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Lack of Acrosome Formation in Mice Lacking a Golgi Protein, GOPC

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Lack of Acrosome Formation in Mice Lacking a Golgi Protein, GOPC Lack of acrosome formation in mice lacking a Golgi protein, GOPC Ryoji Yao*, Chizuru Ito†, Yasuko Natsume*, Yoshinobu Sugitani*, Hitomi Yamanaka*, Shoji Kuretake‡, Kaoru Yanagida‡, Akira Sato‡, Kiyotaka Toshimori†, and Tetsuo Noda*§¶ʈ** *Department of Cell Biology, Japanese Foundation for Cancer Research (JFCR) Cancer Institute, 1-37-1 Kami-Ikebukuro, Toshima-Ku, Tokyo 170-8455, Japan; †Department of Anatomy and Reproductive Cell Biology, Miyazaki Medical Collage, Kiyotake, Miyazaki 889-1692, Japan; ‡Department of Obstetrics and Gynecology, Fukushima Medical Collage, 1 Hikarigaoka, Fukushima 960-1295, Japan; §Department of Molecular Genetics, Tohoku University School of Medicine, 2-1 Seiryo-cho, Aoba-Ku, Sendai 980-8575, Japan; ¶Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 4-1-8 Motomachi, Kawaguchi 332-0012, Japan; and ʈMouse Functional Genomics Research Group, Institute of Physical and Chemical Research (Japan) (RIKEN) Genomic Sciences Center, 214 Maeda-cho, Totsuka-ku, Yokohama, Kanagawa 244-0804, Japan Edited by Kai Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, and approved June 19, 2002 (received for review January 31, 2002) The acrosome is a unique organelle that plays an important role at protein, and propose that GOPC may have a role in vesicle the site of sperm–zona pellucida binding during the fertilization transport from the Golgi apparatus (6). GOPC contains one process, and is lost in globozoospermia, an inherited infertility PDZ domain, two coiled-coil motifs, and two evolutionarily syndrome in humans. Although the acrosome is known to be conserved domains. The PDZ domain of GOPC is required for derived from the Golgi apparatus, molecular mechanisms under- its Frizzled binding, whereas coiled-coil motifs and conserved lying acrosome formation are largely unknown. Here we show that domains are required for its Golgi localization. Charest et al. (7) Golgi-associated PDZ- and coiled-coil motif-containing protein reported that FIG (fused in glioblastoma), a possible human (GOPC), a recently identified Golgi-associated protein, is predom- homologue of GOPC with 92.3% identity, interacted with syn- inantly localized at the trans-Golgi region in round spermatids, and taxin-6, supporting the idea that GOPC may play a role in vesicle male mice in which GOPC has been disrupted are infertile with transport from the Golgi apparatus. In the present study, we have globozoospermia. The primary defect was the fragmentation of investigated the biological role of GOPC by gene targeting to acrosomes in early round spermatids, and abnormal vesicles that generate mice bearing a null allele of GOPC, and found that failed to fuse to developing acrosomes were apparent. In later GOPC-deficient male mice were infertile with globozoospermia. stages, nuclear malformation and an abnormal arrangement of The spermatozoa from GOPC-deficient mice showed complete mitochondria, which are also characteristic features of human lack of acrosomes, which is because of the failure of vesicle globozoospermia, were observed. Interestingly, intracytoplasmic transport from the Golgi apparatus. sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes Materials and Methods CELL BIOLOGY were activated. Thus, GOPC provides important clues to under- Reverse Transcription (RT)-PCR and Northern Blot Analysis. RNA standing the mechanisms underlying spermatogenesis, and the from various mouse tissues was isolated by using a FastTrack kit GOPC-deficient mouse may be a unique and valuable model for (Invitrogen) and cDNA synthesis and PCR were performed by human globozoospermia. using a cDNA cycle kit (Invitrogen). The primers were synthe- sized based on the sequences of exons 2 and 3. The sequences of primers were as follows: exon 2 primer, 5Ј-CCACTCAGCT- he acrosome is a unique structure of the mature spermato- TCAGCTTCATGCC-3Ј; exon 3 primer, 5Ј-AGCCTGGAGAA- zoon, which plays an important role at the site of sperm–zona T CAGCTATGTGCC-3Ј. pellucida binding during the fertilization process. The biogenesis ϩ For Northern blot analysis, 4 ␮g of poly(A) RNA was of the acrosome takes place during the initial phase of spermatid separated in a 1% formaldehyde/agarose gel and transferred to development, when numerous proacrosomic granules are Duralon-UV nylon membrane (Stratagene) according to the formed from trans-Golgi stacks and accumulate in the concave manufacturer’s instructions. The filter was hybridized with region near the trans-Golgi stacks—i.e., medulla. The small the 0.9-kb [␣-32P]dCTP-labeled GOPC cDNA encompassing the granules fuse with each other to form a single large acrosomic PDZ domain. granule that associates with the nuclear envelope (1). During the subsequent cap phase, the acrosome increases its size and begins Immunoblot Analysis. Tissue extracts were prepared by homoge- to spread over the anterior nuclear pole. The nucleus changes its nization in Nonidet P-40 buffer (20 mM Tris⅐HCl, pH 8.0͞137 shape during the subsequent acrosomic phase, which is followed mM NaCl͞1% Nonidet P-40͞10% glycerol) supplemented with by caudal migration of mitochondria during the maturation 1 mM phenylmethylsulfonyl fluoride, 1 ␮g͞ml leupeptin, 1 phase. Although the morphogenic change of the acrosome in ␮g͞ml aprotinin, and 0.5 mM DTT in a Dounce homogenizer. spermatogenesis has been well documented, its molecular basis The homogenates were centrifuged at 13,000 rpm for 15 min and is still largely unknown. protein concentrations were determined by the Bio-Rad protein Globozoospermia (also called round-headed spermatozoa) is assay. Twenty-five micrograms of protein lysates was separated a human infertility syndrome caused by spermatogenesis defects in SDS͞PAGE and electrotransferred to nitrocellulose. Anti- (2–4). The most prominent feature of globozoospermia is the serum against GOPC has been described (6). The bound anti- malformation of the acrosome, and, in the most severe cases, the body was detected by ECL Western blotting detection reagents acrosome is totally absent. Globozoospermia is also character- (Amersham Pharmacia Biotech). ized by abnormal nuclear shape as well as abnormal arrangement of the mitochondria of the spermatozoon (5). Although globo- zoospermia is thought to be an inherited disorder (2), the This paper was submitted directly (Track II) to the PNAS office. etiology of globozoospermia is not known. Abbreviations: GOPC, Golgi-associated PDZ- and coiled-coil motif-containing protein; We have identified mouse Golgi-associated PDZ- and coiled- RT-PCR, reverse transcription–PCR; ICSI, intracytoplasmic sperm injection. coil motif-containing protein (GOPC) as a Frizzled-interacting **To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.162027899 PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 11211–11216 Downloaded by guest on September 30, 2021 Gene Targeting. A genomic clone encompassing exons 2, 2b, 3, and 4 of GOPC was isolated from the 129 genomic library and a targeting vector in which nLacZ and floxed pMC1neo poly(A) replaced exons 2, 2b, and 3 was constructed. A nLacZ fragment was inserted in frame in the PvuI site of exon 2, and the DT-A fragment was ligated at the 3Ј end of the targeting vector for negative selection. The targeting vector was linearized by NotI digestion before electroporation into J1 ES cells. Of 274 G418- resistant clones, 5 clones had undergone homologous recombi- nation as determined by Southern blot analysis. Two clones were injected into C57BL͞6 blastocysts, resulting in the birth of male chimeric mice. Germ-line transmission of the disrupted GOPC allele was achieved by mating with C57BL͞6 females. Immunohistochemistry. Adult testes from wild-type and GOPCϪ/Ϫ mice were fixed with 4% paraformaldehyde (PFA) by perfusion Fig. 1. Structure and expression of GOPC. (a) Structures of GOPC␣ and and immersed for 20 hr at 4°C. The testes were rinsed in GOPC␤. The white box indicates the position at which the 8-aa insertion is phosphate buffer (PB), washed through a sucrose gradient (10-, found in GOPC␤. CR, conserved region; CC, coiled-coil motif; PDZ, PDZ domain. 20-, and 30% in PB), and embedded in OCT compound (Miles, (b) Northern blot analysis of GOPC. Aliquots (4 ␮g) of poly(A) were probed with GOPC cDNA. Lanes: 1, brain; 2, lung; 3, heart; 4, liver; 5, kidney; 6, testis. Elkhart, IN). Frozen sections were immunostained with anti- ϩ GOPC serum, followed by Cy3-conjugated anti-rabbit IgG (Cap- (c) RT-PCR analysis of GOPC␣ and GOPC␤. One microgram of poly(A) RNA pel). Nuclei were stained with 4Ј,6-diamidino-2-phenylindole. were used for RT-PCR analysis. The fragments sized 201 bp and 177 bp represent GOPC␣ and GOPC␤, respectively. 1, brain; 2, liver; 3, kidney; 4, testis. To isolate spermatogenic cells, testes were removed, dissected (d) Immunoblot analysis of GOPC. Twenty-five-microgram aliquots of total with scissors, and digested with collagenase followed by trypsin lysates were analyzed with the anti-GOPC antibody. Lanes: 1, cerebrum; 2, (8). Cells were washed several times, plated onto poly-L-lysine cerebellum; 3, liver; 4, kidney; 5, testis. coated coverslips, fixed, and stained as detailed above. Conventional Transmission Electron Microscopy. Adult testes were polyvinylpyrrolidone (Sigma). A spermatozoon was drawn, tail fixed with 2% glutaraldehyde in 0.2 M cacodylate buffer. After first, into the pipette and injected. Where indicated, oocytes washing in the same buffer, the tissues were cut into small pieces, were stimulated electrically 30 min after ICSI by a single, square immersed in the same fixative for 2 hr at 4°C, rinsed, and then dc pulse (1.5 kV͞cm, 100 ␮s). Sperm-injected oocytes were fixed with OsO4. Thereafter, the samples were dehydrated incubated in HTF medium for 6 h and then cultured in KSOM through a graded ethanol series, and then embedded in Epon (K simplex optimized median) until injected oocytes were 812. Ultrathin sections were cut on an ultramicrotome (model developed to the blastocyst stage.
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