of spermiogenesis [2, 4]. The present studies have examined the distribution of the protein product of this developmentally regulated, male germ cell-specific gene. We have used antibodies specific for GAPD-S to define the temporal and spatial location of 834

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this glycolytic isozyme in mouse germ cells during spermatogenesis and in mature sperm. MATERIALS AND METHODS Tissue and Cell Preparations CD-1 mice were obtained from Charles River Laboratories (Raleigh, NC). New Zealand White rabbits were obtained from Covance Research Products (Denver, PA). Testes, cauda epididymal sperm, and skeletal muscle were collected from adult mice. Fibrous sheaths were prepared from cauda epididymal sperm by the procedure of Olson et al. [34] with modifications as described previously [35]. Round spermatids and condensing spermatids were isolated from adult mouse testes by sequential enzymatic dissociation and unit gravity sedimentation [36, 37]. Pachytene spermatocytes were isolated in a similar fashion from testes of 17-day-old mice [38]. Sertoli cells were isolated from 17-day-old mice as described previously [39]. Protocols for experiments involving animals were approved by the NIEHS Animal Care and Use Committee and are in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.

FIG. 1. Peptides used to generate antisera specific for GAPD-S and somatic GAPD. Antisera specific for GAPD-S and somatic GAPD were prepared against peptide sequences in the regions shown. Peptides A and B correspond to regions from GAPD-S that differ substantially in amino acid sequence from those of somatic GAPD, and do not include the conserved amino acids that form the NADI binding pocket or the thioester substrate binding site. Peptide A contains 37 amino acids corresponding to amino acids 159-194 in the GAPD-S sequence (KGNVEHKNGQLVVDNLEINTYQCKDPKEIPWSSIGNP). Peptide B contains 14 amino acids corresponding to amino acids 293-306 (PSKKDWRGGRGAHQ). Peptide C corresponds to the region of somatic GAPD that is homologous to peptide A in GAPD-S; 24 of 37 amino acids are different (NGTVKAENGKLVINGKPITIFQERDPTNIKWGEAGAE).

Recombinant Protein Production Recombinant GAPD-S protein was prepared using the QiaExpress system (Qiagen, Santa Clarita, CA). A Gapd-s expression cassette was constructed with a Gapd-s cDNA generated by polymerase chain reaction amplification with 30-mer primers incorporating a 5' BamHI site and a 3' Bgl II site (upper primer, taggagagGGATCCgtgggtatcaatggatt; lower primer, ggccttttAGATCTcttctctcggctaaac). The insert was cloned into the BamHI and Bgl II sites of the pQE16 expression vector. Protein expression was induced by the addition of isopropylthiolgalactoside (10 mM) to mid-log phase bacterial cultures. The expressed protein contained an initial sequence of Met-Arg-Ser-Gly derived from the cloning vector. The first GAPD-S derived residue was Val08; therefore, the proline-rich region of GAPD-S was eliminated. The remainder of the protein consisted of the GAPD-S sequence through Lys 438 plus a carboxyl-terminal sequence (Arg-Ser-[His] 6). The formation of insoluble inclusion bodies was minimized by growing bacterial cultures at 25°C. After isopropylthiolgalactoside induction, bacteria were harvested by centrifugation at 3000 x g for 30 min and lysed in an 8 M urea solution, pH 8.0 (8 M urea, 100 mM NaH 2PO 4, 10 mM Tris). Unsolubilized material was removed by centrifugation. Recombinant GAPD-S protein was purified by nickel-agarose column chromatography according to the manufacturer's instructions. Protein was eluted with a pH step gradient using 8 M urea at pH 6.3, pH 5.9, and pH 4.5. Approximately 90% of recombinant GAPD-S protein eluted at pH 5.9, and those fractions were used in subsequent experiments. Antisera Production Peptides derived from the predicted sequence of mouse Gapd-s and rat somatic Gapd were synthesized by the Protein Chemistry Laboratory in the Department of Chemistry, University of North Carolina at Chapel Hill. Peptide A contains amino acids 158-194 and peptide B contains amino acids 293-306 of mouse GAPD-S (Fig. 1). Peptide C contains amino acids 55-91 of rat somatic GAPD and corresponds in location to the region of GAPD-S that contains

peptide A (Fig. 1). Although the mouse GAPD sequence was unknown at the time, it has since been determined that 35 of the 37 amino acids in peptide C are identical in rat and mouse GAPD. Peptides were synthesized with amino groups at both ends for conjugation to bovine thyroglobulin with carbodiimide by the method of Culler and Negro-Vilar [40, 41]. New Zealand White rabbits were immunized with conjugated peptide in complete Freund's adjuvant and boosted with conjugated peptide in incomplete Freund's adjuvant after 10 days and monthly thereafter. Antisera were collected 7-10 days following each immunization, and antibody titers were determined by ELISA. Monoclonal antibody ATC was generated by immunizing BALB/c mice with dissociated testis cells from an adult Fisher rat as described previously [35, 42]. This antibody recognizes a polyproline epitope in a major Mr 67 000 protein of the mouse fibrous sheath [43]. Spent medium collected from cultures of hybridoma cells producing the IgMclass ATC antibody was used without further purification. Immunohistochemistry Testes were collected from adult mice, fixed in Bouin's solution, dehydrated in ethanol, embedded in paraffin, and sectioned at 8 m. Sections were incubated with GAPD-S antiserum Al or B1, either unblocked or blocked with antigenic peptide (1 pg/ml), followed by biotinylated goat anti-rabbit IgG and horseradish peroxidase (HRP)-conjugated avidin according to the manufacturer's instructions (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Immunocomplexes were detected using 3,3'-diaminobenzidine tetrahydrochloride (Aldrich Chemical Company, Milwaukee, WI) as the chromogen. Slides were counterstained with Gill's hematoxylin (Fisher Scientific, Pittsburgh, PA). Photomicrographs were taken with Ektachrome 160T film (Eastman Kodak, Rochester, NY).) Indirect Immunofluorescence (11F) Antisera Al and B1, raised against two different peptides derived from sequences unique to GAPD-S and not

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PLATE I. FIG. 2. GAPD-S protein is first detected in condensing spermatids. When mouse testis sections were immunostained with antiserum Al, GAPD-S protein was first detected in step 12-13 spermatids (a). Staining intensity increased in steps 14-16 of spermatids. Spermatid flagella in the seminiferous tubule lumen also stained intensely with the GAPD-S-specific antiserum. Immunoreactivity of antiserum Al was completely blocked by preincubation with 1 tg/ml peptide A (b). Numbers indicate steps of spermatids nearest each tubule lumen. a) x250, b) X125 (reproduced at 94%).

GAPD-S IN MOUSE SPERMATOGENESIS found in somatic GAPD, were employed to determine the cellular location of GAPD-S in sperm. The presence of somatic GAPD was assayed using antiserum C1. IIF was performed as previously described [44]. Briefly, washed cauda epididymal sperm were fixed by dilution with an equal volume of 3.7% formaldehyde solution (Mallinckrodt Chemical, Paris, KY) in PBS for 10-15 min; free aldehyde groups were blocked with 50 mM glycine in PBS. Sperm were permeabilized with methanol for 1 min at -20°C. After nonspecific reactivity was blocked with 10% goat serum in PBS (Life Technologies, Grand Island, NY) for 15 min at room temperature, sperm were incubated with the primary antibody for 2 h at 37C. Primary antibodies used were antisera Al, B1, and C1 (1:2000) or the same antisera that had been blocked previously by incubation with the antigenic peptide at 100 jgg/ml for 1-16 h at 4°C. After extensive washing in PBS, sperm were incubated for 1 h at 370C with fluorescein isothiocyanate-labeled, affinity-purified goat anti-rabbit IgG (Cappel/Organon Teknika Corp., West Chester, PA) diluted 1:100. Paired phase-contrast and fluorescence photomicrographs were taken with Ektachrome P800/1600 color reversal film (Eastman Kodak) at ASA 800. SDS-PAGE and Western Blot Analysis Isolated germ cells, Sertoli cells, washed epididymal sperm, and isolated fibrous sheaths were extracted with equal volumes of double-strength sample buffer (0.125 M Tris [pH 6.8], 4% SDS, 20% glycerol) supplemented with protease inhibitors (10 tig/ml each aprotinin, leupeptin, and para-aminobenzamidine). Dithiothreitol was added to a final concentration of 40 mM, and samples were boiled for 3-5 min. Testis and muscle tissues were placed in singlestrength sample buffer plus protease inhibitors and sonicated (Tissue Tearor; Biospec Products, Inc., Racine, WI) for approximately 1 min; residual tissue was removed by centrifugation. Protein samples were stored frozen at -20°C until use. Protein concentrations were determined by a Bradford protein assay (Sigma, St. Louis, MO) with BSA as the standard. Proteins were separated on 10% polyacrylamide slab gels (Bio-Rad, Hercules, CA) according to Laemmli [45] and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA) by the method of Towbin et al. [46]. Nonspecific reactivity of these blots was blocked by incubation with 4% nonfat dry milk in PBS for 1-2 h at room temperature or overnight at 4°C. Duplicate blots were incubated either with antiserum C1 (diluted 1:2000) or with C1 antiserum that had been previously blocked by incubation overnight at 4°C with the antigenic peptide at 100 ig/ml. Presumably, because of the enhanced sensitivity of chemiluminescence detection, a

FIG. 3. GAPD-S is localized in the principal piece of the sperm tail. Sperm from the cauda epididymis were fixed with 1.8% formaldehyde and permeabilized with methanol prior to immunostaining with GAPD-S antisera. Paired phase-contrast (a, c, e) and epifluoresence (b,d, f) photomicrographs of the same field are shown after staining with antiserum Al (a, b), antiserum Al blocked with peptide A (c,d), or antiserum C1 (e,f. GAPD-S immunoreactivity was restricted to the principal piece of the sperm flagellum (b) and was blocked completely by preincubation with peptide A (d). No somatic GAPD was detected in cauda sperm by immunostaining with antiserum C1, specific for somatic GAPD (f). However, epididymal epithelial cells present in the sperm preparation did react with antiserum C1 (arrows, f). a-f) x500 (reproduced at 94%).

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higher concentration of peptide was necessary to block antibody reactivity on blots than on tissue sections. The blots were stripped and subsequently probed with antiserum Al (diluted 1:4000) or with blocked Al antiserum. Antiserum B1 (diluted 1:20 000) was used as an additional probe for GAPD-S. Monoclonal antibody ATC, diluted 1:10, was used to assess the presence of an N-terminal polyproline domain in fibrous sheath proteins and truncated recombinant GAPD-S. After washing in Tris-buffered saline (TBS) containing 0.2% Tween (Bio-Rad), blots were incubated for 30-60 min at room temperature with the secondary antibody, either HRP-labeled goat anti-rabbit IgG (diluted 1: 10 000; Santa Cruz Biotechnology, Santa Cruz, CA) or HRP-labeled goat anti-mouse IgM (diluted 1:10000; Kirkegaard and Perry, Gaithersburg, MD). Reactivity was detected by enhanced chemiluminescence (ECL Western Blot Kit; Amersham, Arlington Heights, IL). Values for molecular weight were determined by averaging results from three gels. RESULTS Delayed Expression of GAPD-S Protein To determine when the GAPD-S protein is synthesized during spermatogenesis, GAPD-S-specific antisera were used to immunostain mouse testis sections. Although peak steady-state levels of Gapd-s mRNA are reached by step 9 of spermiogenesis [4], the GAPD-S protein was first detected in steps 12-13 condensing spermatids with both Al (Fig. 2a) and B1 (not shown) antisera. Staining intensity increased during steps 14-16 of spermiogenesis. Flagella of condensing spermatids in the lumen of the seminiferous tubules were stained intensely with both GAPD-S antisera. Residual bodies remaining in the tubules at stage IX of the seminiferous cycle were also stained (not shown). Immunoreactivity of the Al antiserum was completely blocked by preincubation with 1 ig/ml peptide A (Fig. 2b). GAPD-S Protein Localizes to the Principal Piece of the Sperm Tail The localization of GAPD-S in sperm was examined by IIE. Fixed and permeabilized sperm from the cauda epididymis were immunostained with antisera specific for GAPDS or somatic GAPD. Paired phase-contrast and epifluorescence photomicrographs of the same field after staining with antiserum Al (Fig. 3, a and b), antiserum Al blocked with peptide A (Fig. 3, c and d), or antiserum C1 (Fig. 3, e and f) are shown. GAPD-S immunoreactivity was restricted to the principal piece of the sperm flagellum (Fig. 3b) and was blocked completely by preincubation with peptide A (Fig. 3d). Immunostaining with GAPD-S antiserum B 1 gave identical results (not shown). No somatic GAPD was detected in cauda sperm with antiserum C1, specific for the somatic isozyme (Fig. 3f). This antiserum did stain somatic cells present in the sperm preparation (arrows, Fig. 3f). Comparison of GAPD-S and Somatic GAPD by Western Blot Analysis To compare the expression of GAPD-S with that of somatic GAPD and to confirm specificity of the antisera, proteins extracted from mouse somatic tissues and germ cells were subjected to Western blot analysis. GAPD-S was detected in adult testis (T), condensing spermatids (C), and sperm (Z), but not in skeletal muscle (M), Sertoli cells (S), pachytene spermatocytes (P), or round spermatids (R) (Fig.

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FIG. 4. GAPD-S protein migrates at a higher-than-predicted molecular weight. Proteins extracted from several mouse tissues and cells were subjected to Western blot analysis. The blots were incubated with antiserum Al at a dilution of 1:4000 (a), with antiserum Al preincubated with peptide A (b), with antiserum C1 at a dilution of 1:4000 (c), or with antiserum C1 preincubated with peptide C (d). Exposure times were 3 min (a,b) or 3 30 sec (c,d). Molecular weights (x 10- ) are indicated on the left. GAPD-S (a)was detected in adult testis (T), condensing spermatids (C), and sperm (Z), but not in skeletal muscle (M), Sertoli cells (S), pachytene spermatocytes (P), or round spermatids (R). GAPD-S migrated with a molecular weight of 69 200. Additional proteins of smaller size were detected in sperm extracts. Reactivity of antiserum Al was completely blocked by preincubation with peptide A (b). Somatic GAPD (arrow, c) was detected in skeletal muscle (M) and Sertoli cells (S). Reactivity of antiserum Cl with somatic GAPD migrating at Mr 36 000 was blocked by preincubation with peptide C (d).

4a). Although the calculated molecular weight for GAPDS is 47 445, it migrated with a molecular weight of 69 200. Additional proteins migrating at Mr 55 200 and 32 500 were detected in sperm (lane Z). Although not visible in this exposure, another protein migrating at Mr 27 500 was also observed on immunoblots with antiserum Al (see Fig. 5). Reactivity of antiserum Al was blocked by preincubation with peptide A (Fig. 4b). Somatic GAPD (arrow, Fig. 4c) migrated at Mr 36 000, equivalent to its calculated molecular weight, and was detected in skeletal muscle (M) and Sertoli cells (S). After a longer exposure (2 min instead of 30 sec), a faint GAPD band at Mr 36 000 was detected in testis, but not in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm (not shown). Although several nonspecific bands were present in blots probed with both unblocked and blocked antiserum C1, the reactivity of this antiserum with somatic GAPD migrating at Mr 36 000 was blocked by preincubation with peptide C (Fig. 4d). Fibrous Sheaths Contain GAPD-S The fibrous sheath is a major cytoskeletal structure of sperm and is located in the principal piece of the flagellum [47]. To determine whether this structure contains GAPD-

S, as suggested by immunofluorescence results, proteins from mouse sperm (Z) and isolated fibrous sheaths (F) were subjected to Western blot analysis with antiserum Al, antiserum B1, or monoclonal antibody ATC (Fig. 5). Similar protein profiles were observed in sperm and isolated fibrous sheaths with antisera Al and B1. Proteins of Mr 69 200 and 55 200 were recognized by all three antibodies. Although proteins of Mr 32 500 and 27 500 were observed after immunoblotting with antisera Al and B1, these proteins were not detected with monoclonal antibody ATC. This antibody has been shown to recognize a polyproline epitope in proteins of the mouse fibrous sheath [43]. A Proline-Rich Domain Results in Anomalous Migration of GAPD-S Because proline-rich domains have been reported to cause anomalous migration of proteins in SDS-PAGE [4851], we investigated whether the proline-rich N-terminal domain of GAPD-S was responsible for the migration of the protein at Mr 69 200 rather than at the calculated molecular weight of 47 445. Recombinant GAPD-S protein that lacks this proline-rich domain was analyzed on Western blots with antisera Al and B1 and monoclonal antibody ATC. As shown in lane X of Figure 5, the truncated re-

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FIG. 5. Isolated fibrous sheaths contain GAPD-S. Proteins from mouse sperm (Z) and isolated fibrous sheaths (F)were processed for immunoblot analysis. Blots were incubated with antiserum Al (1:4000), antiserum B1 (1:20 000), or monoclonal antibody ATC (1:10). Molecular weights (x 10 3) are indicated on the right. Proteins with molecular weights of 69 200 and 55 200 (marked by *) were recognized by all three antibodies in extracts of sperm (Z) and isolated fibrous sheaths (F). Proteins of M, 32 500 and 27 500 were detected in sperm (Z) and fibrous sheaths (F) with antisera Al and B1, but not with monoclonal antibody ATC. Recombinant GAPD-S protein that lacks the proline-rich domain (lane X) migrates with a molecular weight of 38 250 and was detected with antiserum Al and B1. Monoclonal antibody ATC, which recognizes a polyproline epitope, does not react with this recombinant GAPD-S.

combinant GAPD-S protein migrated at Mr 38 250 rather than at Mr 69 200, as observed for GAPD-S from sperm and fibrous sheaths (lanes Z and F, respectively), when probed with GAPD-S-specific antisera Al or B 1. Recombinant GAPD-S, lacking the proline-rich domain, was not detected on immunoblots with monoclonal antibody ATC. DISCUSSION The GAPD-S protein was first detected in step 12-13 condensing spermatids in the mouse. In previous Northern blot analyses, the 1.5-kilobase Gapd-s mRNA was present in both round and condensing spermatids but was not detected in pachytene spermatocytes or Sertoli cells [2]. In situ hybridization corroborated these findings; Gapd-s transcripts first appeared during the cap phase of spermiogenesis (steps 4-7) and reached maximum levels in step 9-11 spermatids. Transcripts were maintained at this level in step 12-15 spermatids and then abruptly disappeared from step 16 spermatids just prior to spermiation [4]. Furthermore, we did not detect somatic GAPD in isolated germ cells by Western blot analysis. The lack of detectable GAPD or GAPD-S protein in pachytene spermatocytes or round spermatids is consistent with the low levels of GAPD enzyme activity reported for isolated rat germ cells at these stages of spermatogenesis [14, 15, 52]. The apparent delay between reaching peak steady-state levels of Gapd-s mRNA at step 9, as determined by in situ hybridization [4], and initial detection of the protein by immunostaining at step 12 suggests that Gapd-s is subject to translational regulation. Although the mechanism responsible for translational control of Gapd-s is unknown, several RNA-binding proteins have been implicated in the posttranscriptional regulation of gene expression in spermatids. These include testis/brain RNA-binding protein (TB-RBP) [53], Tenr [54], Prbp [55], Spnr [56], PABPt [57, 58], the Y-box proteins p54/p56 [59, 60], and MSY1 [61]. The 5' untranslated region of Gapd-s mRNA contains an H element to which TB-RBP could bind and cause repression of translation [53]. Whether one or more of these

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RNA-binding proteins may be involved in regulating translation of GAPD-S awaits further investigation. Since glycolysis is required for fertilization [17] but is ineffective in round spermatids [10, 13, 15], this metabolic pathway apparently becomes capable of activation during the late stages of spermatid differentiation, sperm maturation in the epididymis, or capacitation in the female reproductive tract. Thus far, glycolysis has not been examined during the later stages of spermiogenesis when GAPD-S protein accumulates. In some mammalian species, the rates of glycolysis [62, 63] and GAPD activity [27] are greater in sperm recovered from the cauda epididymis than in sperm from the caput epididymis. Furthermore, chloro-analogues of glyceraldehyde 3-phosphate that reduce fertility inhibit GAPD activity to a greater extent in cauda sperm than in caput sperm [27]. These earlier studies were carried out before it was known that a spermatogenic cell-specific form of GAPD is present in condensing spermatids and sperm. We have hypothesized that unique structural and functional features of GAPD-S are responsible for increased glycolysis in sperm and for the reproductive toxicity of chloro-analogues of glyceraldehyde 3-phosphate at low doses that do not affect somatic GAPD [1-3]. In mouse sperm isolated from the cauda epididymis, GAPD-S immunoreactivity was confined to the principal piece of the flagellum. The fibrous sheath is the major structural component that defines the principal piece, while the outer dense fibers and axoneme extend into other regions of the flagellum. Western blot analysis demonstrated that GAPD-S is present in fibrous sheaths isolated with 1% Triton X-100, 0.6 M potassium thiocyanate, 4 M urea, and 2 mM dithiothreitol, indicating that this protein is tightly associated with the fibrous sheath. Recently, Westhoff and Kamp [64] demonstrated that GAPD is bound to the fibrous sheath of boar sperm. Another glycolytic enzyme, lactate dehydrogenase-C, has been localized to the principal piece of the fox sperm flagellum [65]. The type 1 hexokinase isozyme (mHK1-S) that is unique to spermatogenic cells is also localized mainly in the principal piece of mouse sperm [66]. Previously, seven glycolytic enzymes, including GAPD and lactate dehydrogenase, were shown to be tightly associated with rabbit sperm following lysis with hypotonic solution [67]. It is important to note that these studies were done before the existence of the sperm-specific isozyme GAPD-S was known and therefore after that experiments were not designed to distinguish between GAPD and GAPD-S. Studies of somatic cells and tissues indicate that glycolytic enzymes form reversible associations with the cytoskeleton and other cellular structures and that these interactions modulate enzyme activities [68]. We have hypothesized that glycolytic enzymes might be sequestered by binding to structural components of sperm [1]. The association of these enzymes with the fibrous sheath may be similar to that reported for glycolytic enzymes with actin filaments in skeletal muscle [69], and this juxtaposition might be important for rapid generation of ATP through glycolysis. The localization of GAPD-S in the fibrous sheath is consistent with a role in regulating sperm motility. GAPD-S migrates more slowly than expected on SDS polyacrylamide gels, as has been reported for other prolinerich proteins [48-51]. The major immunoreactive protein migrated with a molecular weight of 69 200, rather than at the calculated molecular weight of 47 445. Recombinant GAPD-S protein that lacked the proline-rich 105-amino acid N-terminal region migrated at Mr 38 250, similarly to somatic GAPD. Deletion of 105 amino acids, correspond-

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ing to a peptide of approximately Mr 10 000, resulted in a Mr 30 950 reduction. These findings indicate that the proline-rich N-terminal region of GAPD-S is probably responsible for the slower-than-expected migration of the fulllength protein. Multiple protein bands were recognized on Western blots with GAPD-S antisera, most notably in sperm and isolated fibrous sheaths, despite the presence of protease inhibitors during extractions. Only the larger proteins (Mr 69 200 and 55 200) contain the N-terminal proline-rich domain of GAPD-S detected by monoclonal antibody ATC. These results suggest that the smaller 32 500 and 27 500 proteins recognized by antisera Al and B1 result from processing that eliminates the proline-rich region. Furthermore, the ATC antibody was used to isolate the original Gapd-s cDNA from an expression vector library prepared with mouse spermatogenic cell mRNA [2]. These results strongly suggest that the fibrous sheath protein previously named ATC [35] is probably GAPD-S. Proline-rich domains have a high potential for mediating protein-protein interactions [70] and appear to play a role in cellular localization and the formation of functional protein complexes [71]. It is possible that the proline-rich region of GAPD-S serves as an anchor to the fibrous sheath, and perhaps to other glycolytic enzymes, to facilitate the production of ATP in the sperm flagellum. In summary, we have found that GAPD-S is first detected in step 12-13 spermatids, well after maximum steady-state levels of Gapd-s mRNA are present, suggesting that Gapd-s is translationally regulated. Additionally, GAPD-S is localized to the fibrous sheath of the sperm flagellum. Sequestration of glycolytic enzymes adjacent to the motile apparatus of the sperm may be important for generation of energy to fuel hyperactivated motility required for fertilization. ACKNOWLEDGMENTS The authors would like to acknowledge the valuable contributions of Dr. Edward Shatte, Paula Brown, and K. Michelle Cobb to these studies. We are particularly grateful to William Willis for the preparation of mouse sperm fibrous sheaths. We also thank Drs. Patricia Saling, Jonathan Lindzey, and Bayard Storey for their helpful comments on the manuscript.

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