Sperm chromatin proteomics identifies evolutionarily conserved fertility factors

Abstract

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.

Figure 1. Abundance-correlated subtractive proteomic strategy to identify spermatogenesis-enriched proteins

a, Gametogenesis in C. elegans. Upper panels illustrate the progression of germ-cell nuclei in a wild-type male gonad (left) and in one arm of a wild-type bilaterally symmetric hermaphrodite gonad (right). Lower panels show corresponding DAPI-stained nuclei from dissected and fixed gonads. Visually distinct stages of gametogenesis are labelled. b, Proteomic strategy. Spermatogenic chromatin was isolated from germ nuclei of adult XO males derived from the him-8(e1489) X chromosome non-disjunction mutant. Oogenic chromatin was isolated from germ cells of fer-1(hc1) hermaphrodites. fer-1(hc1) mutants produce defective sperm, causing XX animals to be functional females in which some of the oocytes mature, are ovulated unfertilized, and undergo endoreduplication. Spermatogenic and oogenic chromatin was subjected to MudPIT for identification of associated proteins. Subtractive analysis of chromatin proteins identified 132 abundant spermatogenesis-enriched proteins.

Figure 2. Identification of spermatogenic chromatin-associated proteins by proteomic analysis

Immunolocalization of spermatogenic proteins show chromatin association in mature sperm and during meiotic divisions. Sperm meiotic stage: P–D, pachytene to diakinesis transition; M-1, metaphase 1; A-1, anaphase 1; M-2, metaphase 2; MS, mature sperm. Oogenesis: Oo Dia, oocyte diakinesis. DNA is shown in red, antibody staining in green. a–e, Spermatogenesis-specific chromatin-associated proteins (proteins were not detected during any stage of oogenesis): a, SPE-11; b, GSP-3 and GSP-4; c, SMZ-1 and SMZ-2; d, SPCH-1, SPCH-2 and SPCH-3; e, HTAS-1. f, g, Spermatogenic localization of nucleolar proteins: f, TOP-1; g, FIB-1 (C. elegans fibrillarin). TOP-1 surrounds mature sperm chromatin (white arrowheads) but FIB-1 does not (white arrows). Nucleolar localization (white arrows) is ubiquitous in spermatogenic and oogenic germ lines (data not shown, and ref. 9). h, i, Spermatogenic localization of P-granule components: h, GLH-1; i, GLH-2. GLH-2 associates with sperm meiotic chromatin (white arrowheads) but GLH-1 does not. Both localize to perinuclear P-granules (yellow arrows) during spermatogenesis and oogenesis (data not shown, and ref. 10).