已婚夫婦中約有10-15﹪的夫妻有不孕症的困擾，其中約有一半是男性因素所造成。 至今仍有25%男性不孕症病患，病因仍不清楚。 近年來於果蠅及老鼠動物模式中，發現許多基因會影響其生殖能力，顯示出遺傳因素可能於物種的不孕有很大的影響，但這些基因在精子發育過程之中的角色，卻只有少數被瞭解。 本研究主要在尋找參與精子發育過程的新穎基因並鑑定其功能。 我們藉由cDNA微陣列晶片，分析不孕症男性與健康男性睪丸中基因表現的差異，經統計分析後得到十個於造精功能障礙較正常睪丸組織間表現量下降之新穎基因，本論文研究的重點是針對其中一個專一表現於睪丸的Septin12基因(SEPT12)進行探討。
SEPT家族屬於具有GTP結合能力的細胞骨架蛋白且參與細胞區室化，曩泡運輸, 細胞分裂與細胞骨架重整。 Septin12基因特異表現於人類與小鼠睪丸組織。 於精子生成過程，SEPT12表現於精子頂體外圍，頸部與精子中節和尾部交接的環狀結構。 於Septin12基因剔除嵌合公鼠為不孕且睪丸重量明顯下降。 於精液分析發現，精子數目,活動力與正常型態精子數目明顯減少。 於嵌合小鼠睪丸切片發現生精作用一致性下降或停置於圓形精細胞階段，並伴隨精細胞凋亡情形增加。 於嵌合小鼠精子同時具有精細胞頂體部破損與尾部彎曲情形。 經由穿透式電子顯微鏡分析也發現鑲嵌小鼠精子頂體部外膜受損，粒線體減少與排列不完整和細胞核有空泡產生。 另於男性不孕症患者睪丸組織中，SEPTIN12 mRNA含量下降。 利用螢光免疫染色發現，精子無力症與精子型態異常的病患，SEPT12表現量有明顯下降的情形。 總括本研究結果證實，SEPT12於哺乳類精子生成與男性不孕症扮演重要的角色。

It is estimated that 10- 15% of couples are infertile and male factors account for about half of the cases. About 25% of the causes of male infertility are still unknown and the majority of infertile cases have defects in spermatogenesis. But large of those spermatogenesis are still unknown. To identify novel genes which are involved in mammalian spermatogenesis, we studied global gene expression patterns in testicular biopsies of men with spermatogenic defects by c-DNA microarrays. We found ten novel genes of which transcripts were significantly decreased in patients with Sertoli cell only syndrome or Maturation arrest. Because the transcripts of Septin12 (SEPT12) are specific express in human and mouse testis. We chose Septin12 gene for further study.
Septins belong to a family of polymerizing GTP binding proteins that are required for many cellular functions, including membrane compartmentalization, vesicle trafficking, mitosis and cytoskeletal remodeling. In this study, we found SEPTIN12, one of the SEPTIN family members, is expressed specifically in the testis. SEPT12 expressed in multiple subcellular compartments during terminal differentiation of post-meiotic germ cells. To elucidate the role of Septin12, we generated 129 ES cells with a Septin12 mutant allele deleted in the exons encoding the N-terminal GTP binding domain. Most chimeras derived from the targeted ES cells were infertile. Semen analysis of the infertile chimeras showed decreased sperm counts, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria and broken acrosome. In humans, the testicular tissues of men with hypo-spermatogenesis or maturation arrest had lower levels of the SEPTIN12 transcripts. In addition, more spermatozoa with abnormal head, neck and tail morphology lacked SEPT12 immunostaining signals compared to spermatozoa with normal appearance. Our data support a critical role of Septin12 during mammalian spermiogenesis.

Abstract in Chinese ----------------------------------------------------------------------------1
Abstract------------------------------------------------------------------------------------------2
Acknowledgements-----------------------------------------------------------------------------4
I. Literature Review----------------------------------------------------------------------------5
1.1 Male infertility and Spermatogenesis -----------------------------------------------5
1.2 Identification of sterility-related genes during mammalian spermatogenesis--6
1.3 The functional roles of Septin from yeasts to human -------------------------6
1.4 The reproductive roles of SEPTs in model organisms ----------------------------8
1.5 Septins and diseases--------------------------------------------------------------------9
1.6 Goals of this work --------------------------------------------------------------------9
II. Materials and Methods-------------------------------------------------------------------11
2.1 Patients----------------------------------------------------------------------------------11
2.2 Testicular tissues-----------------------------------------------------------------------12
2.3 Microarray analysis-------------------------------------------------------------------12
2.4 Novel genes and similarity search---------------------------------------------------13
2.5 Semi-quantitative Reverse Transcription (RT)-PCR------------------------------13
2.6 RT-PCR for expression patterns of novel genes and Septin12-------------------14
2.7 5’ and 3’ Rapid Amplification of cDNA Ends (RACE), genomic database search and sequence alignment --------------------------------------------------15
2.8 Plasmid construction, cell culture, transfection and immunofluorescence ----15
2.9 Separation of the testicular germ cell populations and sperm preparation-----16
2.10 Generation of anti-SEPT12, western blot analysis and immuno fluorescence study-----------------------------------------------------------------------------------17
2.11 Targeting vector and generation of mutant Mice--------------------------------18
2.12 Semen analysis and TUNEL assay------------------------------------------------18
2.13 Electron microscopy----------------------------------------------------------------19
III Results---------------------------------------------------------------------------------------21
3.1 Down-regulated genes in the testicular tissue with spermatogenic defects ---21
3.2 Validation of microarray data for novel genes-------------------------------------21
3.3 Genomic structure and functional domains of novel genes----------------------22
3.4 Expression pattern of novel genes in micee and human-------------------------22
3.5 Developmental stage-dependent expressions of novel genes in mouse testes-23
3.6 Identification of full length Septin12 transcripts by RACE in mammalian --23
3.7 Septin12 is expressesed in the post-meiotic germ cells --------------------------24
3.8 Reproductive phenotypes of the Septin12+/- chimera mice-----------------------25
3.9 Sperm acrosome and tail defects in Septin12+/- chimera mice-------------------26
3.10 Ultrastructural abnormalities of spermatozoa in the Septin12+/- chimeric mice ------------------------------------------------------------------------------------------27
3.11 Dis-origanized annulus/SEPT rings of spermatozoa in the Septin12 ko chimeric mice -------------------------------------------------------------------28
3.12 Altered expression pattern of SEPT12 in human spermatozoa with abnormal morphology---------------------------------------------------------------------------28
IV. Discussions---------------------------------------------------------------------------------30
4.1 Microarrray analysis of the mammalian testis ------------------------------------30
4.2 Testis-enriched genes which when maturated, may be involved in
spermatogenic defect-----------------------------------------------------31
4.3 The functional role of novel sterility-related genes ------------------------------31
4.4 SEPTs in mammalian spermatogenesis --------------------------------------------34
4.5 SEPT 1/4/6/7 complexs and SEPT12 in mouse spermatogenesis---------------34
4.6 SEPTs and microtubulins ------------------------------------------------------------35
4.7 Haploinsufficiency of SEPT12 during spermatogenesis ------------------------36
4.8 The roles of SEPT12 in male infertility --------------------------------------------38
4.9 Characterization of Septin12 gene in mammalian spermiogenesis -------39
VI. References----------------------------------------------------------------------------------41


List of Figures
Figure 1. Microarray analysis of the human testicular tissue-----------------------------55
Figure 2. Semi-quantitative RT-PCR analysis for 5 novel genes which are down- regulated in Sertoli cell only syndrome (SCOS) ------------------------------56
Figure 3. Semi-quantitative RT-PCR analysis for 8 novel genes which are down-regulated in maturation arrest (MA) -------------------------------------57
Figure 4. The genomic structure, functional domains, size of human transcript, total number of amino acids, and chromosomal localization of novel sterile genes ----------------------------------------------------------------------------------------58
Figure 5. RT-PCR analysis for expression of novel sterile genes in different human (A) and mouse organs (B) -------------------------------------------------------------59
Figure 6. Developmentally regulated expression of mouse orthologues----------------60
Figure 7 Schematic presentations of major Septin12 transcripts in mice and human-61
Figure 8 RT-PCR expression profiles of Septin12 in humans and mice ----------------63
Figure 9 Over-expression of Seprin12 form filaments-like structures in 293T cells -64
Figure 10 Expression pattern and localization of the SEPT12 protein -----------------65
Figure 11 Targeted disruption of Septin12 causes male fertility ------------------------66
Figure 12 The reproductive phenotypes of chimeras (I) ---------------------------------68
Figure 13. The reproductive phenotypes of chimeras (II) -------------------------------70
Figure 14. Electron microscopy shows defects in sperm acrosome and mitochondrial architecture in the chimeric mice -----------------------------------------------71
Figure 15. Dis-localization of SEPT7 in Septin12 Ko chimeric mice sperm ---------72
Figure 16. The expression of SEPTIN12 decreases in infertile men --------------------73
Figure 17. Decreased expression level of SEPT12 in spermatozoa with abnormal morphology -------------------------------------------------------------------------74
Figure18. A working model of SEPT12 during spermiogenesis-------------------------75












List of Tables
Table1. The number of genes which are significantly down-regulated in the testicular samples ------------------------------------------------------------------------------76
Table2. List of testis-enriched genes --------------------------------------------------------77
Table3. List of novel genes -------------------------------------------------------------------78
Table4. List of novel genes primer sequences for RT-PCR ------------------------------79
Table5. List of Septin12 specific primer sequences for RACE and Real-time RT
-PCR-------------------------------------------------------------------------------------80
Table6. The percentages of abnormal spermatozoa from the cauda epidydimis of mice ------------------------------------------------------------------------------------------81
Publications----------------------------------------------------------------------82

1.WHO. 1992. World Health Organization: Laboratory manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. . 3-21 pp.
2.Carlsen, E., Giwercman, A., Keiding, N., and Skakkebaek, N.E. 1992. Evidence for decreasing quality of semen during past 50 years. BMJ 305:609-613.
3.Silber, S.J. 2000. Evaluation and treatment of male infertility. Clin Obstet Gynecol 43:854-888.
4.Matzuk, M.M., and Lamb, D.J. 2002. Genetic dissection of mammalian fertility pathways. Nat Cell Biol 4 Suppl:s41-49.
5.Cooke, H.J., and Saunders, P.T. 2002. Mouse models of male infertility. Nat Rev Genet 3:790-801.
6.Hirsh, A. 2003. Male subfertility. BMJ 327:669-672.
7.Ferlin, A., Raicu, F., Gatta, V., Zuccarello, D., Palka, G., and Foresta, C. 2007. Male infertility: role of genetic background. Reprod Biomed Online 14:734-745.
8.Matzuk, M.M., and Lamb, D.J. 2008. The biology of infertility: research advances and clinical challenges. Nat Med 14:1197-1213.
9.Palermo, G., Joris, H., Devroey, P., and Van Steirteghem, A.C. 1992. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340:17-18.
10.Hughes, V. 2008. Geneticists crack the code of infertility. Nat Med 14:1174.
11.Siddiq, F.M., and Sigman, M. 2002. A new look at the medical management of infertility. Urol Clin North Am 29:949-963.
12.Kumar, R., Gautam, G., and Gupta, N.P. 2006. Drug therapy for idiopathic male infertility: rationale versus evidence. J Urol 176:1307-1312.
13.Wolgemuth, D.J., Laurion, E., and Lele, K.M. 2002. Regulation of the mitotic and meiotic cell cycles in the male germ line. Recent Prog Horm Res 57:75-101.
14.Vogt, P.H. 2004. Molecular genetics of human male infertility: from genes to new therapeutic perspectives. Curr Pharm Des 10:471-500.
15.Okabe, M., Ikawa, M., and Ashkenas, J. 1998. Male infertility and the genetics of spermatogenesis. Am J Hum Genet 62:1274-1281.
16.Boivin, J., Bunting, L., Collins, J.A., and Nygren, K.G. 2007. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Hum Reprod 22:1506-1512.
17.Schultz, N., Hamra, F.K., and Garbers, D.L. 2003. A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc Natl Acad Sci U S A 100:12201-12206.
18.Pang, A.L., Taylor, H.C., Johnson, W., Alexander, S., Chen, Y., Su, Y.A., Li, X., Ravindranath, N., Dym, M., Rennert, O.M., et al. 2003. Identification of differentially expressed genes in mouse spermatogenesis. J Androl 24:899-911.
19.Guo, R., Yu, Z., Guan, J., Ge, Y., Ma, J., Li, S., Wang, S., Xue, S., and Han, D. 2004. Stage-specific and tissue-specific expression characteristics of differentially expressed genes during mouse spermatogenesis. Mol Reprod Dev 67:264-272.
20.Juneja, S.C., and van Deursen, J.M. 2005. A mouse model of familial oligoasthenoteratozoospermia. Hum Reprod 20:881-893.
21.Osada, N., Hida, M., Kusuda, J., Tanuma, R., Hirata, M., Suto, Y., Hirai, M., Terao, K., Sugano, S., and Hashimoto, K. 2002. Cynomolgus monkey testicular cDNAs for discovery of novel human genes in the human genome sequence. BMC Genomics 3:36.
22.Ehmcke, J., Simorangkir, D.R., and Schlatt, S. 2005. Identification of the starting point for spermatogenesis and characterization of the testicular stem cell in adult male rhesus monkeys. Hum Reprod 20:1185-1193.
23.Fox, M.S., Ares, V.X., Turek, P.J., Haqq, C., and Reijo Pera, R.A. 2003. Feasibility of global gene expression analysis in testicular biopsies from infertile men. Mol Reprod Dev 66:403-421.
24.Green, J.A., Robins, J.C., Scheiber, M., Awadalla, S., and Thomas, M.A. 2001. Racial and economic demographics of couples seeking infertility treatment. Am J Obstet Gynecol 184:1080-1082.
25.Hall, P.A., Jung, K., Hillan, K.J., and Russell, S.E. 2005. Expression profiling the human septin gene family. J Pathol 206:269-278.
26.Hartwell, L.H. 1971. Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp Cell Res 69:265-276.
27.Gladfelter, A.S., Pringle, J.R., and Lew, D.J. 2001. The septin cortex at the yeast mother-bud neck. Curr Opin Microbiol 4:681-689.
28.Nguyen, T.Q., Sawa, H., Okano, H., and White, J.G. 2000. The C. elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis. J Cell Sci 113 Pt 21:3825-3837.
29.Hime, G.R., Brill, J.A., and Fuller, M.T. 1996. Assembly of ring canals in the male germ line from structural components of the contractile ring. J Cell Sci 109 ( Pt 12):2779-2788.
30.Peterson, E.A., Kalikin, L.M., Steels, J.D., Estey, M.P., Trimble, W.S., and Petty, E.M. 2007. Characterization of a SEPT9 interacting protein, SEPT14, a novel testis-specific septin. Mamm Genome 18:796-807.
31.Kartmann, B., and Roth, D. 2001. Novel roles for mammalian septins: from vesicle trafficking to oncogenesis. J Cell Sci 114:839-844.
32.Ihara, M., Kinoshita, A., Yamada, S., Tanaka, H., Tanigaki, A., Kitano, A., Goto, M., Okubo, K., Nishiyama, H., Ogawa, O., et al. 2005. Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa. Dev Cell 8:343-352.
33.Kissel, H., Georgescu, M.M., Larisch, S., Manova, K., Hunnicutt, G.R., and Steller, H. 2005. The Sept4 septin locus is required for sperm terminal differentiation in mice. Dev Cell 8:353-364.
34.Xue, J., Tsang, C.W., Gai, W.P., Malladi, C.S., Trimble, W.S., Rostas, J.A., and Robinson, P.J. 2004. Septin 3 (G-septin) is a developmentally regulated phosphoprotein enriched in presynaptic nerve terminals. J Neurochem 91:579-590.
35.Son, J.H., Kawamata, H., Yoo, M.S., Kim, D.J., Lee, Y.K., Kim, S., Dawson, T.M., Zhang, H., Sulzer, D., Yang, L., et al. 2005. Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons. J Neurochem 94:1040-1053.
36.Dent, J., Kato, K., Peng, X.R., Martinez, C., Cattaneo, M., Poujol, C., Nurden, P., Nurden, A., Trimble, W.S., and Ware, J. 2002. A prototypic platelet septin and its participation in secretion. Proc Natl Acad Sci U S A 99:3064-3069.
37.Tsang, C.W., Fedchyshyn, M., Harrison, J., Xie, H., Xue, J., Robinson, P.J., Wang, L.Y., and Trimble, W.S. 2008. Superfluous role of mammalian septins 3 and 5 in neuronal development and synaptic transmission. Mol Cell Biol 28:7012-7029.
38.Joo, E., Tsang, C.W., and Trimble, W.S. 2005. Septins: traffic control at the cytokinesis intersection. Traffic 6:626-634.
39.Kinoshita, M., and Noda, M. 2001. Roles of septins in the mammalian cytokinesis machinery. Cell Struct Funct 26:667-670.
40.Kinoshita, M., Kumar, S., Mizoguchi, A., Ide, C., Kinoshita, A., Haraguchi, T., Hiraoka, Y., and Noda, M. 1997. Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures. Genes Dev 11:1535-1547.
41.Surka, M.C., Tsang, C.W., and Trimble, W.S. 2002. The mammalian septin MSF localizes with microtubules and is required for completion of cytokinesis. Mol Biol Cell 13:3532-3545.
42.Spiliotis, E.T., Kinoshita, M., and Nelson, W.J. 2005. A mitotic septin scaffold required for Mammalian chromosome congression and segregation. Science 307:1781-1785.
43.Zhu, M., Wang, F., Yan, F., Yao, P.Y., Du, J., Gao, X., Wang, X., Wu, Q., Ward, T., Li, J., et al. 2008. Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization. J Biol Chem 283:18916-18925.
44.Ono, R., Ihara, M., Nakajima, H., Ozaki, K., Kataoka-Fujiwara, Y., Taki, T., Nagata, K., Inagaki, M., Yoshida, N., Kitamura, T., et al. 2005. Disruption of Sept6, a fusion partner gene of MLL, does not affect ontogeny, leukemogenesis induced by MLL-SEPT6, or phenotype induced by the loss of Sept4. Mol Cell Biol 25:10965-10978.
45.Barral, Y., Mermall, V., Mooseker, M.S., and Snyder, M. 2000. Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast. Mol Cell 5:841-851.
46.Takizawa, P.A., DeRisi, J.L., Wilhelm, J.E., and Vale, R.D. 2000. Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. Science 290:341-344.
47.Dobbelaere, J., and Barral, Y. 2004. Spatial coordination of cytokinetic events by compartmentalization of the cell cortex. Science 305:393-396.
48.Longtine, M.S., DeMarini, D.J., Valencik, M.L., Al-Awar, O.S., Fares, H., De Virgilio, C., and Pringle, J.R. 1996. The septins: roles in cytokinesis and other processes. Curr Opin Cell Biol 8:106-119.
49.Mino, A., Tanaka, K., Kamei, T., Umikawa, M., Fujiwara, T., and Takai, Y. 1998. Shs1p: a novel member of septin that interacts with spa2p, involved in polarized growth in saccharomyces cerevisiae. Biochem Biophys Res Commun 251:732-736.
50.Greenbaum, M.P., Yan, W., Wu, M.H., Lin, Y.N., Agno, J.E., Sharma, M., Braun, R.E., Rajkovic, A., and Matzuk, M.M. 2006. TEX14 is essential for intercellular bridges and fertility in male mice. Proc Natl Acad Sci U S A 103:4982-4987.
51.Greenbaum, M.P., Ma, L., and Matzuk, M.M. 2007. Conversion of midbodies into germ cell intercellular bridges. Dev Biol 305:389-396.
52.Hall, P.A., and Russell, S.E. 2004. The pathobiology of the septin gene family. J Pathol 204:489-505.
53.Russell, S.E., McIlhatton, M.A., Burrows, J.F., Donaghy, P.G., Chanduloy, S., Petty, E.M., Kalikin, L.M., Church, S.W., McIlroy, S., Harkin, D.P., et al. 2000. Isolation and mapping of a human septin gene to a region on chromosome 17q, commonly deleted in sporadic epithelial ovarian tumors. Cancer Res 60:4729-4734.
54.Kalikin, L.M., Sims, H.L., and Petty, E.M. 2000. Genomic and expression analyses of alternatively spliced transcripts of the MLL septin-like fusion gene (MSF) that map to a 17q25 region of loss in breast and ovarian tumors. Genomics 63:165-172.
55.Kinoshita, A., Kinoshita, M., Akiyama, H., Tomimoto, H., Akiguchi, I., Kumar, S., Noda, M., and Kimura, J. 1998. Identification of septins in neurofibrillary tangles in Alzheimer's disease. Am J Pathol 153:1551-1560.
56.Kuhlenbaumer, G., Hannibal, M.C., Nelis, E., Schirmacher, A., Verpoorten, N., Meuleman, J., Watts, G.D., De Vriendt, E., Young, P., Stogbauer, F., et al. 2005. Mutations in SEPT9 cause hereditary neuralgic amyotrophy. Nat Genet 37:1044-1046.
57.Sugino, Y., Ichioka, K., Soda, T., Ihara, M., Kinoshita, M., Ogawa, O., and Nishiyama, H. 2008. Septins as diagnostic markers for a subset of human asthenozoospermia. J Urol 180:2706-2709.
58.Lhuillier, P., Rode, B., Escalier, D., Lores, P., Dirami, T., Bienvenu, T., Gacon, G., Dulioust, E., and Toure, A. 2009. Absence of annulus in human asthenozoospermia: Case Report. Hum Reprod.
59.Kuo, P.L., Wang, S.T., Lin, Y.M., Lin, Y.H., Teng, Y.N., and Hsu, C.C. 2004. Expression profiles of the DAZ gene family in human testis with and without spermatogenic failure. Fertil Steril 81:1034-1040.
60.Yang, Z.W., Wreford, N.G., and de Kretser, D.M. 1990. A quantitative study of spermatogenesis in the developing rat testis. Biol Reprod 43:629-635.
61.Yan, W., Rajkovic, A., Viveiros, M.M., Burns, K.H., Eppig, J.J., and Matzuk, M.M. 2002. Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper. Mol Endocrinol 16:1168-1184.
62.Yeh, Y.C., Yang, V.C., Huang, S.C., and Lo, N.W. 2005. Stage-dependent expression of extra-embryonic tissue-spermatogenesis-homeobox gene 1 (ESX1) protein, a candidate marker for X chromosome-bearing sperm. Reprod Fertil Dev 17:447-455.
63.Lin, Y.M., Chen, C.W., Sun, H.S., Tsai, S.J., Hsu, C.C., Teng, Y.N., Lin, J.S., and Kuo, P.L. 2001. Expression patterns and transcript concentrations of the autosomal DAZL gene in testes of azoospermic men. Mol Hum Reprod 7:1015-1022.
64.Cheng, Y.S., Kuo, P.L., Teng, Y.N., Kuo, T.Y., Chung, C.L., Lin, Y.H., Liao, R.W., Lin, J.S., and Lin, Y.M. 2006. Association of spermatogenic failure with decreased CDC25A expression in infertile men. Hum Reprod 21:2346-2352.
65.Cho, C., Willis, W.D., Goulding, E.H., Jung-Ha, H., Choi, Y.C., Hecht, N.B., and Eddy, E.M. 2001. Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet 28:82-86.
66.Nagata, K., Kawajiri, A., Matsui, S., Takagishi, M., Shiromizu, T., Saitoh, N., Izawa, I., Kiyono, T., Itoh, T.J., Hotani, H., et al. 2003. Filament formation of MSF-A, a mammalian septin, in human mammary epithelial cells depends on interactions with microtubules. J Biol Chem 278:18538-18543.
67.Kinoshita, M., Field, C.M., Coughlin, M.L., Straight, A.F., and Mitchison, T.J. 2002. Self- and actin-templated assembly of Mammalian septins. Dev Cell 3:791-802.
68.Nagata, K., Asano, T., Nozawa, Y., and Inagaki, M. 2004. Biochemical and cell biological analyses of a mammalian septin complex, Sept7/9b/11. J Biol Chem 279:55895-55904.
69.Martinez, C., Sanjuan, M.A., Dent, J.A., Karlsson, L., and Ware, J. 2004. Human septin-septin interactions as a prerequisite for targeting septin complexes in the cytosol. Biochem J 382:783-791.
70.Kremer, B.E., Haystead, T., and Macara, I.G. 2005. Mammalian septins regulate microtubule stability through interaction with the microtubule-binding protein MAP4. Mol Biol Cell 16:4648-4659.
71.Swanson, W.J., and Vacquier, V.D. 2002. The rapid evolution of reproductive proteins. Nat Rev Genet 3:137-144.
72.Hong, S., Choi, I., Woo, J.M., Oh, J., Kim, T., Choi, E., Kim, T.W., Jung, Y.K., Kim, D.H., Sun, C.H., et al. 2005. Identification and integrative analysis of 28 novel genes specifically expressed and developmentally regulated in murine spermatogenic cells. J Biol Chem 280:7685-7693.
73.Buckland, P.R. 2004. Allele-specific gene expression differences in humans. Hum Mol Genet 13 Spec No 2:R255-260.
74.Turek, P.J., Ljung, B.M., Cha, I., and Conaghan, J. 2000. Diagnostic findings from testis fine needle aspiration mapping in obstructed and nonobstructed azoospermic men. J Urol 163:1709-1716.
75.Coy, J.F., Dubel, S., Kioschis, P., Thomas, K., Micklem, G., Delius, H., and Poustka, A. 1996. Molecular cloning of tissue-specific transcripts of a transketolase-related gene: implications for the evolution of new vertebrate genes. Genomics 32:309-316.
76.Sapiro, R., Kostetskii, I., Olds-Clarke, P., Gerton, G.L., Radice, G.L., and Strauss, I.J. 2002. Male infertility, impaired sperm motility, and hydrocephalus in mice deficient in sperm-associated antigen 6. Mol Cell Biol 22:6298-6305.
77.Yen, P.H., Chai, N.N., and Salido, E.C. 1996. The human autosomal gene DAZLA: testis specificity and a candidate for male infertility. Hum Mol Genet 5:2013-2017.
78.Erickson, R.P., Harper, K., Menge, A., and Lee, C.Y. 1979. Immunological studies of the sperm-specific phosphoglycerate kinase-2 of mice. J Reprod Immunol 1:185-191.
79.Burmester, S., and Hoyer-Fender, S. 1996. Transcription and translation of the outer dense fiber gene (Odf1) during spermiogenesis in the rat. A study by in situ analyses and polysome fractionation. Mol Reprod Dev 45:10-20.
80.Kolk, A.H., and Samuel, T. 1975. Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa. Biochim Biophys Acta 393:307-319.
81.Munier, A., Serres, C., Kann, M.L., Boissan, M., Lesaffre, C., Capeau, J., Fouquet, J.P., and Lacombe, M.L. 2003. Nm23/NDP kinases in human male germ cells: role in spermiogenesis and sperm motility? Exp Cell Res 289:295-306.
82.Teng, Y.N., Lin, Y.M., Lin, Y.H., Tsao, S.Y., Hsu, C.C., Lin, S.J., Tsai, W.C., and Kuo, P.L. 2002. Association of a single-nucleotide polymorphism of the deleted-in-azoospermia-like gene with susceptibility to spermatogenic failure. J Clin Endocrinol Metab 87:5258-5264.
83.Aoki, V.W., Liu, L., and Carrell, D.T. 2005. Identification and evaluation of a novel sperm protamine abnormality in a population of infertile males. Hum Reprod 20:1298-1306.
84.Tanaka, H., Miyagawa, Y., Tsujimura, A., Matsumiya, K., Okuyama, A., and Nishimune, Y. 2003. Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations. Mol Hum Reprod 9:69-73.
85.Bourne, H.R. 1997. How receptors talk to trimeric G proteins. Curr Opin Cell Biol 9:134-142.
86.Goto, M., and Eddy, E.M. 2004. Speriolin is a novel spermatogenic cell-specific centrosomal protein associated with the seventh WD motif of Cdc20. J Biol Chem 279:42128-42138.
87.Ito, S., Sakai, A., Nomura, T., Miki, Y., Ouchida, M., Sasaki, J., and Shimizu, K. 2001. A novel WD40 repeat protein, WDC146, highly expressed during spermatogenesis in a stage-specific manner. Biochem Biophys Res Commun 280:656-663.
88.Nie, D.S., Xiang, Y., Wang, J., Deng, Y., Tan, X.J., Liang, Y.H., and Lu, G.X. 2005. Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis. Biochem Biophys Res Commun 328:1010-1018.
89.Xue, J.C., and Goldberg, E. 2000. Identification of a novel testis-specific leucine-rich protein in humans and mice. Biol Reprod 62:1278-1284.
90.Turner, A.J., Matsas, R., and Kenny, A.J. 1985. Are there neuropeptide-specific peptidases? Biochem Pharmacol 34:1347-1356.
91.Ghaddar, G., Ruchon, A.F., Carpentier, M., Marcinkiewicz, M., Seidah, N.G., Crine, P., Desgroseillers, L., and Boileau, G. 2000. Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family. Biochem J 347:419-429.
92.Carpentier, M., Guillemette, C., Bailey, J.L., Boileau, G., Jeannotte, L., DesGroseillers, L., and Charron, J. 2004. Reduced fertility in male mice deficient in the zinc metallopeptidase NL1. Mol Cell Biol 24:4428-4437.
93.Frew, I.J., Hammond, V.E., Dickins, R.A., Quinn, J.M., Walkley, C.R., Sims, N.A., Schnall, R., Della, N.G., Holloway, A.J., Digby, M.R., et al. 2003. Generation and analysis of Siah2 mutant mice. Mol Cell Biol 23:9150-9161.
94.Liu, Z., Oughtred, R., and Wing, S.S. 2005. Characterization of E3Histone, a novel testis ubiquitin protein ligase which ubiquitinates histones. Mol Cell Biol 25:2819-2831.
95.Dev, K.K., van der Putten, H., Sommer, B., and Rovelli, G. 2003. Part I: parkin-associated proteins and Parkinson's disease. Neuropharmacology 45:1-13.
96.Beckmann, J.S., Maurer, F., Delorenzi, M., and Falquet, L. 2005. On ubiquitin ligases and cancer. Hum Mutat 25:507-512.
97.Dickins, R.A., Frew, I.J., House, C.M., O'Bryan, M.K., Holloway, A.J., Haviv, I., Traficante, N., de Kretser, D.M., and Bowtell, D.D. 2002. The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice. Mol Cell Biol 22:2294-2303.
98.Ono, T., Slaughter, G.R., Cook, R.G., and Means, A.R. 1989. Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein. J Biol Chem 264:2081-2087.
99.Means, A.R., Cruzalegui, F., LeMagueresse, B., Needleman, D.S., Slaughter, G.R., and Ono, T. 1991. A novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene. Mol Cell Biol 11:3960-3971.
100.Wu, J.Y., and Means, A.R. 2000. Ca(2+)/calmodulin-dependent protein kinase IV is expressed in spermatids and targeted to chromatin and the nuclear matrix. J Biol Chem 275:7994-7999.
101.Field, C.M., and Kellogg, D. 1999. Septins: cytoskeletal polymers or signalling GTPases? Trends Cell Biol 9:387-394.
102.Zhu, M., Wang, F., Yan, F., Yao, P.Y., Du, J., Gao, X., Wang, X., Wu, Q., Ward, T., Li, J., et al. 2008. Septin 7 interacts with CENP-E and is required for its kinetochore localization. J Biol Chem.
103.Hsu, S.C., Hazuka, C.D., Roth, R., Foletti, D.L., Heuser, J., and Scheller, R.H. 1998. Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 20:1111-1122.
104.Ding, X., Yu, W., Liu, M., Shen, S., Chen, F., Cao, L., Wan, B., and Yu, L. 2008. GTP Binding Is Required for SEPT12 to Form Filaments and to Interact with SEPT11. Mol Cells 25:385-389.
105.Ding, X., Yu, W., Liu, M., Shen, S., Chen, F., Wan, B., and Yu, L. 2007. SEPT12 interacts with SEPT6 and this interaction alters the filament structure of SEPT6 in Hela cells. J Biochem Mol Biol 40:973-978.
106.Vega, I.E., and Hsu, S.C. 2003. The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. Neuroreport 14:31-37.
107.Schmidt, K., and Nichols, B.J. 2004. Functional interdependence between septin and actin cytoskeleton. BMC Cell Biol 5:43.
108.Low, C., and Macara, I.G. 2006. Structural analysis of septin 2, 6, and 7 complexes. J Biol Chem 281:30697-30706.
109.Moreno, R.D., and Schatten, G. 2000. Microtubule configurations and post-translational alpha-tubulin modifications during mammalian spermatogenesis. Cell Motil Cytoskeleton 46:235-246.
110.Moritz, M., and Agard, D.A. 2001. Gamma-tubulin complexes and microtubule nucleation. Curr Opin Struct Biol 11:174-181.
111.Manandhar, G., Schatten, H., and Sutovsky, P. 2005. Centrosome reduction during gametogenesis and its significance. Biol Reprod 72:2-13.
112.Yan, W., Ma, L., Burns, K.H., and Matzuk, M.M. 2004. Haploinsufficiency of kelch-like protein homolog 10 causes infertility in male mice. Proc Natl Acad Sci U S A 101:7793-7798.
113.Liu, Y., Yao, Z.X., Bendavid, C., Borgmeyer, C., Han, Z., Cavalli, L.R., Chan, W.Y., Folmer, J., Zirkin, B.R., Haddad, B.R., et al. 2005. Haploinsufficiency of cytochrome P450 17alpha-hydroxylase/17,20 lyase (CYP17) causes infertility in male mice. Mol Endocrinol 19:2380-2389.
114.Xu, B., Hao, Z., Jha, K.N., Zhang, Z., Urekar, C., Digilio, L., Pulido, S., Strauss, J.F., 3rd, Flickinger, C.J., and Herr, J.C. 2008. Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency. Dev Biol 319:211-222.
115.Zhang, Z., Kostetskii, I., Moss, S.B., Jones, B.H., Ho, C., Wang, H., Kishida, T., Gerton, G.L., Radice, G.L., and Strauss, J.F., 3rd. 2004. Haploinsufficiency for the murine orthologue of Chlamydomonas PF20 disrupts spermatogenesis. Proc Natl Acad Sci U S A 101:12946-12951.