ok1374 heterozygotes were more adversely affected than it129 homozygotes. Most of the phenotypes observed in the it129 homozygous animals are highly penetrant, making enhancement difficult to detect. However, data from the complementation assay for sperm-specific sterility strongly suggest a more severe defect in it129/ok1374 animals. Cross-fertilization of sterile it129 homozy- gous hermaphrodites by wild type males yields progeny with high viability (96%; see Figure 1C). In contrast, cross-fertilization of sterile it129/ok1374 hermaphrodites produces embryos with very low viability (6%; Figure 4C). Furthermore, the same data demonstrate that the number of fertilized embryos is significantly lower for it129/ok1374 heterozygotes than it129 homozygotes (10 vs. 48, respectively). Sperm are normally the limiting gamete for fertilization in C. elegans, but these results suggest that oocyte production might be defective in it129/ok1374 hermaphrodites. Therefore, we examined the gonads of these strains directly by DAPI staining. Germ cell development in C. elegans proceeds distally to proximally within the gonad, and is most readily distinguishable by changes in nuclear morphology [45]. In hermaphrodites, the proximal arm of the wild-type adult gonad contains a row of individual oocytes whose nuclei are arrested at diakinesis of meiosis I. Our analysis indicates that the germ lines of it129 homozygotes are similar to wild type hermaphrodites, and the proximal gonad contains morphologically normal oocytes whose six diakinetic bivalents are easily seen (Figure 4D, top and bottom left panels). In contrast, the germ lines of it129/ok1374 animals show an increased population of germ cells and a concommitant reduction in the number of oocytes in the proximal arm of the gonad. This defect in oogenesis is variable; some germ lines appear largely normal, while in other examples oocytes are absent and have been completely supplanted by an excess number of germ cells (as in Figure 4D, top and bottom second panels). A similar phenotype has been reported for mutations in a number of genes that govern the proliferation vs. meiosis decision, such as glp-1 [46]. In addition, we also observed a spermatogenesis defect in the germ line of males. Wild-type adult males accumulate large numbers of highly condensed spermatid nuclei within the seminal vesicle. The it129 homozygous males likewise contain an abundance of compact spermatid nuclei (Figure 4D, top and bottom third panels). However, the seminal vesicle of it129/ok1374 males contain relatively few nuclei that also appear larger or less condensed than spermatid nuclei (Figure 4D, rightmost top and bottom panels). In both hermaphrodites and males, the mitotic and pachytene regions of the germ line in the distal gonad appear normal (albeit occasionally reduced in size). These results suggest that the differentiation of gametes in both sexes is impaired in the it129/ok1374 heterozygous mutants, but with opposite effects depending upon the type of gamete: males possess fewer spermatids than normal, while hermaphrodites contain an excess of germ cell nuclei rather than oocytes. Gamete-specific differences in proliferation and differentiation have been reported previously. For example, loss of gld-1 causes germ line overproliferation only in hermaphrodites undergoing oogenesis [47], while loss of puf-8 causes overproliferation only in sperm-producing germ lines [48]. Transgene rescue of it129 with the wild type uba-1 gene further confirmed its identification. Initial attempts at rescue by germ line microinjection indicated that worms might be exquisitely sensitive to the dosage of this gene. Control injections with the rol-6 marker [49] produced numerous F1 rolling progeny with stable transmis- sion in subsequent generations. In contrast, coinjection of uba-1 with rol-6 at typical concentrations resulted in low brood sizes with very few F1 rollers and no stably transmitting lines, suggestive of transgene toxicity. To reduce the gene dosage, the concentration of UBA-1 in C. elegans Development uba-1 DNA was decreased relative to rol-6 and genomic N2 DNA was also included in the injections. At the lowest concentration tested, four of sixteen stably transmitting lines exhibited partial rescue of both sperm-specific sterility and embryonic lethality at the restrictive temperature. Therefore, the wild type uba-1 transgene is able to complement the it129 mutation. Expression of a uba-1::GFP reporter transgene has been reported in a variety of somatic tissues but not the germ line [50], although a functional role for UBA-1 in this tissue is indicated by the mutant phenotype. Transgenes are often silenced within the germ line, so in situ hybridization was employed to detect transcription of the endogenous uba-1 gene within the gonad. Abundant expression was detected in germ cells that had initiated meiosis in wild type hermaphrodites (during sperm and oocyte production) and males (Figure 5). Signal intensity appeared to be higher during oocyte production; this observation was confirmed by comparing fem- 1(hc17) hermaphrodites, which make only oocytes, to fem-3(q23) hermaphrodites, which make only sperm. Peak expression occurs at pachytene of the first meiotic division, is absent immediately afterwards, and is detected again in late oogenesis. This pattern is more apparent in the fem-1(hc17) gonad, which is from an older adult than the wild type hermaphrodite. Sequence determination of the uba-1 coding region from the it129-bearing strain revealed the molecular lesion. A single nucleotide substitution was detected that converts the proline at position 1024 to serine (Pro1024Ser, Figure 4B). The complete structure of E1 ubiquitin-activating enzyme has not yet been determined, but X-ray crystal structures of the activating enzymes for ubiquitin-like proteins SUMO and NEDD8 are available [51,52]. The proline residue that is mutated in uba-1(it129) maps near the active site where the ubiquitin moiety is predicted to be covalently attached to the E1 protein. On the basis of the structural data, the Pro1024Ser mutation might be expected to alter catalytic activity of the enzyme. In vivo Defects in Ubiquitination and Embryogenesis The uba-1 gene encodes the only known E1 ubiquitin-activating enzyme in C. elegans, so a defect in its activity is predicted to impair subsequent steps in the enzymatic cascade and cause an overall decrease in the level of ubiquitination on substrate proteins. We tested this hypothesis directly by using ubiquitin-specific antibodies to assess the amount of ubiquitination in worm protein lysates. To control for variations in ubiquitination activity at different stages of development, we extracted protein from age-synchronized young adult hermaphrodites shifted as L3 larvae. Since these uba-1(it129) animals are infertile due to sperm-specific sterility, we included as an additional control a strain containing spe-26(it112) (a temperature-sensitive, sperm-specific sterile mutation). Western blots show a significant reduction in the amount of ubiquitin signal in uba-1(it129) protein extracts compared to wild-type and spe- 26(it112) controls (Figure 6). Note that the level of ubiquitin in the high molecular weight region of the blot is particularly diminished, presumably reflecting a substantial reduction in the amount of poly-ubiquitinated substrates. Therefore, the uba-1(it129) mutation exhibits an in vivo decrease in protein ubiquitination. Reduced ubiquitination is predicted to adversely impact proteasomal degradation of target proteins. Well-characterized roles for ubiquitin-mediated proteolysis in C. elegans occur during the early events of embryogenesis. The anaphase-promoting complex (APC) is an E3 ligase that is required for degradation of the meiotic inhibitor securin [53]. Complete loss of APC activity results in metaphase arrest of the one-celled embryo [19]. The uba-1(it129) mutation does not produce the one-celled arrest caused by loss of APC activity, but instead mimics the PLoS Genetics | www.plosgenetics.org 7 July 2008 | Volume 4 | Issue 7 | e1000131