Sperm chromatin proteomics identifies evolutionarily conserved fertility factors.

Male infertility is a long-standing enigma of significant medical concern. The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure. Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility. Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility. Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis; proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin; prioritization of sperm proteins based on abundance; and subtraction of common proteins to eliminate general chromatin and meiotic factors. Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis. Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility. Unexpected roles in spermatogenesis were also detected for factors involved in other processes. Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility. Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.

Chromosome cohesion is regulated by a clock gene paralogue TIM-1.

Faithful transmission of the genome requires that a protein complex called cohesin establishes and maintains the regulated linkage between replicated chromosomes before their segregation. Here we report the unforeseen participation of Caenorhabditis elegans TIM-1, a paralogue of the Drosophila clock protein TIMELESS, in the regulation of chromosome cohesion. Our biochemical experiments defined the C. elegans cohesin complex and revealed its physical association with TIM-1. Functional relevance of the interaction was demonstrated by aberrant mitotic chromosome behaviour, embryonic lethality and defective meiotic chromosome cohesion caused by the disruption of either TIM-1 or cohesin. TIM-1 depletion prevented the assembly of non-SMC (structural maintenance of chromosome) cohesin subunits onto meiotic chromosomes; however, unexpectedly, a partial cohesin complex composed of SMC components still loaded. Further disruption of cohesin activity in meiosis by the simultaneous depletion of TIM-1 and an SMC subunit decreased homologous chromosome pairing before synapsis, revealing a new role for cohesin in metazoans. On the basis of comparisons between TIMELESS homologues in worms, flies and mice, we propose that chromosome cohesion, rather than circadian clock regulation, is the ancient and conserved function for TIMELESS-like proteins.

Sperm chromatin: fertile grounds for proteomic discovery of clinical tools.

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 development and motility are regulated by PP1 phosphatases in Caenorhabditis elegans.

Sperm from different species have evolved distinctive motility structures, including tubulin-based flagella in mammals and major sperm protein (MSP)-based pseudopods in nematodes. Despite such divergence, we show that sperm-specific PP1 phosphatases, which are required for male fertility in mouse, function in multiple processes in the development and motility of Caenorhabditis elegans amoeboid sperm. We used live-imaging analysis to show the PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) are required to partition chromosomes during sperm meiosis. Postmeiosis, tracking fluorescently labeled sperm revealed that both male and hermaphrodite sperm lacking GSP-3/4 are immotile. Genetic and in vitro activation assays show lack of GSP-3/4 causes defects in pseudopod development and the rate of pseudopodial treadmilling. Further, GSP-3/4 are required for the localization dynamics of MSP. GSP-3/4 shift localization in concert with MSP from fibrous bodies that sequester MSP at the base of the pseudopod, where directed MSP disassembly facilitates pseudopod contraction. Consistent with a role for GSP-3/4 as a spatial regulator of MSP disassembly, MSP is mislocalized in sperm lacking GSP-3/4. Although a requirement for PP1 phosphatases in nematode and mammalian sperm suggests evolutionary conservation, we show PP1s have independently evolved sperm-specific paralogs in separate lineages. Thus PP1 phosphatases are highly adaptable and employed across a broad range of sexually reproducing species to regulate male fertility.

Elucidating gene regulatory mechanisms for sperm function through the integration of classical and systems approaches in C. elegans.

From worms to mammals, successful spermatogenesis depends on a gene expression profile that balances activating and repressive mechanisms. Besides developmental control of specific spermatogenic genes, male fertility requires temporal shifts in global gene expression and dramatic changes in chromatin structure and condensation. Recent studies are beginning to elucidate the molecular processes that both drive these temporal changes in gene expression and underlie fertility. In this review, we provide an overview of relevant C. elegans studies that have laid the groundwork for modern approaches. Next, we highlight recent studies that investigate how gene expression in C. elegans is modulated during spermatogenesis. These studies use large-scale genomic profiling in combination with bioinformatics, genetics, biochemistry, and in vitro methods to target specific stages or processes during sperm formation. Such studies are beginning to elucidate the multiple layers of gene regulation required during spermatogenesis, i.e., transcriptional, post-transcriptional, and epigenetic. Moreover, knowledge of how C. elegans coordinately regulates gene expression during spermatogenesis promises to provide key insights into parallel processes in mammals that are vital for fertility.

Epigenetic processes implemented during spermatogenesis distinguish the paternal pronucleus in the embryo.

Though spermatozoon and egg contribute an equal share of nuclear DNA content to the newly formed embryo, there are inherent epigenetic differences between the paternal and maternal pronuclei in early cleavage stage embryos. Information about how to decipher sperm DNA in the embryo is established via sperm-specific DNA packaging that occurs during spermatogenesis. In addition to protamines, paternal factors that package sperm DNA distinctly from oocyte or somatic DNA include histones and their modifications, histone variants, chromatin-binding proteins, and non-coding RNAs. These evolutionarily conserved factors play interconnected roles in heterochromatin formation, gene regulation, and maintenance of genome integrity, which influence key processes after fertilization. This review focuses on recent developments from genomic and proteomic studies in model organisms showing that components closely associated with sperm DNA contribute to embryonic survival. These advances may reveal important insights into the treatment of infertility and use of assisted reproductive technologies.