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) TRY-5 in seminal fluid interacting with SWM-1 and (ii) an unknown signal both seminal fluid and in the hermaphrodite interacting with the SPE-8 group complex. Mutations in the
spe-8 group genes (
spe-8, -12, -19, -27, -29) result in spermatids that cannot activate in unmated hermaphrodites. We focus on one likely target of the SPE-8 complex, the SPE-6 serine- threonine kinase. Here, we test the hypothesis that active SPE-6 maintains the spermatid stage, and that activation is achieved through downregulation of SPE-6. A suppressor screen from Sam Ward's lab with
spe-27(
it132) generated numerous suppressor mutations that restore partial fertility. Our work examines the suppressor mutations recovered in
spe-6. Null
spe-6 mutations stall spermatogenesis at the transition from primary to secondary spermatocytes. In contrast, the
spe-6 suppressor mutations cause premature spermatid activation, bypassing SPE-8 group signaling. The alleles we focus on are:
hc166,
hc176,
hc188,
hc190,
zq10,
zq11, and
zq18. These mutations likely reduce function and are scattered across the gene, suggesting that lower of SPE-6 function initiates activation. One mutation,
zq11, is an allele specific suppressor of
spe27(it132).
zq11 does not suppress
spe-27(
it110), indicating that SPE-6 and SPE-27 interact physically and suggest that SPE-6 is a target of SPE-8 group signaling. Two other
spe-6 alleles have intronic mutations. The
hc190 and
zq18 alleles are G to A substitutions of the 1st and 5th bases, respectively, of the 5' end of the intron a highly-conserved site for splicing. These conditional mutations cause premature sperm activation and partial mRNA loss, although normal splices were present for both mutants at 25 deg C. Thus, loss of SPE-6 function through a simple drop of transcript abundance is sufficient to cause premature activation. Thus, SPE-6 function is necessary to maintain the spermatid stage. We have used CRISPR/Cas9 to induce similar intronic mutations in
spe-44. However, neither mutation in
spe-44 produces a phenotype. We are now attempting to create both mutations in the same intron.