SPE-44 Implements Sperm Cell Fate

The sperm/oocyte decision in the hermaphrodite germline of Caenorhabditis elegans provides a powerful model for the characterization of stem cell fate specification and differentiation. The germline sex determination program that governs gamete fate has been well studied, but direct mediators of cell-type-specific transcription are largely unknown. We report the identification of spe-44 as a critical regulator of sperm gene expression. Deletion of spe-44 causes sperm-specific defects in cytokinesis, cell cycle progression, and organelle assembly resulting in sterility. Expression of spe-44 correlates precisely with spermatogenesis and is regulated by the germline sex determination pathway. spe-44 is required for the appropriate expression of several hundred sperm-enriched genes. The SPE-44 protein is restricted to the sperm-producing germline, where it localizes to the autosomes (which contain sperm genes) but is excluded from the transcriptionally silent X chromosome (which does not). The orthologous gene in other Caenorhabditis species is similarly expressed in a sex-biased manner, and the protein likewise exhibits autosome-specific localization in developing sperm, strongly suggestive of an evolutionarily conserved role in sperm gene expression. Our analysis represents the first identification of a transcriptional regulator whose primary function is the control of gamete-type-specific transcription in this system

The deletion of a sperm-specific transcription factor causes cell cycle and sperm differentiation defects in Caenorhabditis elegans

Germ cells are highly dynamic in that they undergo several dramatic transitions throughout their lives. They begin as highly proliferative stem cells. Eventually, they become mitotically proliferating germ cell precursors. In Caenorhabditis elegans, these precursor cells do not even have a specified cell fate. Only after the cells move past the mitotic zone do the precursor cells enter meiosis and initiate the sexual specification process. As the gametes move through meiosis, sex-specific proteins are transcribed and translated. These proteins contribute to the differentiation pathway that creates a functional, haploid gamete. The regulation of these sperm-specific genes and proteins is not well understood. To further understand the process of how gametes progress through the early stages of meiosis, we examined a deletion mutation in the gene for a sperm-specific transcription factor, SPE-44. This deletion causes catastrophic failure of both the meiotic and differentiation pathways of spermatogenesis. spe-44 mutants fail to create functional sperm, as their cells arrest in M phase. Instead of sperm, spe-44 gonads contain terminal cells of varying size and composition. Smaller, older cells contain supernumerary microtubule asters and a variable amount of DNA. Phospho-histone H3, an M-phase marker, persists in the terminal, small cells of the mutant. As for sperm differentiation, spe-44 mutants contain the Major Sperm Protein (MSP) but fail to assemble it into paracrystals after synthesis. The membranous organelles (MOs), Golgi-derived and nematode sperm-specific organelles which associate with the assembled MSP paracrystals, mislocalize within the small cells of the mutant and fail to dock on the cell cortex. During wildtype spermatogenesis, the SPE-44 protein itself localizes to the chromatin of the developing spermatocytes during pachytene and disappears in the karyosome stage. Microarray data from Harold Smith of the NIH show that spe-44 mutants do not express many of the sperm-enriched genes that are necessary for sperm production in C. elegans. SPE-7, a nematode sperm-specific protein, is among the gene products not present in the spe-44 mutant. SPE-44 appears to function as the link between sperm specification and sperm production; it is the first sperm-specific transcriptional regulator in the C. elegans germ line to be described

Expression and localization of <i>spe-44</i> mRNA.

<p>A. Quantification of <i>spe-44</i> expression by qRT-PCR. Transcript levels in each sample were normalized against actin as an internal control; the scale was arbitrarily set at one for the <i>fem-1</i> L3 value. Mean values and standard deviations are from triplicate samples of the indicated stage and genotype. ND, not determined. Genotypes from left to right are wild-type hermaphrodites, wild-type males, <i>fem-3(gf)</i> hermaphrodites, <i>fem-1(lf)</i> hermaphrodites, and <i>fog-1(lf)</i> hermaphrodites. B. <i>In situ</i> hybridization for <i>spe-44</i> transcript (arrow) in dissected <i>fem-3(gf)</i> L3 gonad. C. Same gonad stained with DAPI. D. <i>In situ</i> hybridization for <i>spe-44</i> in dissected <i>fem-1(lf)</i> L4 gonad.</p

<i>spe-44</i> conservation in <i>Caenorhabditis</i> species.

<p>A. Tree indicating the relationship of the SPE-44 orthologs from the indicated species. CLUSTALW2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002678#pgen.1002678-Larkin1" target="_blank">[81]</a> was used to perform multiple sequence alignment of complete amino acid sequences for the following proteins: Wormbase accession numbers CN01523 (<i>C. brenneri</i>; abbreviated Cbrn), RP47564 (<i>C. remanei</i>; Crem), CBP08202 (<i>C. briggsae</i>; Cbrg), CE05305 (<i>C. elegans</i>; Celg), and JA05534 (<i>C. japonica</i>; Cjap). Also included were the paralogous GMEB-3 from <i>C. elegans</i> (accession number CE31443; Celg GMEB-3), and human GMEB1 as an outgroup (NCBI accession NP_077808; Hs GMEB1). Branch length reflects the degree of similarity. B. Sex-biased expression. Relative read number from RNA-Seq data; female/hermaphrodite values were normalized to one for each species. <i>Celg, C. elegans; Crem, C. remanei; Cbrn, C. brenneri, Cjap, C. japonica</i>. C. Conservation of <i>spe-44</i> upstream sequences. Blue indicates conserved sequence elements; asterisks indicate identical nucleotides. D. SPE-44 protein localization in <i>C. remanei</i>. Dissected male gonad co-stained with anti-SPE-44 antibody (green) and DAPI (red). Higher magnification (inset) reveals exclusion from a single DNA focus, presumably X (arrow).</p

Summary of microarray results and transcriptional activation by SPE-44.

<p>A–B. Summary of microarray results. Venn diagrams of the genes that were down-regulated (A) or up-regulated (B) in <i>spe-44</i> mutant males compared to the categorization (sperm-enriched, oocyte-enriched, or germline intrinsic) defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002678#pgen.1002678-Reinke2" target="_blank">[23]</a>. C. Transcriptional activation by SPE-44. Expression of <i>lacZ</i> reporters containing the indicating promoter plus <i>spe-44</i> under repression (−) or induction (+). ß-galactosidase activity quantified by ONPG assay for triplicate samples under each condition.</p

Spermatogenesis defects in <i>spe-44</i> males.

<p>A–B. Isolated gonads from wild-type (A) and <i>spe-44</i> (B) males visualized by DIC. Indicated are pachytene (p), karyosome (k), and meiotically dividing (d) spermatocytes, and spermatids (sp). C–E. Sperm spreads from wild-type (C) and <i>spe-44</i> (D–E) males. C. Wild-type spermatogenesis with spermatocytes in anaphase I (ana I), anaphase II (ana II), post-meiotic budding division (bd), and mature spermatids (sp). D. <i>spe-44</i> spermatogenesis with aberrantly dividing spermatocytes. E. Enlarged image of <i>spe-44</i>. F–G. Isolated gonads from wild-type (F) and <i>spe-44</i> (G) males co-stained with anti-tubulin (green) and DAPI (blue). Labeling as in panels A–B. Additionally indicated are residual bodies from the budding division (bd), and a mix of terminally arrested and dying spermatocytes (*). H. Higher magnification of individual sperm from <i>spe-44</i> males. Top row, DAPI staining to highlight nuclear morphologies; bottom row, merge of DAPI (blue) and tubulin (green). See text for labeling.</p

Pattern of SPE-44 protein expression and localization.

<p>A. Wild-type adult male gonad co-stained with anti-SPE-44 (green) and DAPI (red). Insert at higher magnification shows details of nuclear morphologies during the appearance/disappearance of SPE-44 labeling. Arrow indicates unlabeled DNA. B. Individual nuclei from DAPI-stained wild-type male gonads that were co-stained with anti-SPE-44 only (S44), anti-dimethylated histone H3 (Lys9) antibody only (H3K9), or both antibodies. Top row, DAPI; middle row, antibody (Ab); bottom row, merged image. C. Wild-type male gonad stained with anti-SPE-44 (green), anti-MSP (blue), and DAPI (red). D–F. Wild-type hermaphrodite gonads stained with anti-SPE-44 (green) and DAPI (red). D. L4 larval stage. E. Adult stage. F. Spermatogenesis to oogenesis transition at the L4/adult molting stage. Insert shows details of the first oocyte nuclei interspersed with a few last SPE-44-staining nuclei. The pachytene region (p) is indicated for orientation.</p

Organelle assembly defects in <i>spe-44</i> sperm.

<p>A–B. Wild-type (A) and <i>spe-44</i> (B) spermatocytes co-stained with anti-MSP (green) and DAPI (blue, white). Shown at right are individual sperm at higher magnification in metaphase I (MI), metaphase II (MII), the budding division (bd), mature spermatids (sp), and arrested spermatocytes (arrested). C–D. Wild-type (C) and <i>spe-44</i> (D) gonads co-stained with MO-specific 1CB4 antibody (red) and DAPI (blue).</p

Known sperm genes among SPE-44 targets.

<p>Known sperm genes among SPE-44 targets.</p

Cell cycle defects in <i>spe-44</i> sperm.

<p>A–B. Wild-type (A) and <i>spe-44</i> (B) male gonads co-stained with DAPI (blue) and anti-phosphorylated histone H3 Ser10 (red). C–D. Sperm spreads from wild-type (C) and <i>spe-44</i> (D) males stained with antibody MPM-2 (green) and DAPI (blue). Labeling as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002678#pgen-1002678-g002" target="_blank">Figure 2C</a>. Additionally indicated are metaphase I (MI), metaphase II (MII), and arrested spermatocytes (arrest).</p