UBA-1 in C. elegans Development Figure 7. OMA-1::GFP expression. A) Adult hermaphrodites expressing the oma-1::GFP integrated transgene. Shown are examples of wild type and uba-1(it129) animals that contain one and four one-celled embryos, respectively. B) Frequency of one-celled embryos in the uterus. The number of one-celled embryos per gonad arm were counted for wild type (N = 40) and uba-1(it129) (N = 34) hermaphrodites. C) Distribution of one-celled embryos in the meiotic, pronuclear, and mitotic stages of development, as visualized by OMA-1::GFP. doi:10.1371/journal.pgen.1000131.g007 it’s difficult to speculate on the molecular mechanisms responsible for the observed lethality. In other instances, the uba-1 mutant phenotype suggests a previously unidentified role for ubiquitination. Body size in C. elegans is governed by a canonical TGF-ß signal transduction pathway that initiates with the DBL-1 ligand [56,57]. Components of the TGF-ß pathway in other organisms are known to be regulated by ubiquitin conjugation [58]. Different ubiquitin modifications produce antagonistic effects on signal transduction: mono-ubiquitination of Co-Smad stabilizes the protein and promotes signaling, while poly-ubiquitination of R-Smad leads to its proteasomal degradation and down-regulation of signaling. Given the effects of the uba-1 mutation on C. elegans body size, it seems likely that components of the DBL-1/TFG-ß pathway are similarly regulated by ubiquitin. The sperm-specific sterility of uba-1(it129), coupled with the recent identification of spe-16 as an E3 ubiquitin ligase homolog (Steve L’Hernault, personal communication), indicate a previously uncharacterized role for ubiquitin in C. elegans spermatogenesis. Ubiquitination is known to be essential for sperm function in a wide variety of organisms, and roles in mammalian spermatogen- esis include regulation of the meiotic cell cycle, histone modification and chromatin remodeling, protein sorting during sperm differentiation, and quality control for defective sperm [59– 62]. In C. elegans, early events like meiosis appear unaffected by the uba-1(it129) mutation, suggesting that the infertility of these morphologically normal spermatozoa is due to a later defect in sperm development. In a manner analogous to mammalian sperm, ubiquitination in C. elegans might function in protein sorting as the spermatids divide from the residual body. Errors in this process are known to adversely affect sperm function: mutation of spe-15, which encodes a myosin homolog, impairs the asymmetric segregation of proteins during spermatid budding and causes sperm-specific sterility [63]. Alternatively, ubiquitination might promote proteasomal degradation of a protein that inhibits fertilization, and decreased activity of UBA-1 would lead to inappropriate persistence of the proposed inhibitor. Spermatid activation and downstream events occur in the absence of new protein synthesis, so degradation of pre-existing component(s) is a plausible mechanism of regulation. Another possibility is that uba- 1(it129) infertility might reflect a role for ubiquitin-mediated proteolysis in the sperm-oocyte interaction. Fertilization in ascidians is mediated by an extracellular enzyme from sperm that conjugates ubiquitin to a sperm receptor on the egg surface, leading to its degradation via the proteasome [64]. Ongoing analysis is designed to determine if one (or more) of these hypotheses is correct. Multiple E3 ligases are involved in formation of the reproduc- tive structures of the male tail, so the defects observed in uba-1 mutant males might arise from impairment of one or more known PLoS Genetics | www.plosgenetics.org 10 July 2008 | Volume 4 | Issue 7 | e1000131