A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.
References
[1]
McLachlan RI, de Kretser DM (2001) Male infertility: the case for continued research. Med J Aust 174: 116–117.
[2]
Cedenho AP (2007) Evaluation of the subfertile male. In: Oehninger S, Kruger T, editors. Male Infertility, Diagnosis and Treatment. UK: Inferma UK Ltd. 117–140.
[3]
Jarow JP, Espeland MA, Lipshultz LI (1989) Evaluation of the azoospermic patient. J Urol 142: 62–65.
[4]
World Health Organization (2010) World Health Organization Labororatory Manual for the Examination and Processing of Human Semen. Geneva, World Health Organization.
[5]
Coddington CC, Oehninger SC, Olive DL, Franken DR, Kruger TF, et al. (1994) Hemizona index (HZI) demonstrates excellent predictability when evaluating sperm fertilizing capacity in in vitro fertilization patients. J Androl 15: 250–254.
[6]
Esterhuizen AD, Franken DR, Lourens JG, van Rooyen LH (2001) Clinical importance of zona pellucida-induced acrosome reaction and its predictive value for IVF. Hum Reprod 16: 138–144.
[7]
Liu DY, Baker HW (2003) Disordered zona pellucida-induced acrosome reaction and failure of in vitro fertilization in patients with unexplained infertility. Fertil Steril 79: 74–80.
[8]
Nixon B, Aitken RJ, McLaughlin EA (2007) New insights into the molecular mechanisms of sperm-egg interaction. Cell Mol Life Sci 64: 1805–1823.
[9]
Reid AT, Redgrove K, Aitken RJ, Nixon B (2011) Cellular mechanisms regulating sperm-zona pellucida interaction. Asian J Androl 13: 88–96.
Nixon B, Bielanowicz A, McLaughlin EA, Tanphaichitr N, Ensslin MA, et al. (2009) Composition and significance of detergent resistant membranes in mouse spermatozoa. J Cell Physiol 218: 122–134.
[12]
Nixon B, Mitchell LA, Anderson AL, McLaughlin EA, O’Bryan M K, et al. (2011) Proteomic and functional analysis of human sperm detergent resistant membranes. J Cell Physiol 226: 2651–2665.
[13]
Biggers JD, Whitten WK, Whittingham DG (1971) The culture of mouse embryo in vitro. In: Daniel JCJ, editor. Methods in Mammalian Embryology. San Francisco, CA: Freeman Press. pp. 86–116.
[14]
Mitchell LA, Nixon B, Aitken RJ (2007) Analysis of chaperone proteins associated with human spermatozoa during capacitation. Mol Hum Reprod 13: 605–613.
[15]
Schagger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199: 223–231.
[16]
Dun MD, Smith ND, Baker MA, Lin M, Aitken RJ, et al.. (2011) The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction. J Biol Chem.
[17]
Redgrove KA, Anderson AL, Dun MD, McLaughlin EA, O’Bryan MK, et al. (2011) Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae. Dev Biol 356: 460–474.
[18]
Baba T, Niida Y, Michikawa Y, Kashiwabara S, Kodaira K, et al. (1994) An acrosomal protein, sp32, in mammalian sperm is a binding protein specific for two proacrosins and an acrosin intermediate. J Biol Chem 269: 10133–10140.
[19]
Dube C, Leclerc P, Baba T, Reyes-Moreno C, Bailey JL (2005) The proacrosin binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm. J Androl 26: 519–528.
[20]
Luconi M, Porazzi I, Ferruzzi P, Marchiani S, Forti G, et al. (2005) Tyrosine phosphorylation of the a kinase anchoring protein 3 (AKAP3) and soluble adenylate cyclase are involved in the increase of human sperm motility by bicarbonate. Biol Reprod 72: 22–32.
[21]
Turner RM, Eriksson RL, Gerton GL, Moss SB (1999) Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82. Mol Hum Reprod 5: 816–824.
[22]
Baker MA, Aitken RJ (2009) Proteomic insights into spermatozoa: critiques, comments and concerns. Expert Rev Proteomics 6: 691–705.
[23]
Baker MA, Reeves G, Hetherington L, Aitken RJ (2010) Analysis of proteomic changes associated with sperm capacitation through the combined use of IPG-strip pre-fractionation followed by RP chromatography LC-MS/MS analysis. Proteomics 10: 482–495.
[24]
Danshina PV, Geyer CB, Dai Q, Goulding EH, Willis WD, et al. (2010) Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice. Biol Reprod 82: 136–145.
[25]
Iguchi N, Tobias JW, Hecht NB (2006) Expression profiling reveals meiotic male germ cell mRNAs that are translationally up- and down-regulated. Proc Natl Acad Sci U S A 103: 7712–7717.
[26]
Hereng TH, Elgstoen KB, Cederkvist FH, Eide L, Jahnsen T, et al. (2011) Exogenous pyruvate accelerates glycolysis and promotes capacitation in human spermatozoa. Hum Reprod 26: 3249–3263.
[27]
De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, et al. (2009) DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress. Biol Reprod 81: 517–524.
[28]
Hammoud SS, Nix DA, Hammoud AO, Gibson M, Cairns BR, et al. (2011) Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men. Hum Reprod 26: 2558–2569.
[29]
Simon L, Castillo J, Oliva R, Lewis SE (2011) Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes. Reprod Biomed Online 23: 724–734.
[30]
Huszar G, Jakab A, Sakkas D, Ozenci CC, Cayli S, et al. (2007) Fertility testing and ICSI sperm selection by hyaluronic acid binding: clinical and genetic aspects. Reprod Biomed Online 14: 650–663.
[31]
Huszar G, Ozkavukcu S, Jakab A, Celik-Ozenci C, Sati GL, et al. (2006) Hyaluronic acid binding ability of human sperm reflects cellular maturity and fertilizing potential: selection of sperm for intracytoplasmic sperm injection. Curr Opin Obstet Gynecol 18: 260–267.
[32]
Huszar G, Sbracia M, Vigue L, Miller DJ, Shur BD (1997) Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios. Biol Reprod 56: 1020–1024.
[33]
Huszar G, Stone K, Dix D, Vigue L (2000) Putative creatine kinase M-isoform in human sperm is identifiedas the 70-kilodalton heat shock protein HspA2. Biol Reprod 63: 925–932.
[34]
Ergur AR, Dokras A, Giraldo JL, Habana A, Kovanci E, et al. (2002) Sperm maturity and treatment choice of in vitro fertilization (IVF) or intracytoplasmic sperm injection: diminished sperm HspA2 chaperone levels predict IVF failure. Fertil Steril 77: 910–918.
[35]
Dun MD, Smith ND, Baker MA, Lin M, Aitken RJ, et al. (2011) The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction. J Biol Chem 286: 36875–36887.
[36]
Carmona E, Weerachatyanukul W, Soboloff T, Fluharty AL, White D, et al. (2002) Arylsulfatase a is present on the pig sperm surface and is involved in sperm-zona pellucida binding. Dev Biol 247: 182–196.
[37]
Kimura M, Kim E, Kang W, Yamashita M, Saigo M, et al. (2009) Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization. Biol Reprod 81: 939–947.
[38]
Lathrop WF, Carmichael EP, Myles DG, Primakoff P (1990) cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals. J Cell Biol 111: 2939–2949.
[39]
Tantibhedhyangkul J, Weerachatyanukul W, Carmona E, Xu H, Anupriwan A, et al. (2002) Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding. Biol Reprod 67: 212–219.
[40]
Sabeur K, Cherr GN, Yudin AI, Primakoff P, Li MW, et al. (1997) The PH-20 protein in human spermatozoa. J Androl 18: 151–158.
[41]
Dix DJ, Allen JW, Collins BW, Poorman-Allen P, Mori C, et al. (1997) HSP70–2 is required for desynapsis of synaptonemal complexes during meiotic prophase in juvenile and adult mouse spermatocytes. Development 124: 4595–4603.
[42]
Mori C, Nakamura N, Dix DJ, Fujioka M, Nakagawa S, et al. (1997) Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp 70–2 knockout mice. Dev Dyn 208: 125–136.
[43]
Zhu D, Dix DJ, Eddy EM (1997) HSP70–2 is required for CDC2 kinase activity in meiosis I of mouse spermatocytes. Development 124: 3007–3014.
[44]
Govin J, Caron C, Escoffier E, Ferro M, Kuhn L, et al. (2006) Post-meiotic shifts in HSPA2/HSP70.2 chaperone activity during mouse spermatogenesis. J Biol Chem. 281: 37888–37892.
[45]
Xu H, Kongmanas K, Kadunganattil S, Smith CE, Rupar T, et al. (2011) Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells. J Lipid Res 52: 2187–2197.
[46]
Baba D, Kashiwabara S, Honda A, Yamagata K, Wu Q, et al. (2002) Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg. J Biol Chem 277: 30310–30314.
