Thesis

Premature Sperm Activation Prevented by spe-6 in the Nematode, Caenorhabditis elegans

Spermatogenesis pauses only at the penultimate stage, the spermatid. The pause ends when an external signal activates the spermatid into a spermatozoon. Activation involves two pathways: (i) the try-5 pathway, named because the trypsin-like protein TRY-5, in the seminal fluid activates spermatids during mating; and (ii) the spe-8 pathway, named because the SPE-8 group complex transduces the activation signal within the sperm after the cells receive an unknown activation signal. Mutations in the spe-8 group genes (spe-8, -12, -19, -27, -29, and -43) result in spermatids that cannot activate in unmated hermaphrodites. We focus on one target of the SPE-8 complex, SPE-6 a serine-threonine kinase protein. Here, we test the hypothesis that active SPE-6 maintains the spermatid stage, and loss of SPE-6 function results in activation. A suppressor screen from Samuel Ward’s laboratory with spe-27(it132ts) generated numerous suppressor mutations that restore partial fertility. The only gene to be hit multiple times in the screen was spe-6. Previously known null spe-6 mutations stall spermatogenesis at the transition from primary to secondary spermatocytes. In contrast, most spe-6 suppressor mutations cause premature spermatid activation. Five suspected alleles of spe-6 were examined through complementation crosses and sequencing; three of five were confirmed to be spe-6 alleles. The alleles uncovered: zq10, zq11, and zq18 are compared to known spe-6 alleles: hc166, hc187, hc188, and hc190 to get a broader view of spe-6. All alleles except zq11 were able to suppress the sterility of spe-29(it127). Additionally, mutant males had active sperm in their seminal vesicles, while wild type males never harbor active sperm. These suppressor mutations were located in various locations with respect to the 3D structure of the protein, and two others were intronic and altered a splice junction, likely reducing transcript availability. These results suggest that a reduction in SPE-6 can be brought about through loss of function point mutations or loss of transcripts, with the effect that sperm activate prematurely, bypassing the need for the spe-8 group pathway signaling. The unique allele zq11 is an allele specific suppressor of spe-27(it132ts): it does not suppress spe-27(it110) or spe-29(it127). Allele specific suppression vi provides genetic evidence that SPE-6 and SPE-27 proteins interact physically and suggests that SPE-6 is a target of SPE-8 group signaling. The two splicing mutations were replicated in spe-44 using CRISPR/Cas9 in an effort to induce conditional mutation in this important gene. However, neither mutation in spe-44 produced a phenotype.

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