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a Department of Urology, Osaka University Medical School, Suita City, Osaka 565-0871, Japan
b Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka 565-0871, Japan
c Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka 565-0871, Japan
d First Department of Anatomy, Miyazaki Medical College, Kiyotake Town, Miyazaki 889-1692, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of the sperm mitochondria is a major specific event in spermiogenesis. The sperm mitochondria are specifically rearranged around the developed flagellum. The energy metabolism in mitochondria is important for their flagella movement and sperm viability. Phosphatidylcholine (PC) is the major constituent phospholipid in both the inner and the outer membrane of mammalian mitochondria [4]. Recently, endogenous PC has appeared to be a good candidate as a substrate for energy metabolism in sea urchin spermatozoa [5]. Moreover, PC accelerated the in vitro development of human acrosomal responsiveness [6]. The metabolism of PC may be an important part of sperm motility in mammals. However, the mechanisms by which PC is imported to mitochondria and those of acrosomal responsiveness are largely unclear.
Here we report a novel gene specifically expressed in haploid germ cells isolated from a subtracted cDNA library of mouse testis [7]. A cDNA-encoded phospatidylcholine transfer protein (PCTP)-like protein (PCTP-L) was present predominantly at the flagella in elongated spermatids through to sperm. The stage-specific expression of PCTP-L and specific localization in sperm flagellum suggest that it has specific roles in sperm maturation or fertilization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total RNA was extracted from the testis of 35-day-old C57BL/6 mouse with guanidine thiocyanate followed by CsCl centrifugation; polyadenylated RNA was then selected by oligo(dT)-cellulose chromatography [8]. The corresponding cDNA library was prepared as described by Gubler and Hoffmann [9] with some modifications; this method is called the linker-primer method [8]. The cDNA fragments were directionally inserted between the NotI (dephosphorylated) and BglII sites of pAP3neo vector. The ligated DNAs were electroporated into MC1061A cells as previously described [10]. The complexity of the cDNA library obtained was about 4 x 106 colony-forming units. The strategy for preparation of the subtracted cDNA library was described by Tanaka et al. [2] and Kobori et al. [11]. The library was generated by subtracting mouse testis mRNA 17 days old, just before spermiogenesis starts, from cDNAs of 35-day-old mouse testis containing all kinds of differentiated germ cells.
Plasmid DNA of each clone randomly selected from the subtracted cDNA library was screened by Northern blot analysis using mRNAs of testes taken from 17- and 35-day-old mice [7]. We named the clones expressed exclusively in 35-day-old testis haploid germ cell-specific sequence tags "Transcript Increased in Spermiogenesis (TISP)". One of these, TISP-81, showed significant homology with murine PCTP in the GenBank, EMBL, and DDBJ databases. To isolate the complete cDNA of TISP-81, MC1061A cells carrying a 35-day-old mouse testis cDNA library were selected with a 0.5-kilobase (kb) EcoRI-NotI fragment of the TISP-81 cDNA.
Northern Blot Analysis and DNA Sequencing
Freshly removed organs of an adult mouse (C57BL/6 strain) were homogenized in Trizol reagent (Gibco/BRL, Grand Island, NY). Germ and other somatic cells of the testes were prepared as described in our previous report [12]. Briefly, tunica albuginea was removed from each testis. Then seminiferous tubules were placed in 0.02 M Hepes and 0.1% collagenase and gently unraveled with forceps. The tubule suspension was left standing to precipitate tubule fragments. The supernatant containing separated cells was filtrated through a nylon mesh (NBC Industries Co., Ltd., Tokyo, Japan) and centrifuged. The precipitant was used as a Leydig cell fraction. Remaining tubules were dispersed in PBS containing 1 mM EDTA to remove residual Leydig cells. Tubules were cut into small fragments with a knife, transferred to a conical tube, and washed by pipetting in PBS containing 1 mM EDTA. The conical tube was left standing; the supernatant was filtrated through a nylon mesh and used as a germ cell fraction. The remaining sedimented tubules were vigorously pipetted a few times. The sample was then left standing. The sedimented sample was used as a tubule fraction (containing mainly Sertoli cells).
Total RNA was extracted according to the manufacturer's recommendation and quantified by optical density measurement. RNA samples containing 2.2 M formaldehyde were subjected to electrophoresis in a 1.0% agarose gel containing 0.66 M formaldehyde. RNAs were transferred to a nitrocellulose membrane filter in 20-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate). After baking for 2 h at 80°C, the membrane was preincubated in a solution containing 50% formamide, 4-strength SSC, 5-strength Denhardt's solution, 0.2% SDS, and 120 µg/ml of denatured sonicated salmon sperm DNA at 42°C for 12 h. Hybridization was performed with the 32P-labeled full-length pctp-l cDNA probe prepared by BcaBest random primer kit (Takara, Shiga, Japan) under the same conditions of preincubation for 24 h. The membrane was washed twice with a solution of 0.2-strength SSC and 0.1% SDS at 55°C for 30 min. Signals of the bands were detected by Image Analyzer (Fuji Film, Tokyo, Japan).
Dideoxy-chain-termination sequencing reactions [13] were performed with fluorescent dye-labeled primers and thermal cycle sequencing kits purchased from Li-Cor. The reaction products were analyzed by Model 4000 (Li-Cor, Lincoln, NE). GenBank, EMBL, DDBJ, Swiss-Prot, and PIR data banks were searched for homology with the isolated cDNA or deduced amino acid sequence.
In Situ Hybridization
Antisense digoxigenin (DIG)-labeled RNA was used for in situ hybridization. Testis of an adult mouse (C57BL/6) was directly frozen in OTC embedding compound (Tissue-Tek; Sakura Finetechnical Co., Ltd., Tokyo, Japan) and sectioned. The resulting cryostat sections (7 µm) were placed on a Superfrost micro-slide glass with silane coating (Matsunami Glass Ind. Ltd., Osaka, Japan). The sections were dried and fixed in a solution of 4% paraformaldehyde, 0.5% glutaraldehyde, and 0.5 M sodium phosphate buffer (pH 7.4). A pctp-l cDNA insert was subcloned into pBluescript II SK(-) at EcoRI and EcoRV sites. An antisense probe was generated by transcription of an EcoRI digest with T7 RNA polymerase, and a sense probe by transcription of EcoRV digest with T3 RNA polymerase. Probes were labeled with DIG-UTP (Boehringer Mannheim, Mannheim, Germany). In situ hybridization was performed as described previously [14, 15]. After hybridization, the bound probe was detected by incubating with anti-DIG-Fab fragments conjugated with alkaline phosphatase (Boehringer Mannheim), followed by a color reaction involving 4-nitro blue tetrazolium chloride (Boehringer Mannheim) and X-phosphate (5-bromo-4-chloro-3-indolyl-phosphate; Boehringer Mannheim). Sections were contrasted with 1% methyl green staining solution (Muto Pure Chemicals, Ltd., Tokyo, Japan) and examined under a microscope.
Preparation of Antiserum
The partial pctp-l cDNA fragment was subcloned into SmaI site of pGEX-2 vector and expressed as a glutathione S-transferase fusion protein [16]. The fusion protein was expressed in Escherichia coli by isopropyl beta-D-thiogalactopyranoside induction and purified with glutathione-agarose beads (Sigma Chemical Co. Ltd., Tokyo, Japan). Polyclonal antiserum was prepared by injection of the above antigen followed by booster injections at 2-wk intervals, 7 times in total, to Japanese white rabbits. The antiserum was mixed with acetone-fixed mouse spleen powder overnight at 4°C and centrifuged for 10 min at 13 000 x g. The clear upper layer in the centrifuge tube was used for Western blotting and immunohistochemical analyses.
Immunoblotting
Freshly removed organs of an adult mouse (C57BL/6 strain) were resuspended at 4°C with a lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, and 1 mM EDTA. After homogenization, samples were centrifuged at 13 000 x g for 20 min at 4°C to remove insoluble debris. Protein concentration was determined by the method of Bradford [17] with BSA as a standard. Each extract containing approximately 100 µg of protein was subjected to SDS-PAGE [18]. After electrophoresis, proteins were transferred to Immobilon filters (Millipore, Bedford, MA). The filters were blocked with 5% nonfat dry milk for 1 h at room temperature. The blocked filters were reacted with anti-PCTP-L rabbit antiserum diluted 1:100 in TBS for 12 h at 4°C, washed 3 times with 0.05% Tween 20 in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 5 min each, and incubated with anti-rabbit immunoglobulins (Ig) conjugated with peroxidase (1:500; Amersham Pharmacia Biotech, Tokyo, Japan) for 1 h at room temperature. After further washing under the same conditions, reactive bands were visualized by development with POD staining kit (Wako Pure Chem. Ind. Ltd., Osaka, Japan).
Immunohistochemistry
Testes were immersed in OTC embedding compound (Tissue-Tek) for frozen tissue specimens and frozen at -20°. Sections 10 µm in thickness were prepared by using a cryomicrotome (Microm HM 500OM, Woodstock, CT) and were fixed with 100% methanol at -20°C for 10 min. After blocking with 5% nonfat dry milk for 1 h at room temperature, each section was incubated with anti-PCTP-L rabbit antiserum diluted 1:500 in PBS for 12 h at 4°C. Then sections were reacted with anti-rabbit Ig conjugated with horseradish peroxidase (Amersham Pharmacia Biotech) for 1 h at room temperature and stained with 0.05% diaminobenzidine in 50 mM Tris-HCl (pH. 7.4) plus 0.3% H2O2. Some of the sections were counterstained with methyl green and examined under a microscope.
Immunocytochemistry
Sperm suspensions obtained from cauda epididymidis were placed onto a Superfrost micro-slide glass with silane coating (Matsunami) and treated with PBS on ice for 30 min. For indirect immunofluorescent staining, the slides were incubated with anti-PCTP-L rabbit antiserum diluted 1:500 in PBS for 12 h at 4°C. Then the slides were treated with fluorescein-linked anti-rabbit donkey Ig (Amersham Pharmacia Biotech) for 2 h at room temperature, washed with PBS, and observed under a fluorescent microscope (Olympus BX50, Tokyo, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To elucidate the mechanism of spermiogenesis, we prepared a cDNA library of adult mouse testis-subtracted mRNA of infant testis (17 days of age) [7], in which the cDNA clones showing haploid-specific expression were concentrated because haploid germ cells develop after the age of 17 days. Various clones randomly selected from the subtracted cDNA library were screened by Northern blot analysis using mRNA of testes taken from 17- and 35-day-old mice [7]. One of the clones, TISP-81, showed specific expression in haploid cells and significant homology with PCTP. To obtain the complete cDNA clone, 2 x 106 colonies from a pAP3neo mouse testis cDNA library were screened with the 32P-labeled 0.5-kb EcoRI-NotI fragment of the TISP-81 cDNA, and 25 positive clones were independently isolated. The three clones having the longest cDNA insert, about 1.4 kilobase pairs, were sequenced. They all had the same nucleotide sequences and one long open reading frame that started with a putative start codon at nucleotide 325 and terminated with a putative stop codon at 1198. The complete nucleotide and deduced amino acid sequences are shown in Figure 1 (DDBJ accession no. AB031550). The stop codon was located at nucleotide 7 upstream of the ATG sequence at 325, which we assumed to be the translation initiation codon of the 873 cDNA. The cDNA encoded 291 amino acids and contained a 3' untranslated region of 195 nucleotides with a poly(A)+ tail of about 18 bases, following a consensus AATAAA polyadenylation signal at position 1348.
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We did not find any genes having high homology with the cDNA by computer-mediated homology search of nucleotide sequences in GenBank, EMBL, and DDBJ. The deduced amino acid sequence of the cDNA showed, however, 28% identity and 49% similarity with mouse PCTP [19] and some homology with bovine and rat PCTP [20, 21]. The sequence alignment of bovine, rat, and mouse PCTP amino acids is shown in Figure 2. We named this newly isolated gene PCTP-like protein.
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Northern Blotting Analysis and In Situ Hybridization of the pctp-l Gene in the Testis
Messenger RNA expression of the pctp-l gene was detected by Northern blotting as a transcript of 1.4 kb, not only strongly in the testis, but also weakly in the liver, kidney, and intestine. No transcript was detected in the brain, heart, lung, spleen, muscle, or ovary (Fig. 3A). During male germ cell development, the transcript was not detected in the neonatal mouse testis until 17 days of age. Then significant signals were detected from age 23 days through to adulthood (Fig. 3B). Northern blotting of mRNA from fractionated mouse testicular cells (i.e., the germ, Sertoli, and Leydig cells) [12] showed that pctp-l gene was exclusively expressed in the germ cells (Fig. 3C). These results indicated that the expression of the pctp-l gene occurred only in germ cells in a developmentally regulated manner.
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To determine the developmental stages of the germ cells expressing pctp-l mRNA, in situ hybridization analysis was performed. Specific staining was detected with the antisense probe exclusively in the round and elongated spermatid layers of the seminiferous tubules (Fig. 4A), but not in the spermatids at the final maturation phase (step 15–16) (Fig. 4C).
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Western Blot Analysis and Immunohistochemical Identification of PCTP-L Protein
Western blot analysis revealed a protein band with a molecular weight of approximately 40 000 in the liver, testis, and sperm, but no positive signal in other tissues such as brain, heart, lung, spleen, or even kidney and intestine (Fig. 5A). During male germ cell development, PCTP-L was detected first in the 23-day-old mouse testis, and the signal increased with age (Fig. 5B).
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Immunohistochemical examinations of the adult mouse testis and epididymal sperm using the anti-PCTP-L polyclonal antibody were performed. The expression of PCTP-L protein was detected first in the elongated spermatids at the flagella during spermatogenesis (Fig. 6), and a positive signal was also observed at the head and tail of the epididymal sperm (Fig. 7). These observations were in good agreement with the results of Western blot analysis, indicating that PCTP-L was a novel differentiation-associated molecule specifically expressed after the differentiation of haploid spermatids reached the elongated step.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we constructed a haploid germ cell-specific cDNA library by subtracting mRNA of 17-day-old mouse testes from the cDNAs of 35-day-old testes [7] and achieved the identification and characterization of a novel gene isolated from the library [7]. Nucleotide sequence analysis of the cDNA and computer-assisted homology search showed no homologous genes registered in cDNA banks. However, the deduced amino acid sequence showed approximately 28% identity and 49% similarity with mouse PCTP [19] (Fig. 2). We named this newly isolated gene pctp-like protein gene (pctp-l).
Northern blot analyses showed that pctp-l mRNA was detected as a transcript of 1.4 kb not only strongly in the testis, but also weakly in the liver, kidney, and intestine. As to the testis, pctp-l mRNA was expressed only in germ cells (Fig. 3C), and the gene expression occurred in round and elongated spermatids. The expression of PCTP-L protein in kidney and intestine deviated slightly from the expression of the mRNA. This may be due to the sensitivity of detection in Western blotting or to a difference in stability of the protein. PCTP-L was detected in elongated and final maturation phase spermatids (Fig. 6B). In contrast, pctp-l mRNA was strongly detected in round and elongated spermatids (Fig. 4A) and not in the spermatids at the final maturation phase (Fig. 4C). These observations taken together indicate that the expression of PCTP-L protein must be regulated by posttranscriptional control. PCTP-L protein in testis was predominantly detected at the flagella of elongated spermatids, with a strong signal also found at the tail of epididymal sperm (Fig. 7).
Phospholipid is a principal component of plasma membrane, and phospholipid transfer activity was initially discovered in the membrane-free cytosol of rat liver [21]. This activity is attributable to a number of different phospholipid transfer proteins, the most prominent of which is the PCTP highly specific for PC [20]. PC is the major constituent phospholipid in both the inner and the outer membrane of mammalian mitochondria [25]. The terminal steps of the de novo synthesis of PC occur primarily on the cytoplasmic surface of the endoplasmic reticulum. Consequently, the biogenesis of mitochondria requires efficient import of PC from the endoplasmic reticulum. Several mechanisms for the intracellular transport of phospholipids have been proposed [26]. One of the steps in the import of PC in mitochondria is transmembrane movement across the outer membrane [4]. PCTP may also be essential for membrane biogenesis through delivering PC to sites of cell growth.
In marine animals such as fish and sea urchin, spermatozoa cannot use exogenous substrates for energy production because the seminal plasma is diluted by sea water. It has been indicated that sea urchin spermatozoa obtain energy for swimming through the oxidation of endogenous phospholipids [27]. It was also found, after incubation of sea urchin spermatozoa with sea water, that the content of PC decreased, with no change in the levels of other phospholipids [28]. PC is thus possibly a substrate for energy metabolism in sea urchin spermatozoa. In mammalian spermatozoa, sugar in seminal plasma has been postulated as responsible for the motility [29]. As to mouse sperm, glucose is required for hyperactivated motility of sperm [30] and for fertilization in vitro [31]. Without seminal plasma, however, mammalian spermatozoa can maintain motility under aerobic conditions [32]. During incubation, the endogenous phospholipid content diminishes with metabolism [33]. Thus, mammalian spermatozoa are also capable of using phospholipids for energy metabolism. A previous study [34] indicated that enzymes for energy metabolism are located in flagella in mammalian spermatozoa.
We have isolated pctp-l gene, and the gene product was expressed in the flagella of mouse sperm (Fig. 7). Although glucose is a main substrate for energy production in mouse sperm, the sperm was also shown to have the potential to use PC. PCTP-L may play a key role in energy metabolism using such an endogenous substrate.
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FOOTNOTES |
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1 Correspondence: Yoshitake Nishimune, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita City, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp 
Accepted: January 20, 2000.
Received: September 13, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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