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.

Sex is determined in Caenorhabditis elegans by the ratio of X chromosomes to the sets of autosomes, the X:A signal. A set of genes called X signal elements (XSEs) communicates X-chromosome dose by repressing the masculinizing sex determination switch gene xol-1 (XO lethal) in a dose-dependent manner. xol-1 is active in 1X:2A embryos (males) but repressed in 2X:2A embryos (hermaphrodites). Here we showed that the autosome dose is communicated by a set of autosomal signal elements (ASEs) that act in a cumulative, dose-dependent manner to counter XSEs by stimulating xol-1 transcription. We identified new ASEs and explored the biochemical basis by which ASEs antagonize XSEs to determine sex. Multiple antagonistic molecular interactions carried out on a single promoter explain how different X:A values elicit different sexual fates. XSEs (nuclear receptors and homeodomain proteins) and ASEs (T-box and zinc finger proteins) bind directly to several sites on xol-1 to counteract each other's activities and thereby regulate xol-1 transcription. Disrupting ASE-and XSE-binding sites in vivo recapitulated the misregulation of xol-1 transcription caused by disrupting cognate signal element genes. XSE-and ASE-binding sites are distinct and nonoverlapping, suggesting that direct competition for xol-1 binding is not how XSEs counter ASEs. Instead, XSEs likely antagonize ASEs by recruiting cofactors with reciprocal activities that induce opposite transcriptional states. Most ASE-and XSE-binding sites overlap xol-1's -1 nucleosome, which carries activating chromatin marks only when xol-1 is turned on. Coactivators and corepressors tethered by proteins similar to ASEs and XSEs are known to deposit and remove such marks. The concept of a sex signal comprising competing XSEs and ASEs arose as a theory for fruit flies a century ago. Ironically, while the recent work of others showed that the fly sex signal does not fit this simple paradigm, our work shows that the worm signal does.

It has been proposed that retinoic acid receptors (RARs) and thyroid hormone receptors (TRs) both bind to AGGTCA “half-site” sequences, but distinguish their different target genes by recognizing different half-site spacings. We report here that artificial DNA binding sites based on these AGGTCA half-sites confer high affinity, but poor specificity, and that spacing alone does not account for the divergent DNA recognition properties of TRs and RARs. Instead, we have determined that the non-consensus half-sites that are present in naturally occurring RAR and TR target genes play a crucial role in defining receptor DNA recognition specificity, and work together with flanking sequences and half-site spacing to produce receptor-specific DNA binding in vitro. We also provide evidence that auxiliary proteins in cells generate an additional layer of receptor-specific target gene recognition, in part by destabilizing the binding of nuclear receptors to the “wrong” response elements.

Sex is determined in C. elegans by a chromosome-counting mechanism that tallies X chromosome dose relative to the sets of autosomes, the X:A ratio. A group of genes on X called X signal elements (XSEs) communicates X chromosome number by repressing the activity of the master sex-determination switch gene xol-1 in a dose-dependent manner. xol-1 is repressed by transcriptional and posttranscriptional mechanisms and is inactive in XX animals (hermaphrodite) but active in XO animals (male). Prior to our work, the nature of the autosomal signal and its target(s) were unknown. Here we show the signal includes discrete, trans-acting autosomal signal elements (ASEs) that counter XSEs to coordinately control both sex determination and dosage compensation. sea-1, the first autosomal signal element, encodes a T-box transcription factor that opposes XSEs by activating transcription of xol-1. Hence, xol-1 integrates both X and autosomal signals to determine sexual fate.