Chu DS et al. (2013) Adv Exp Med Biol "Spermatogenesis."

Chu DS, Shakes DC

[Adv Exp Med Biol, 2013]

During spermatogenesis, pluripotent germ cells differentiate to become efficient delivery vehicles to the oocyte of paternal DNA. Though male and female germ cells both undergo meiosis to produce haploid complements of DNA, at the same time they also each undergo distinct differentiation processes that result in either sperm or oocytes. This review will discuss our current understanding of mechanisms of sperm formation and differentiation in Caenorhabditis elegans gained from studies that employ a combination of molecular, transcriptomic, and cell biological approaches. Many of these processes also occur during spermatogenesis in other organisms but with differences in timing, molecular machinery, and morphology. In C. elegans, sperm differentiation is implemented by varied modes of gene regulation, including the genomic organization of genes important for sperm formation, the generation of sperm-specific small RNAs, and the interplay of specific transcriptional activators. As sperm formation progresses, chromatin is -systematically remodeled to allow first for the implementation of differentiation programs, then for sperm-specific DNA packaging required for transit of paternal genetic and epigenetic information. Sperm also exhibit distinctive features of -meiotic progression, including the formation of a unique karyosome state and the centrosomal-based segregation of chromosomes during symmetric meiotic -divisions. Sperm-specific organelles are also assembled and remodeled as cells complete -meiosis and individualize in preparation for activation, morphogenesis, and the acquisition of motility. Finally, in addition to DNA, sperm contribute specific cellular factors that contribute to successful embryogenesis.

Chu DS (2018) PLoS Biol "Zinc: A small molecule with a big impact on sperm function."

Chu DS

[PLoS Biol, 2018]

Zinc is an essential mineral, but our understanding of its uses in the body is limited. Capitalizing on approaches available in the model system Caenorhabditis elegans, Zhao and colleagues show that zinc transduces a signal that induces sperm to become motile. This is an enigmatic process because sperm in all sexually-reproducing animals are transcriptionally inactive. Zinc levels inside sperm are regulated by an evolutionarily conserved zinc transporter called Zrt- and Irt-like Protein Transporter 7.1 (ZIPT-7.1). This zinc transporter localizes to intracellular organelles, suggesting that it primarily controls zinc levels by releasing zinc into the cytoplasm from internal stores rather than importing it from the external environment. The zinc released within cells acts as a messenger in a signaling pathway to promote mobility acquisition. These studies reveal an important role for zinc as an intracellular second messenger that generates physiological changes vital for sperm motility and fertility.

Chu DS et al. (2006) Nature "Sperm chromatin proteomics identifies evolutionarily conserved fertility factors."

Wu TF, Nix P, Yates Iii JR, Ralston EJ, Meyer BJ, Liu H, Chu DS

[Nature, 2006]

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.

Chu DS et al. (2002) Genes Dev "A molecular link between gene-specific and chromosome-wide transcriptional repression."

Kuo AF, Meyer BJ, Chu DS, Dawes HE, Lieb JD, Chan RC

[Genes Dev, 2002]

Gene-specific and chromosome-wide mechanisms of transcriptional regulation control development in multicellular organisms. SDC-2, the determinant of hermaphrodite fate in Caenorhabditis elegans, is a paradigm for both modes of regulation. SDC-2 represses transcription of X chromosomes to achieve dosage compensation, and it also represses the male sex-determination gene her-1 to elicit hermaphrodite differentiation. We show here that SDC-2 recruits the entire dosage compensation complex to her-1, directing this X-chromosome repression machinery to silence an individual, autosomal gene. Functional dissection of her-1 in vivo revealed DNA recognition elements required for SDC-2 binding, recruitment of the dosage compensation complex, and transcriptional repression. Elements within her-1 differed in location, sequence, and strength of repression, implying that the dosage compensation complex may regulate transcription along the X chromosome using diverse recognition elements that play distinct roles in repression.

DS Chu et al. (1999) International C. elegans Meeting "Gene and chromosome specific localization of SDC-1 directs sexual fate and implements dosage compensation."

BJ Meyer, DM Lapidus, DS Chu

[International C. elegans Meeting, 1999]

The SDC-1 protein coordinately regulates the sex determination and dosage compensation pathways in C. elegans . Genetic evidence indicates that SDC-1 works in concert with SDC-2 and SDC-3 to direct sexual fate and reduce X expression in hermaphrodites. SDC-1 is a 139 kD protein that contains seven putative zinc finger DNA binding domains. Mutations in sdc-1 cause partial masculinization of most XX animals but have no effect on males (XO). This defect is caused by aberrant regulation of the male-specific her-1 gene (Villeneuve and Meyer 1987, Cell , 48:25-37) and results in elevated her-1 transcript levels in sdc-1 mutant hermaphrodites (Trent et al. 1991, Mech. Dev. , 34:43-56). We asked if SDC-1 binds directly to the her-1 promoter to repress its transcription. To do so, we created extrachromosomal arrays carrying multiple copies of (1) the her-1 promoter region, (2) transgenes encoding a GFP-LacI fusion protein and (3) lacO , the binding site for the LacI repressor protein. Arrays were visualized by GFP fluorescence through confocal microscopy. Using anti-SDC-1 antibodies, we found that SDC-1 colocalizes to the her-1 promoter. SDC-2 and SDC-3 also localize to the her-1 promoter (see abstract by Dawes, H. et al.) indicating that SDC-1 works in combination with SDC-2 and SDC-3 to repress her-1 transcription directly. sdc-1 mutants also disrupt dosage compensation, causing elevated X-linked gene transcripts and dumpy and egg-laying defective phenotypes. Interestingly, though mutations in all other dosage compensation genes affect the abundance or localization of the dosage compensation complex, sdc-1 mutations fail to perturb the complex. By immunostaining, we found that SDC-1 localizes to the X chromosome and colocalizes with other dosage compensation complex members such as SDC-3 and DPY-28. Therefore, SDC-1 may directly modulate the activity of the dosage compensation complex once assembled on X rather than contributing to its assembly or localization.

Shorrock, M. et al. (2011) International Worm Meeting "Characterizing the paternal RNA contribution of C. elegans sperm."

Chu, D.S., Wu, T., Shorrock, M.

[International Worm Meeting, 2011]

Distinct transcriptional programs during gamete formation specify the differentiation of sperm and oocytes. In particular, while RNA is transcribed during meiosis and most of spermatogenesis, mature sperm become largely transcriptionally silent in later spermatogenesis as DNA is tightly compacted and cytoplasmic components are removed. However, a portion of RNA is retained in mature sperm. These paternal transcripts are delivered alongside DNA to the developing embryo, and have recently been implicated in successful fertilization and embryo development. Despite their potential contribution to early development, the RNA content of mature sperm is poorly characterized and their functional significance remains largely unexplored. Using C. elegans, we are characterizing the RNA content of sperm. To do this, we first compared the profiles of RNA isolated from sperm, embryos, and whole worms. We purified RNA from mature sperm, embryo, and whole worm. As expected, we found embryo and whole worm expression profiles, determined by quantitative electrophoretic mobility, were dominated by two peaks characteristic of ribosomal RNAs. Conversely, mature sperm lacked discrete ribosomal peaks and had a comparatively diverse transcript population. These results verify that mature sperm retain RNA but are able to remove abundant ribosomal RNAs. This supports that sperm possess a set of RNAs distinct from somatic cells. We are now conducting microarray and next-generation sequencing analysis of RNA isolated from mature sperm to identify the specific paternal transcripts that are delivered to the embryo.

Huang, W. et al. (2019) International Worm Meeting "Features of histone H2A variant HIS-35 incorporation before and after fertilization."

Shorrock, M., Chu, D.S., Corpuz, C., Huang, W.

[International Worm Meeting, 2019]

To successfully pass down genetic material to the next generation, proper germ cell and embryo development is essential. These developmental processes are facilitated by proper gene regulation that is under the control of core histone proteins and their respective histone variants. By exchanging core histones with their variants, chromatin structure is remodeled to alter DNA accessibility to target genes. Previous research has identified 3 histone H2A variants namely, HTZ-1, HTAS-1, and HIS-35 in C. elegans.HTZ-1 is an evolutionary conserved H2A variant that is vital for development. Studies revealed parent derived HTZ-1 is removed from both maternal and paternal genome shortly after fertilization. This is in contrast to the sperm-specific H2A variant HTAS-1, which is retained in the paternal genome after fertilization. These results suggest how maternal and paternal-contributed H2A variants can be processed differently after fertilization. However, the role and processing of HIS-35 is unknown. Preliminary his-35 mutant studies revealed a 35% reduction in progeny when compared to the wildtype. In addition, following a GFP tagged his-35 strain has shown HIS-35 incorporation in spermatogenic, oogenic germline and fertilized embryos. These results suggested that HIS-35 can be involved in both fertility and embryogenesis. I hypothesize that HIS-35 regulate fertility associated genes during germ cell development. To assess HIS-35's role in fertility, we utilized a strain expressing HIS-35::GFP to examine HIS-35 incorporation throughout germline progression. Our results showed that HIS-35 colocalizes with H3K4me2, a mark of active transcription but not with H3K9me2, a repressive mark. This indicated that HIS-35 may play a role in regulating target genes needed for proper germ cell development. To assess HIS-35's role during embryogenesis, we tracked maternally derived (matHIS-35) and paternally derived HIS-35 (patHIS-35) separately during embryo development. Our results showed differences in HIS-35 incorporation between matHIS-35 and patHIS-35. Following 1 to 2 cell stage embryos, patHIS-35 was removed from the paternal genome shortly after fertilization. At the same time, matHIS-35 incorporates into the paternal genome and was retained in the maternal genome. Following 4 cell to late stage embryos, matHIS-35 incorporates into chromatin throughout embryogenesis. However, patHIS-35 was only detected starting at late stage embryo. These results suggested parent specific regulatory roles of HIS-35 post fertilization. This study provides further evidence that histone variants are removed and incorporated distinctly in the new embryo to maximize fertility.

Hinojosa Paiz, J. et al. (2019) International Worm Meeting "Illuminating gene regulatory roles of the sperm specific histone variant, HTAS-1."

Tabuchi, T., Chu, D.S., Rechtsteiner, A., Singh, S., Hinojosa Paiz, J.

[International Worm Meeting, 2019]

A main contributor to infertility is improper spermatogenesis. For proper progression of sperm development, correct gene regulatory patterns, which are governed by specific proteins, are vital. Histone variants are among these specialized proteins. Resembling fraternal twins, histone variants differ slightly in amino acid sequence from their core histone counterpart yet have alternative tissue specific effects on gene regulation. Our model organism, Caenorhabditis elegans, expresses four different histone H2A variants, one being the sperm specific HTAS-1 variant. Previous studies conducted on C. elegans have demonstrated that HTAS-1 is incorporated into chromatin late during male spermatogenesis and mutant htas-1 strains generate less progeny. Preliminary immunostaining data suggests HTAS-1 possesses a gene regulatory role as it colocalizes on autosomes and is absent on the silenced X chromosome. Yet, it is still unknown where HTAS-1 specifically localizes on genomic DNA and whether HTAS-1 directly regulates genes it binds in chromatin. To investigate where on DNA HTAS-1 is present, we conducted ChIP-seq on wild type mature sperm. We identified genomic sites of HTAS-1 chromatin integration, 26.5% of which are sperm specific genes. From this subset of sperm genes, we then asked whether these genes fall within genes that are potentially regulated by HTAS-1. To explore which genes are regulated by HTAS-1, I will perform RNA-seq on isolated mRNAs from mutant htas-1 and wild type male germlines. From RNA-seq data, I expect to find genes that are both up and down regulated when comparing mutant ?htas-1 ??and wild type transcript levels. Subsequently, to investigate if HTAS-1 regulates sperm specific genes it directly binds, I will perform smFISH on mutant htas-1 and wild type worms. From smFISH data, I expect to find the abundance of gene transcripts to increase or decrease with time proposing HTAS-1's role in the establishment of correct gene expression patterns, which also supports why it might not be found on the X chromosome. Findings from this study will help reveal potential gene targets for further investigation that are essential for proper male spermatogenesis. Knowledge on what genes HTAS-1 regulates and how it regulates these genes can give insight to how a histone variant is necessary for male fertility.

Morrison, Kayleigh N. et al. (2021) MicroPubl Biol

Morrison, Kayleigh N., Ragle, James Matthew, Shakes, Diane C., Uyehara, Christopher M., Ward, Jordan D.

[MicroPubl Biol, 2021]

During the process of cell differentiation, specific cytoskeletal proteins can sequentially assemble into a wide variety of diverse molecular superstructures. Nematode spermatogenesis provides a powerful system for studying these transitions since sperm-specific transcription ceases prior to the meiotic divisions and translation ceases shortly thereafter (Chu and Shakes, 2013). Therefore, structural transitions that follow the meiotic divisions must be carried out by the remodeling of already synthesized proteins. The Major Sperm Protein (MSP) is a nematode-specific cytoskeletal element whose polymerization dynamics drive the pseudopod-based motility of the activated sperm (Roberts, 2005). In C. elegans, MSP additionally functions as the extracellular signaling molecule for triggering both ovulation and oocyte maturation (Miller et al., 2003). MSP is highly abundant in sperm, where it reaches 10-15% of total and 40% of soluble cellular protein (Roberts 2005). Within developing spermatocytes, MSP is packaged into fibrous bodymembranous organelle (FB-MO) complexes (Fig. 1A, Roberts et al., 1986). By assembling into paracrystalline FBs, MSP is both sequestered away from the critical meiotic processes of chromosome segregation and cytokinesis while also being packaged for efficient segregation into spermatids during the post-meiotic partitioning process (Chu and Shakes 2013, Nishimura and LHernault, 2010, Price et al., 2021). Following the meiotic divisions and sperm individualization, FBs disassemble, and MSP disperses as dimers throughout the spermatid cytoplasm (Fig. 1A). When sperm activate to form motile spermatozoa, MSP polymerization within the pseudopod drives the motility of the crawling sperm (Chu and Shakes, 2013). Thus, MSP exists in at least three distinct molecular states: 1) in highly organized paracrystalline FBs within developing spermatocytes 2) as unpolymerized dimers within spermatids, and 3) in dynamically polymerizing filaments and fibers within crawling spermatozoa.

Shakes, D.C. et al. (2009) International Worm Meeting "Features of sperm cell differentiation that alter the meiotic program C. elegans."

Shakes, D.C., Noritake, A., Chu, D.S., LaPrade, K., Wu, J, Sadler, P.L., Moore, L.L.

[International Worm Meeting, 2009]

The fundamental process of meiosis underlies two differentiation programs that occur at different rates and generate vastly different cell types, sperm and oocytes. There is a limited understanding in any organism, including C. elegans, regarding how sperm or oocyte specification either coordinates with or modifies meiosis to give rise to these disparate cell types. We have conducted an in-depth analysis of sperm cell formation to understand how gamete-specific features influence meiotic events and progression. Our work has produced a detailed timeline of late meiotic prophase during spermatogenesis in C. elegans. This study is unique in that it defines a broad set of cytological and molecular landmarks that inter-relates changes in chromosome morphology and dynamics with germ cell cellularization, subcellular organelle disassembly, spindle formation, and meiotic cell cycle transitions to accurately stage nuclear progression. This analysis has uncovered differences in sperm meiotic chromatin composition and morphology compared to oocyte meiosis. Nuclei progressing past pachynema undergo distinct morphological changes after the incorporation of sperm nuclear basic proteins into sperm chromatin. Most strikingly, C. elegans spermatogenesis includes a previously undescribed extended stage when chromosomes form a constricted mass within an intact nuclear envelope. This karyosome stage, which is a common feature of meiosis in many organisms, follows desynapsis and is largely transcriptionally inactive. However it is highly dynamic, as multiple cell signaling pathways are sequentially activated during this stage and in an order that is distinct from that of diakinetic oocytes. Also in contrast to developing oocytes, spermatocytes exhibit centrosome-directed microtubule dynamics that are distinct in both their timing and morphology. Correspondingly, we observe that kinetochore structures that mediate chromosome segregation also exhibit sperm-specific features. Overall, several of these gamete-specific features effectively increase the efficiency and pace of meiotic progression during sperm formation. These studies identify specific features of the meiotic program that differ in sperm and oocyte meiosis, revealing that the underlying molecular machinery required for meiosis is differentially regulated in each sex.