REVIEW Proteomics and the genetics of sperm chromatin condensation

Rafael Oliva 1,2 and Judit Castillo 1,2

Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell. Proteomics allows the identification of the proteins that compose the spermatogenic cells and the study of their function. The recent developments in mass spectrometry (MS) have markedly increased the throughput to identify and to study the sperm proteins. Catalogs of thousands of testis and spermatozoan proteins in human and different model species are becoming available, setting up the basis for subsequent research, diagnostic applications and possibly the future development of specific treatments. The present review intends to summarize the key genetic and chromatin changes at the different stages of spermatogenesis and in the mature sperm cell and to comment on the presently available proteomic studies.

Asian Journal of Andrology (2011) 13, 24–30; doi:10.1038/aja.2010.65; published online 1 November 2010

Keywords: epigenetic; imprinting; protamine; proteome; spermatozoa

INTRODUCTION

Spermatogenesis involves extremely marked cellular, genetic and chromatin changes resulting in the generation of the highly specialized sperm cell (Figure 1). Spermatogonial stem cells replicate and differentiate into primary spermatocytes that undergo genetic recombination to give rise to haploid round spermatids. 1–4 Round spermatids then undergo a differentiation process called spermiogenesis where marked cellular, epigenetic and chromatin remodeling takes place. 2,5–12 The nucleosomes are disassembled and the histones are removed and replaced by the high positively charged protamines forming tight toroidal complexes, organizing 85–95% of the human sperm DNA (Figure 1). At the cellular level, most of the cytoplasm is removed, and a large flagella and the acrosomal vesicle are assembled (Figure 1). Finally, the spermatozoon undergoes a maturation process through its transit in the epididymis where the chromatin is further compacted through the formation of disulfide bonds and zinc bridges among protamines, and the acquirement of different membrane and cellular functionalities. 13–15 Once in the female tract, the spermatozoon must be capacitated, a process involving many signaling changes and the attainment of hyperactivated motility. 16–19 Before the sperm cell penetrates the oocyte, the sperm–oocyte recognition and the acrosomal reaction must take place. 20 Finally, once in the oocyte, the male pronucleus must undergo another extremely marked chromatin remodeling process where the nucleoprotamine structure is disassembled and a new nucleosomal and chromatin structure is assembled (Figure 1). The accessibility of the spermatozoon has facilitated the study of its composition and mechanisms involved in its function and makes this cell particularly well suited for proteomic analysis. 21 In addition, dissecting the differentiation process of spermatogenesis through proteomic analysis provides important potential biomedical applications in regenerative medicine, 22,23 in the identification of the genetic basis of male infertility, 24–28 in understanding the origin of genetic and epigenetic mutation, 5,9,10,26,29–32 in reproductive toxicology 33 and in the development of potential contraceptive strategies. 34,35 Different studies have investigated the genetic and protein changes and the mechanisms involved in the different stages of spermatogenesis and function of spermatozoa. The present review intends to complement different recent reviews focusing on the proteomics of the mature sperm cell, 21,36–43 on testicular proteomics 44,45 or on the proteomic changes upon epididymal maturation and capacitation. 46 To reach this goal, the structure followed will be to describe the key genetic and chromatin changes at the different stages of spermatogenesis (Figure 1), with indication of the related proteomic studies being performed based on large-scale mass spectrometry (MS) identification of proteins.

TESTICULAR PROTEOMICS: SPERMATOGONIAL STEM CELLS, SPERMATOCYTES AND SPERMATIDS

One of the initial approaches applied to identify proteins present in the different stages of spermatogenesis exploited the changes in cellular abundance during testis development. Using this approach, the two-dimensional (2D) proteome profile changes during mouse testis development led to the identification using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) of 44 proteins with substantial changes in protein abundance during development. 47 Subsequent application of MALDI-TOF/TOF using a similar approach allowed identification of 257 different proteins that clustered into six different expression patterns. 48 A limitation of the analysis of the entire testis is the existence of mixed cellular population including the presence of somatic cells. Therefore, other approaches have been used such as the isolation of the different cellular components of the testis or the study of cultured germ cells.

One of the testicular cells studied using proteomics is the spermatogonial stem cell. 49–51 The interest in the study of the spermatogonial stem cells is multiple. Pathological perturbation of the stem cell is suspected as the origin of certain types of testicular cancers and male infertility, so that identification of the mechanisms involved would facilitate the development of preventive or treatment options. 52,53 In addition, the discovery of pluripotent stem cells within the testis raises important biomedical applications in regenerative medicine. 23 Important issues in stem cell research are the identification of key genes and proteins needed to maintain the pluripotent state or that can be used as markers for their identification. 49 A very elegant application of a comparative proteomics approach has been applied to demonstrate that after comparison of the proteomic profiles of cultured mouse multipotent adult germ line stem cells with embryonic stem cells, only 18 proteins were detected as differentially expressed out of a total of 409 proteins identified using 2D separation of the proteins followed by MS. 22 The interpretation of this result was that the proteomes of multipotent adult germ line stem cells were highly similar to those of embryonic stem cells. 22 A different approach to characterize the proteome of the spermatogonial stem cell exploited the peculiarities of the testis developmental biology in the dogfish Scyliorhinus canicula . 54 These authors isolated, under stereomicroscope and dissection, the testicular germinative zone, highly enriched in spermatogonial stem cells, and used 2D and MALDI-TOF/TOF to identify 16 proteins and to also demonstrate the feasibility of this model to study the stem cell niche. 54 Still, a different set of studies has used testicular cell sorting to obtain enriched cellular fractions with which proteomic analysis is performed. This approach was applied to separate spermatogonia from 9-day-old rats followed by protein 2D analysis and identification of the proteins using MALDI-TOF. 55 More recently, the same group applied a similar procedure on immature and mature rat testis combined with sedimentation at unit gravity or elutriation to obtain highly enriched fractions of spermatogonia, spermatocytes and early spermatids. 56 Subsequently, 2D difference in gel electrophoresis allowed identification of the relative abundance of 1274 proteins of which 265 differed significantly in the three groups of cell types. MALDI-TOF/TOF was then used to identify 123 non-redundant proteins clustering into the clades of mitotic, meiotic and post-meiotic cell types. 56 It is also important to consider the close relationship between the Sertoli cell and the spermatogenic cells. Recently, the effect of the loss of Dicer in the Sertoli cell, required for microRNA biogenesis, on the testicular proteome, has been studied. 57

Once the diploid spermatogonium is committed, it divides mitotically to produce two diploid intermediate cells called primary spermatocytes. Each primary spermatocyte then duplicates its DNA and subsequently undergoes meiosis I to produce two haploid secondary spermatocytes (Figure 1). Very importantly, this stage involves genetic recombination of homologous chromosomes to increase the genetic variability of the gamete. Many of the genetic causes of male infertility stem from meiotic anomalies. For instance, an important proportion of cases of male infertility are due to meiotic arrest. 25,26,28 Also, many chromosomal structural anomalies result in incorrect pairing at meiosis and in the generation of chromosomally unbalanced gametes responsible for embryo lethality or severe anomalies in the offspring. 58 Well-known causes and risk factors of male infertility such as the presence of Y-chromosome microdeletions also result in a variety of phenotypes which may include Sertoli cell-only syndrome, spermatogenic arrest and hypospermatogenesis resulting in azoospermia or oligospermia. 24,27,59 Thus, proteomics, through the indicated above strategies, allows the identification of the proteins involved in the meiotic stage of spermatogenesis with the potential to contribute to the identification of the involved pathogenic mechanisms associated to male infertility in these cases. 47,48,56

After the completion of the meiosis, the haploid round spermatids are generated (Figure 1). Haploid round spermatids are still transcriptionally active. 60 However, another aspect of the biology of the spermatogenesis that deserves consideration is that each cell division from a spermatogonium to a spermatid is incomplete. The cells remain connected to one another by bridges of cytoplasm to allow synchronous development. It has been proposed that these cellular bridges allow the exchange of proteins and gene products so that, even though the round spermatids are genetically haploid, they may express proteins as if they were diploid. 60 The haploid spermatids have been the focus of different proteomic studies. Fluorescence-activated cell sorting sorting has been applied to isolate haploid mouse spermatids followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 2116 proteins, 299 of which were testis specific and 155 were novel. 61 Interestingly, the analysis of the chromosomal distribution of the haploid identified genes showed an underrepresentation of the X chromosome, interpreted as owing to meiotic X-chromosome inactivation, and overrepresentation of chromosome 11 upon expansion of the gene families. 61 In a different approach, the proteomic analysis in testis biopsies of testosterone-treated men allowed the identification of proteins potentially related to the induced testis regression. 62 As an experimental model, the effect of hyperthermia on mouse spermatogenesis has also been studied. 63 The same group also investigated the whole human testis, identifying 462 unique proteins. 64,65 Evidence for protein heterogeneity was concluded upon the identification of 180 different proteins in more than one protein spot. Also, phosphoprotein staining allowed identification of 52 phosphorylated proteins. 65 Proteins related to altered fertility in fish have also been identified using a whole-testis proteomic approach. 66

The proteomic studies during spermiogenesis are highly relevant in the identification of all the proteins involved and the mechanisms of the nucleohistone-to-nucleoprotamine transition, which probably represents the most marked chromatin change that cells may undergo (Figure 1). One of the initial chromatin changes in the nucleohistone-to-nucleoprotamine transition is the incorporation of histone variants. 67–72 Another important early event is histone hyperacetylation that occurs during spermiogenesis just before the nucleosome disassembly. 73–78 It was postulated that histone hyperacetylation and rapid turnover of acetyl groups could rapidly and reversibly expose binding sites in chromatin for subsequent binding of chromosomal proteins. 74 More recently, it was also shown in vitro that histone hyperacetylation facilitated nucleosome disassembly and histone displacement by protamines. 79,80 Also, hyperacetylated nucleosomes were shown to appear in a more relaxed structure upon binding to electron microscopy grids. 80,81 It has been shown that the testis-specific bromo-domain-containing protein binds to hyperacetylated histone-4, triggering a reorganization of the chromatin. 3,10,82,83 Impaired histone-4 hyperacetylation has been detected in infertile patients. 84,85 Once the nucleosomes are disassembled, transition proteins are incorporated. 2,86 Transition proteins are then finally replaced by protamines to form a highly compact nucleoprotamine complex (Figure 1). 2,3,6,7,10,86–90 It is known that protamines are phosphorylated before binding to DNA and that a substantial dephosphorylation takes place concomitant to nucleoprotamine maturation. 2,91–93 The dynamics of binding of the protamines to DNA has also been studied. 94–96 After binding to DNA, the formation of interdisulfide bonds between protamines further stabilizes the nucleoprotamine complex. 15,97 Different models for the structure of nucleoprotamine have been proposed. 98–104 These chromatin changes during spermiogenesis take place in the context of a marked metamorphosis of the sperm cell and shaping of the head and associated structures such as the perinuclear theca and manchette. 8,105 However, despite substantial amount of information available, the identification of the molecular mechanisms governing the nucleohistone-to-nucleoprotamine transition and all the sperm cell changes still requires substantial effort. The available ca