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Cortical Organization by the Septin Cytoskeleton Is Essential for Structural and Mechanical Integrity of Mammalian Spermatozoa

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Cortical Organization by the Septin Cytoskeleton Is Essential for Structural and Mechanical Integrity of Mammalian Spermatozoa View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Developmental Cell, Vol. 8, 343–352, March, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.devcel.2004.12.005 Cortical Organization by the Septin Cytoskeleton Is Essential for Structural and Mechanical Integrity of Mammalian Spermatozoa Masafumi Ihara,1 Ayae Kinoshita,2 Shuichi Yamada,3 cytoskeleton is essential for the structural and me- Hiromitsu Tanaka,4 Ai Tanigaki,5 Ayumi Kitano,1 chanical integrity of mammalian spermatozoa. Motohito Goto,3 Kazutoshi Okubo,6 Hiroyuki Nishiyama,6 Osamu Ogawa,6 Introduction Chiaki Takahashi,7 Shigeyoshi Itohara,8 Yoshitake Nishimune,4 Makoto Noda,7 The septins are a family of polymerizing GTP binding and Makoto Kinoshita1,5,* proteins originally discovered in budding yeast as a 1Biochemistry and Cell Biology Unit group of cell cycle mutants which cause defects in cy- 2 Alzheimer Research Unit tokinesis (Field and Kellogg, 1999). Loss or mutation of HMRO any one of the five septins commonly results in multinu- Kyoto University Graduate School of Medicine clear and multilocular morphology. These mutants lack Kyoto electron-dense circular striations which are normally 3 Institute for Virus Research organized beneath the plasma membrane between a Kyoto University mother cell and the bud (Byers and Goetsch, 1976; Fra- Kyoto zier et al., 1998). These circular striations represent the 4 Department of Science for Laboratory Animal “septin ring,” a subcellular structure which can be visu- Experimentation alized by immunofluorescence or by the expression of Research Institute for Microbial Diseases septins fused to biofluorescent proteins. To date, the Osaka University septin ring has been known to play multiple roles in Suita cytokinesis: it serves as a scaffold for various mitosis- 5 PRESTO associated molecules (Gladfelter et al., 2001) and for Japan Science and Technology Agency positioning the mitotic spindle (Kusch et al., 2002), and Kawaguchi as a diffusion barrier for partitioning membrane do- Japan mains between a mother cell and the bud (Barral et al., 6 Department of Urology 2000; Takizawa et al., 2000). Although such a structure 7 Department of Molecular Oncology and its related functions have not been demonstrated Kyoto University Graduate School of Medicine in higher organisms, requirement of the septin cytoskel- Kyoto eton for cytokinesis seems conserved up to mammals 8 Laboratory for Behavioral Genetics (Neufeld and Rubin 1994; Kinoshita et al., 1997; Nguyen RIKEN Brain Science Institute et al., 2000). Wako Despite the progress of septin biology in yeast, many Japan questions unique to the metazoan septin system re- main unsolved: (1) Septins are abundantly expressed in nondividing cells, but little is known about their roles in Summary postmitotic events (Beites et al., 1999; Adam et al., 2000; Dent et al., 2002; Finger et al., 2003). (2) Although Septins are polymerizing GTP binding proteins re- mammalian septin filaments can self-organize circular quired for cortical organization during cytokinesis bundles both in vitro and in interphase tissue culture and other cellular processes. A mammalian septin cells following actin perturbation (Kinoshita et al., gene Sept4 is expressed mainly in postmitotic neural 2002), the physiological relevance of this unique prop- cells and postmeiotic male germ cells. In mouse and erty has not been established because a similar struc- human spermatozoa, SEPT4 and other septins are ture has not been found in vivo. (3) Although most sep- found in the annulus, a cortical ring which separates tins are believed to function as cytoskeletal proteins, a the middle and principal pieces. Sept4−/− male mice few septin gene products are implicated in mito- are sterile due to defective morphology and motility chondrial functions, apoptosis, and/or tumorigenesis. of the sperm flagellum. In Sept4 null spermatozoa, the For instance, the human and mouse Sept4 genes annulus is replaced by a fragile segment lacking cor- respectively generate polypeptides of 32 and 46 kDa in tical material, beneath which kinesin-mediated in- addition to the larger 48–54 kDa cytoskeletal isoforms. traflagellar transport stalls. The sterility is rescued by The 32 and 46 kDa isoforms are targeted to mito- injection of sperm into oocytes, demonstrating that chondria in tissue culture cells, and the 32 kDa isoform each Sept4 null spermatozoon carries an intact hap- induces cell death when overexpressed (Larisch et al., loid genome. The annulus/septin ring is also disorga- 2000; Takahashi et al., 2003). Messenger RNA for the nized in spermatozoa from a subset of human pa- 32 kDa “proapoptotic” isoform is downregulated during tients with asthenospermia syndrome. Thus, cortical malignant progression of human lymphoma (Elhasid et organization based on circular assembly of the septin al., 2004). Thus, Sept4 is thought to be a potential tu- mor-suppressor gene in humans. However, it has also *Correspondence: [email protected] been claimed that Sept4 is an oncogene whose upreg- Developmental Cell 344 ulation is responsible for human colon cancer (Tanaka normal range (Figure 1C), the Sept4−/− mice infertility et al., 2002). As yet, these contradictory hypotheses is technically attributable not to oligozoospermia (low have not been critically tested for relevance in vivo. sperm concentration), but to terato-asthenospermia To address the puzzling situation regarding mamma- (defective sperm morphology and motility). lian septins, especially SEPT4, we disrupted the Sept4 gene in the mouse. The distinct phenotype observed in Septin Organization in Normal and Sept4 the Sept4 null mice provides answers and clues to Null Spermatozoa some of these remaining questions. In this report, we To address the molecular basis underlying the struc- focus on the discovery of a septin-based circular struc- tural and mechanical defects of the Sept4 null sperma- ture in the spermatozoa of diverse mammals. Here, we tozoa, we examined the subcellular localization of reveal that the septin ring in the mouse spermatozoa SEPT4 and other septin subunits in normal spermato- is essential for cortical organization and intraflagellar zoa. Immunocytochemistry and fluorescence micro- transport, and demonstrate a critical role for the septin scopy analysis detected SEPT4 as a pair of dots be- cytoskeleton in mammalian spermiogenesis and repro- neath the plasma membrane between the middle and duction. principal pieces (Figure 2C). The dots often appeared fused depending on the staining conditions. Three Results other septin subunits, SEPT1/6/7, colocalized to the SEPT4 dots (Figure 2C and Supplemental Figure S2), Sept4 Is Essential for Male Fertility in Mice while Sept2/5/8/10/11/12 and the major cytoskeletal To explore the postmitotic and postmeiotic roles of the proteins, α-tubulin, β-actin, and vimentin, were not con- septin cytoskeleton in vivo, we created mice lacking centrated at the dots (data not shown). On the other Sept4. The Sept4 gene and its products are expressed hand, SEPT1/6/7 were delocalized and dispersed mainly in postmitotic neural cells (Kinoshita et al., 2000) throughout the cytoplasm in Sept4 null spermatozoa and postmeiotic male germ cells (Figure 1A). The (Figure 2D and Supplemental Figure S2). targeting vector was designed to replace the entire coding exons 2–10 with a neo cassette to make a null Defective Cortical Organization in Sept4 allele (Figure 1B). The Sept4+/– and Sept4−/− mice were Null Spermatozoa born at a rate close to the predicted Mendelian fre- Consistently, immuno-electron microscopy (immuno- quency. Elimination of the SEPT4 polypeptides in EM) detected SEPT4 in a submembranous ring of high Sept4−/− tissues was confirmed (Figure 4 and not electron density which is known as the annulus (Figure shown). The Sept4−/− mice had normal health without 3A). By transmission electron microscopy (TEM), the di- spontaneous development of malignancy (solid tumor ameter of the annulus in Sept4+ spermatozoa was 0.58 and hematologic) until up to 10 months old (n > 150), ± 0.09 ␮m (n = 38), and the axonemal 9+2 structure and except that males were completely sterile (Figure 1C). the surrounding outer dense fibers were indistinguish- In contrast, Sept4+/− males and Sept4−/− females were able between Sept4+ and Sept4 null spermatozoa (Fig- fertile and produced normal-size litters. There were no ure 3B). The annulus maintained this constant diameter gross anatomical and histological differences in the with or without rudimentary cytoplasm (“cytoplasmic seminal vesicles, prostates, testes and epididymides droplet”) around the middle piece (Figure 3C). The an- between Sept4+/− and Sept4−/− males (Figure 1C and nulus is in closer proximity to the fibrous sheath than Supplemental Figure S1). Thus, the sterility is not attrib- to mitochondria (Figures 3B and 3C). In Sept4 null sper- utable to developmental abnormality in and/or immatu- matozoa, however, the annulus was never found, and rity of the male reproductive organs. the fibrous sheath often failed to cover a proximal seg- ment of the principal piece (Figure 3B). The defects of Sept4 Is Essential for Structural and Mechanical cortical organization between mitochondria and the fi- Integrity of the Mouse Spermatozoa brous sheath correspond to the narrow segment ob- To elucidate defects underlying the male sterility, we served by DIC microscopy (Figure 2A). Thus, we con- compared mature spermatozoa collected from caudae
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