Sperm Chromatin
2008; Elsevier BV; Volume: 7; Issue: 10 Linguagem: Inglês
10.1074/mcp.r800005-mcp200
ISSN1535-9484
Autores Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoSperm are remarkably complex cells with a singularly important mission: to deliver paternal DNA and its associated factors to the oocyte to start a new life. The integrity of sperm DNA is a keystone of reproductive success, which includes fertilization and embryonic development. In addition, the significance in these processes of proteins that associate with sperm DNA is increasingly being appreciated. In this review, we highlight proteomic studies that have identified sperm chromatin proteins with fertility roles that have been validated by molecular studies in model organisms or correlations in the clinic. Up to 50% of male-factor infertility cases in the clinic have no known cause and therefore no direct treatment. In-depth study of the molecular basis of infertility has great potential to inform the development of sensitive diagnostic tools and effective therapies that will address this incongruity. Because sperm rely on testis-specific protein isoforms and post-translational modifications for their development and function, sperm-specific processes are ideal for proteomic explorations that can bridge the research lab and fertility clinic. Sperm are remarkably complex cells with a singularly important mission: to deliver paternal DNA and its associated factors to the oocyte to start a new life. The integrity of sperm DNA is a keystone of reproductive success, which includes fertilization and embryonic development. In addition, the significance in these processes of proteins that associate with sperm DNA is increasingly being appreciated. In this review, we highlight proteomic studies that have identified sperm chromatin proteins with fertility roles that have been validated by molecular studies in model organisms or correlations in the clinic. Up to 50% of male-factor infertility cases in the clinic have no known cause and therefore no direct treatment. In-depth study of the molecular basis of infertility has great potential to inform the development of sensitive diagnostic tools and effective therapies that will address this incongruity. Because sperm rely on testis-specific protein isoforms and post-translational modifications for their development and function, sperm-specific processes are ideal for proteomic explorations that can bridge the research lab and fertility clinic. Sperm are intricate yet streamlined cells with the underlying purpose of producing a healthy baby. Sperm cells in every sexually reproducing animal are highly specialized delivery vehicles for the chromatin cargo within, which is composed of DNA and its associated proteins. It is clear that sperm chromatin is essential for sperm function and subsequent embryonic development because defects in sperm chromatin are linked to natural reproductive malfunctions like spontaneous abortion as well as assisted reproductive failure (1Boe-Hansen G.B. Fedder J. Ersboll A.K. Christensen P. The sperm chromatin structure assay as a diagnostic tool in the human fertility clinic.Hum. Reprod. 2006; 21: 1576-1582Crossref PubMed Scopus (112) Google Scholar, 2Bungum M. Humaidan P. Axmon A. Spano M. Bungum L. Erenpreiss J. Giwercman A. Sperm DNA integrity assessment in prediction of assisted reproduction technology outcome.Hum. Reprod. 2007; 22: 174-179Crossref PubMed Scopus (461) Google Scholar, 3Cebesoy F.B. Aydos K. Unlu C. Effect of sperm chromatin damage on fertilization ratio and embryo quality post-ICSI.Arch. Androl. 2006; 52: 397-402Crossref PubMed Scopus (25) Google Scholar). These defects can include disrupted DNA integrity caused by genetic mutations, apoptotic DNA fragmentation, or exposure to environmental agents and free radicals (4Andrabi S.M. Mammalian sperm chromatin structure and assessment of DNA fragmentation.J. Assist. Reprod. Genet. 2007; 24: 561-569Crossref PubMed Scopus (36) Google Scholar). Furthermore, disruption of chromatin proteins contributes to decreased fertility (5Belokopytova I.A. Kostyleva E.I. Tomilin A.N. Vorob'ev V.I. Human male infertility may be due to a decrease of the protamine P2 content in sperm chromatin.Mol. Reprod. Dev. 1993; 34: 53-57Crossref PubMed Scopus (0) Google Scholar). Despite this evidence, most clinical assays for sperm chromatin only detect gross defects in DNA integrity (4Andrabi S.M. Mammalian sperm chromatin structure and assessment of DNA fragmentation.J. Assist. Reprod. Genet. 2007; 24: 561-569Crossref PubMed Scopus (36) Google Scholar, 6Angelopoulou R. Plastira K. Msaouel P. Spermatozoal sensitive biomarkers to defective protaminosis and fragmented DNA.Reprod. Biol. Endocrinol. 2007; 5: 36Crossref PubMed Scopus (0) Google Scholar). This, combined with the typical assessments of sperm production, including motility, morphology, and male hormone levels, still result in a striking 30–50% of male-factor infertility cases having no known cause (7Turek P.J. Practical approaches to the diagnosis and management of male infertility.Nat. Clin. Pract. Urol. 2005; 2: 226-238Crossref PubMed Scopus (0) Google Scholar). With 2.1 million couples in the United States facing infertility as of 2002, there is a pressing need for an expanded array of sensitive tests to improve diagnostic capabilities in the clinic (8National Survey of Family Growth (2002) cycle 6, Hyattsville, MD, U. S. Dept. of Health and Human Services. National Center for Health Statistics, 2004Google Scholar). The requirement for more research extends beyond diagnosis, however, because many of the basic cellular mechanisms that underlie male infertility remain unknown. Consequently, virtually no therapies exist that remedy the molecular causes of sperm dysfunction. Rather, prolonged or invasive assisted reproductive technology (ART) 1The abbreviations used are: ART, assisted reproductive technology; ICSI, intracytoplasmic sperm injection; SNBP, sperm nuclear basic protein; IVF, in vitro fertilization; 2D-PAGE, two-dimensional poly-acrylamide gel electrophoresis; LC-MS2, liquid chromatography-tandem mass spectrometry; GO, Gene Ontology; RNAi, RNA interference; MudPIT, Multidimensional Protein Identification Technology; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. 1The abbreviations used are: ART, assisted reproductive technology; ICSI, intracytoplasmic sperm injection; SNBP, sperm nuclear basic protein; IVF, in vitro fertilization; 2D-PAGE, two-dimensional poly-acrylamide gel electrophoresis; LC-MS2, liquid chromatography-tandem mass spectrometry; GO, Gene Ontology; RNAi, RNA interference; MudPIT, Multidimensional Protein Identification Technology; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. procedures and intracytoplasmic sperm injection (ICSI) are used to bypass male infertility, with a modest 42% success rate (9Assisted Reproductive Technology (ART) Report. U. S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics, Hyattsville, MD2004Google Scholar). In addition, the safety of widespread ICSI use has recently been called into question. It is speculated that sperm from infertile patients contain cytologically subtle chromatin abnormalities that can affect the resulting embryo (10D'Occhio M.J. Hengstberger K.J. Johnston S.D. Biology of sperm chromatin structure and relationship to male fertility and embryonic survival.Anim. Reprod. Sci. 2007; 101: 1-17Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 11Georgiou I. Syrrou M. Pardalidis N. Karakitsios K. Mantzavinos T. Giotitsas N. Loutradis D. Dimitriadis F. Saito M. Miyagawa I. Tzoumis P. Sylakos A. Kanakas N. Moustakareas T. Baltogiannis D. Touloupides S. Giannakis D. Fatouros M. Sofikitis N. Genetic and epigenetic risks of intracytoplasmic sperm injection method.Asian J. Androl. 2006; 8: 643-673Crossref PubMed Scopus (0) Google Scholar). Thus it is important to define the epigenetic features of paternal chromatin that can affect future generations. Further knowledge about sperm chromatin protein function can lead to the development of novel therapies that target only sperm, reducing possibilities for side effects or unintended consequences on resulting offspring. Any comprehensive understanding of sperm biology must include proteomic analysis. Sperm are particularly well suited to proteomic approaches. Compared with most other cell types, they are easily isolated from other tissues and fluids. Importantly, sperm development and function rely heavily on sperm-specific protein isoforms of somatic counterparts, as well as the post-translational modification of key sperm proteins (12Caron C. Govin J. Rousseaux S. Khochbin S. How to pack the genome for a safe trip.Prog. Mol. Subcell. Biol. 2005; 38: 65-89Crossref PubMed Google Scholar, 13Sassone-Corsi P. Unique chromatin remodeling and transcriptional regulation in spermatogenesis.Science. 2002; 296: 2176-2178Crossref PubMed Scopus (330) Google Scholar). For example, during spermatogenesis, histone proteins in developing sperm are replaced by testis-specific histone variants important for fertility (14Churikov D. Zalenskaya I.A. Zalensky A.O. Male germline-specific histones in mouse and man.Cytogenet. Genome Res. 2004; 105: 203-214Crossref PubMed Scopus (0) Google Scholar). Also, because de novo transcription in post-meiotic sperm is largely silenced, the cell depends on post-translational modifications to implement subsequent stages of sperm formation, maturation, and activation (15Dadoune J.P. Siffroi J.P. Alfonsi M.F. Transcription in haploid male germ cells.Int. Rev. Cytol. 2004; 237: 1-56Crossref PubMed Scopus (0) Google Scholar). The development of clinical applications arising from the proteomic discovery of sperm proteins and sperm-specific protein isoforms is a budding field, with abundant opportunities for innovation. Of particular promise is the identification of sperm-specific post-translational modifications that are functionally important during and after sperm development. Functional analysis of sperm-specific factors following proteomic identification is crucial to our understanding of male reproduction. Direct experimental approaches can identify relevant variables and reduce experimental complexity. Such strategies are facilitated by the use of model organisms like sea urchins, fish, mice, worms, and flies. Model organisms often have simplified anatomical structures and streamlined developmental programs in comparison to humans. Their male germ cells can be obtained in large quantities in various developmental stages. Though some male reproductive proteins evolve rapidly (16Swanson W.J. Vacquier V.D. The rapid evolution of reproductive proteins.Nat. Rev. Genet. 2002; 3: 137-144Crossref PubMed Scopus (932) Google Scholar), others involved in fundamental processes of sperm development like meiosis are conserved (17Chu D.S. Liu H. Nix P. Wu T.F. Ralston E.J. Yates 3rd, J.R. Meyer B.J. Sperm chromatin proteomics identifies evolutionarily conserved fertility factors.Nature. 2006; 443: 101-105Crossref PubMed Scopus (139) Google Scholar, 18Hackstein J.H. Hochstenbach R. Pearson P.L. Towards an understanding of the genetics of human male infertility: lessons from flies.Trends Genet. 2000; 16: 565-572Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 19Xu E.Y. Lee D.F. Klebes A. Turek P.J. Kornberg T.B. Reijo Pera R.A. Human BOULE gene rescues meiotic defects in infertile flies.Hum. Mol. Genet. 2003; 12: 169-175Crossref PubMed Scopus (96) Google Scholar). As in humans, the chromatin of each of these animals is at its most compact in mature sperm. Ultimately, defining the molecular functions of conserved proteins can aid in identifying biologically relevant clinical markers of human infertility, as well as targets for infertility therapies. One challenge currently facing proteomicists, basic biologists, and clinicians is how to make sense of the vast amount of data being generated through proteomic, genomic, functional, and clinical studies. Only through solving this challenge can the full potential of proteomics be translated into meaningful clinical options for male infertility. This review will highlight a selection of recent advances in our understanding of sperm chromatin biology that allow proteomic data to be interpreted within cellular or functional contexts. These studies include strategies for employing bioinformatics to generate testable hypotheses, increasing the specificity of proteomic analysis, analyzing function in model organisms, and correlating expression changes with detailed observations from the clinic. The proteomics of other sperm subcellular compartments, fluid components, or physiological reproductive structures are presented elsewhere (20Aitken R.J. Baker M.A. The role of proteomics in understanding sperm cell biology.Int. J. Androl. 2007; 30: 1-8PubMed Google Scholar, 21Lefievre L. Bedu-Addo K. Conner S.J. Machado-Oliveira G.S. Chen Y. Kirkman-Brown J.C. Afnan M.A. Publicover S.J. Ford W.C. Barratt C.L. Counting sperm does not add up any more: time for a new equation?.Reproduction. 2007; 133: 675-684Crossref PubMed Scopus (0) Google Scholar). By discussing sperm chromatin-based studies, we illustrate the contributions that chromatin studies can make toward understanding fertility, as well as the need to develop sophisticated molecular tools for the study of male-factor infertility. We also promote communication and integration between lab and clinic when designing and interpreting studies. Sperm are arguably the most specialized cells in the human body (Fig. 1A). As such, a remarkable set of complex events must unfold to ensure the proper development and function of each cell. These include a) configuring chromatin for efficient delivery to the egg and preserving the epigenetic information needed for subsequent zygotic development (22Wu T.F. Chu D.S. Epigenetic processes implemented during spermatogenesis distinguish the paternal pronucleus in the embryo.Reprod. Biomed. Online. 2008; 16: 13-22Abstract Full Text PDF PubMed Google Scholar); b) using a flagellated force-generating tail structure for long-distance travel to the egg (23Suarez S.S. Marquez B. Harris T.P. Schimenti J.C. Different regulatory systems operate in the midpiece and principal piece of the mammalian sperm flagellum.Soc. Reprod. Fertil. Suppl. 2007; 65: 331-334PubMed Google Scholar, 24Suarez S.S. Pacey A.A. Sperm transport in the female reproductive tract.Hum. Reprod. Update. 2006; 12: 23-37Crossref PubMed Scopus (572) Google Scholar); and c) unleashing a series of membrane-associated cellular changes for penetrating the substantial corona and zona pellucida layers surrounding the egg (25Brewis I.A. Moore H.D. Fraser L.R. Holt W.V. Baldi E. Luconi M. Gadella B.M. Ford W.C. Harrison R.A. Molecular mechanisms during sperm capacitation.Hum. Fertil. (Camb.). 2005; 8: 253-261Crossref PubMed Scopus (18) Google Scholar, 26Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Brewis I.A. Moore H.D. Fraser L.R. Holt W.V. Baldi E. Luconi M. Gadella B.M. Ford W.C. Harrison R.A. Signaling pathways involved in sperm capacitation: molecular mechanisms during sperm capacitation.Soc. Reprod. Fertil. Suppl. 2007; 65: 245-259PubMed Google Scholar, 27Tomes C.N. Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Brewis I.A. Moore H.D. Fraser L.R. Holt W.V. Baldi E. Luconi M. Gadella B.M. Ford W.C. Harrison R.A. Molecular mechanisms of membrane fusion during acrosomal exocytosis signaling pathways involved in sperm capacitation: molecular mechanisms during sperm capacitation.Soc. Reprod. Fertil. Suppl. 2007; 65: 275-291PubMed Google Scholar). The sperm cell develops these capacities at distinct time points before fertilization (Fig. 1B). Chromatin changes occur in the testis during meiosis (in which copies of the genome are partitioned into haploid spermatid cells) and spermiogenesis (in which spermatids elongate to form sperm with fully compacted chromatin). Sperm motility is gained in the epididymis upon exit from the testis, and sperm capacitation for penetrating the zona pellucida occurs within the female reproductive tract. These events are largely controlled by post-translational events, for transcription and translation greatly subside as DNA becomes tightly compacted and cytoplasm is jettisoned during spermiogenesis (13Sassone-Corsi P. Unique chromatin remodeling and transcriptional regulation in spermatogenesis.Science. 2002; 296: 2176-2178Crossref PubMed Scopus (330) Google Scholar, 15Dadoune J.P. Siffroi J.P. Alfonsi M.F. Transcription in haploid male germ cells.Int. Rev. Cytol. 2004; 237: 1-56Crossref PubMed Scopus (0) Google Scholar). Progression of subsequent developmental stages is mediated by existing signaling molecules (26Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Brewis I.A. Moore H.D. Fraser L.R. Holt W.V. Baldi E. Luconi M. Gadella B.M. Ford W.C. Harrison R.A. Signaling pathways involved in sperm capacitation: molecular mechanisms during sperm capacitation.Soc. Reprod. Fertil. Suppl. 2007; 65: 245-259PubMed Google Scholar, 27Tomes C.N. Salicioni A.M. Platt M.D. Wertheimer E.V. Arcelay E. Allaire A. Sosnik J. Visconti P.E. Brewis I.A. Moore H.D. Fraser L.R. Holt W.V. Baldi E. Luconi M. Gadella B.M. Ford W.C. Harrison R.A. Molecular mechanisms of membrane fusion during acrosomal exocytosis signaling pathways involved in sperm capacitation: molecular mechanisms during sperm capacitation.Soc. Reprod. Fertil. Suppl. 2007; 65: 275-291PubMed Google Scholar). For example, the phosphatase protein PP1γ is involved in multiple aspects of sperm development, including spermiogenesis and acquisition of sperm motility (28Chakrabarti R. Cheng L. Puri P. Soler D. Vijayaraghavan S. Protein phosphatase PP1 gamma 2 in sperm morphogenesis and epididymal initiation of sperm motility.Asian J. Androl. 2007; 9: 445-452Crossref PubMed Scopus (0) Google Scholar, 29Chakrabarti R. Kline D. Lu J. Orth J. Pilder S. Vijayaraghavan S. Analysis of Ppp1cc-null mice suggests a role for PP1 gamma 2 in sperm morphogenesis.Biol. Reprod. 2007; 76: 992-1001Crossref PubMed Scopus (0) Google Scholar, 30Varmuza S. Jurisicova A. Okano K. Hudson J. Boekelheide K. Shipp E.B. Spermiogenesis is impaired in mice bearing a targeted mutation in the protein phosphatase 1c gamma gene.Dev. Biol. 1999; 205: 98-110Crossref PubMed Scopus (0) Google Scholar). Much is yet unknown regarding the molecular basis of sperm biology, particularly how these drastic changes in cell morphology and function occur in the absence of new protein synthesis. The wide array of changes in chromatin during sperm development provides an ideal platform for proteomic exploration of essential sperm-specific chromatin proteins. During meiosis, sperm chromosomes are segregated in a distinct fashion from that of oocyte chromosomes with unique timing and generated end products (31Hunt P. Hassold T. Sex matters in meiosis.Science. 2002; 296: 2181-2183Crossref PubMed Scopus (310) Google Scholar). After meiosis, sperm DNA experiences extreme chromosome compaction during spermiogenesis. This compaction is mediated by drastic changes at the most fundamental level of DNA packaging where a nucleosomal architecture shifts to a toroidal structure (32Allen M.J. Lee C. Lee J.D. Pogany G.C. Balooch M. Siekhaus W.J. Balhorn R. Atomic force microscopy of mammalian sperm chromatin.Chromosoma. 1993; 102: 623-630Crossref PubMed Scopus (69) Google Scholar). Sperm nuclear basic proteins (SNBPs), which include variants of histone subunits, transition proteins, and protamine proteins, implement this change (33Lewis J.D. Abbott D.W. Ausio J. A haploid affair: core histone transitions during spermatogenesis.Biochem. Cell Biol. 2003; 81: 131-140Crossref PubMed Scopus (0) Google Scholar, 34Lewis J.D. Song Y. de Jong M.E. Bagha S.M. Ausio J. A walk though vertebrate and invertebrate protamines.Chromosoma. 2003; 111: 473-482Crossref PubMed Google Scholar). This transition occurs in a stepwise fashion, replacing somatic histones with testis-expressed histone variants, then transition proteins, and finally protamines (35Braun R.E. Packaging paternal chromosomes with protamine.Nat. Genet. 2001; 28: 10-12Crossref PubMed Google Scholar). Deficits in SNBPs are known to cause male infertility (36Cho C. Willis W.D. Goulding E.H. Jung-Ha H. Choi Y.C. Hecht N.B. Eddy E.M. Haploinsufficiency of protamine-1 or -2 causes infertility in mice.Nat. Genet. 2001; 28: 82-86Crossref PubMed Google Scholar, 37Yu Y.E. Zhang Y. Unni E. Shirley C.R. Deng J.M. Russell L.D. Weil M.M. Behringer R.R. Meistrich M.L. Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4683-4688Crossref PubMed Scopus (214) Google Scholar, 38Zhao M. Shirley C.R. Yu Y.E. Mohapatra B. Zhang Y. Unni E. Deng J.M. Arango N.A. Terry N.H. Weil M.M. Russell L.D. Behringer R.R. Meistrich M.L. Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.Mol. Cell. Biol. 2001; 21: 7243-7255Crossref PubMed Scopus (0) Google Scholar). A growing body of work indicates that these chromatin proteins do not act exclusively to compact sperm DNA. Histone localization and post-translational modification of histones encode epigenetic information that may regulate transcription important for sperm development (22Wu T.F. Chu D.S. Epigenetic processes implemented during spermatogenesis distinguish the paternal pronucleus in the embryo.Reprod. Biomed. Online. 2008; 16: 13-22Abstract Full Text PDF PubMed Google Scholar). They may also serve to mark the heterochromatic state of specific regions of the genome that may be important after fertilization, when somatic histones are incorporated back into paternal chromatin or during subsequent zygotic development (39Ooi S.L. Henikoff S. Germline histone dynamics and epigenetics.Curr. Opin. Cell Biol. 2007; 19: 1-9Crossref Scopus (0) Google Scholar). Thus achieving a deep understanding of the molecular basis of sperm chromatin composition and dynamics will impact multiple levels of fertility biology. Any single component of a system must be understood within the context of the whole. To date, most proteomic studies on human sperm have concentrated on determining the protein content of the whole sperm cell. Estimates of sperm proteome size have been made based on two-dimensional poly-acrylamide gel electrophoresis (2D-PAGE), which creates a visual reference map of the sperm proteome by separating proteins according to isoelectric point and mass to ideally resolve protein isoforms (40Naaby-Hansen S. Waterfield M.D. Cramer R. Proteomics–post-genomic cartography to understand gene function.Trends Pharmacol. Sci. 2001; 22: 376-384Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Using this technique, the number of sperm proteins has been estimated at 1000–2000 (41Martinez-Heredia J. Estanyol J.M. Ballesca J.L. Oliva R. Proteomic identification of human sperm proteins.Proteomics. 2006; 6: 4356-4369Crossref PubMed Scopus (191) Google Scholar, 42Naaby-Hansen S. Flickinger C.J. Herr J.C. Two-dimensional gel electrophoretic analysis of vectorially labeled surface proteins of human spermatozoa.Biol. Reprod. 1997; 56: 771-787Crossref PubMed Scopus (147) Google Scholar). Subsequently, the resolution of 3872 separate protein spots was achieved via multiple 2D-PAGE, each corresponding to a restricted pH range for separation in the first dimension (43Li L.W. Fan L.Q. Zhu W.B. Nien H.C. Sun B.L. Luo K.L. Liao T.T. Tang L. Lu G.X. Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors.Asian J. Androl. 2007; 9: 321-329Crossref PubMed Scopus (44) Google Scholar). Shotgun proteomic approaches have also contributed substantially to sperm proteome characterization (44Johnston D.S. Wooters J. Kopf G.S. Qiu Y. Roberts K.P. Analysis of the human sperm proteome.Ann. N. Y. Acad. Sci. 2005; 1061: 190-202Crossref PubMed Scopus (92) Google Scholar, 45Baker M.A. Reeves G. Hetherington L. Muller J. Baur I. Aitken R.J. Identification of gene products present in Triton X-100 soluble and insoluble fractions of human spermatozoa lysates using LC-MS/MS analysis.Proteomics Clin. Appl. 2007; 1: 524-532Crossref PubMed Scopus (146) Google Scholar). Human sperm proteins were identified after proteolytic digestion and high-resolution liquid chromatography to separate peptides before tandem mass spectral identification (LC-MS2). To decrease sample complexity, sperm proteins were divided into Triton X-100 soluble or insoluble fractions, then further separated via one-dimensional SDS-PAGE before protease treatment and LC-MS2. Repeated rounds of LC-MS2 identified peptides corresponding to 1760 and 1056 proteins, respectively; though the identified protein list is only available for the latter (44Johnston D.S. Wooters J. Kopf G.S. Qiu Y. Roberts K.P. Analysis of the human sperm proteome.Ann. N. Y. Acad. Sci. 2005; 1061: 190-202Crossref PubMed Scopus (92) Google Scholar, 45Baker M.A. Reeves G. Hetherington L. Muller J. Baur I. Aitken R.J. Identification of gene products present in Triton X-100 soluble and insoluble fractions of human spermatozoa lysates using LC-MS/MS analysis.Proteomics Clin. Appl. 2007; 1: 524-532Crossref PubMed Scopus (146) Google Scholar). Such proteomic technologies are powerful and have yielded remarkably large lists of proteins. However, each still has its challenges, which include the detection of low abundance proteins (46Ahn N.G. Shabb J.B. Old W.M. Resing K.A. Achieving in-depth proteomics profiling by mass spectrometry.ACS Chem. Biol. 2007; 2: 39-52Crossref PubMed Scopus (38) Google Scholar). For 2D-PAGE, the excision and mass spectrometric characterization of spots to assign protein identity can be laborious. For example, Li et al. (43Li L.W. Fan L.Q. Zhu W.B. Nien H.C. Sun B.L. Luo K.L. Liao T.T. Tang L. Lu G.X. Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors.Asian J. Androl. 2007; 9: 321-329Crossref PubMed Scopus (44) Google Scholar) verified the protein identities of 16 of 3872 spots, whereas Martinez-Heredia et al. (41Martinez-Heredia J. Estanyol J.M. Ballesca J.L. Oliva R. Proteomic identification of human sperm proteins.Proteomics. 2006; 6: 4356-4369Crossref PubMed Scopus (191) Google Scholar) identified 131 of 1000 spots, providing just a glimpse of the proteins and their functions (47de Mateo S. Martinez-Heredia J. Estanyol J.M. Domiguez-Fandos D. Vidal-Taboada J.M. Ballesca J.L. Oliva R. Marked correlations in protein expression identified by proteomic analysis of human spermatozoa.Proteomics. 2007; 7: 4264-4277Crossref PubMed Scopus (107) Google Scholar). While shotgun proteomic techniques can give a more thorough picture, the proteolytic digestion of whole proteins into individual peptides en masse may obfuscate detection of multiple isoforms or combinations of post-translational modifications of any one protein. The difference in the number of protein species detected via 2D-PAGE (3872) and the number of protein identities resolved by shotgun proteomics (1056) may attest to differences in technology. For example, the smaller number of proteins detected by shotgun proteomics may reflect that there are fewer proteins and protein isoforms in sperm because mature sperm have eliminated almost all cytoplasm. The larger number of protein spots detected by 2D-PAGE may suggest that there are more post-translationally modified versions of the proteins present. The development of new or combined proteomic approaches leading to more comprehensive identification of the sperm proteome places a full appreciation of the complexity of post-translational regulation within our grasp. The elucidation of the sperm proteome provides a rich source of raw material for bioinformatic investigation. The use of Gene Ontology (GO) classifications to generate putative functional or subcellular localization groups provides a general snapshot of the proteome (48Lan N. Montelione G.T. Gerstein M. Ontologies for proteomics: towards a systematic definition of structure and function that scales to the genome level.Curr. Opin. Chem. Biol. 2003; 7: 44-54Crossref PubMed Scopus (0) Google Scholar). In addition to GO analysis, more sophisticated classification methods can be employed to reveal meaningful patterns. For example, analysis of the sperm proteome of Drosophila melanogaster has generated new hypotheses about germline gene regulation and chromatin organization (49Dorus S. Busby S.A. Gerike U. Shabanowitz J. Hunt D.F. Karr T.L. Genomic and functional evolution of the Drosophila melanogaster sperm proteome.Nat. Genet. 2006; 38: 1440-1445Crossref PubMed Scopus (192) Google Scholar). First, shotgun mass spectrometric analysis leading to the identification of 381 proteins in Drosophila sperm was performed. By analyzing the chromosomal distribution of corresponding genes, Dorus et al. (49Dorus S. Busby S.A. Gerike U. Shabanowitz J. Hunt D.F. Karr T.L. Genomic and functional evolution of the Drosophila melanogaster sperm proteome.Nat. Genet. 2006; 38: 1440-1445Crossref PubMed Scopus (192) Google Scholar) determined that sperm proteins are underrepresented on the X chromosome. This result is consistent with the finding from DNA microarray analysis that male-expressed genes are also underrepresented on X (50Parisi M. Nuttall R. Edwards P. Minor J. Naiman D. Lu J. Doctolero M. Vainer M. Chan C. Malley J. Eastman S. Oliver B. A survey of ovary-, testis-, and soma-biased gene expression in Drosophila melanogaster adults.Genome Biol. 2004; 5: R40Crossref PubMed Google Scholar). Sub-chromosomal clustering analysis also shows that individual chromosomes have regions with
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