Proper condensation of DNA into sperm ensures fertility and proper transmission of genetic information. Sperm DNA is highly condensed in order to package the entire genome into very small sperm. Using a subtractive proteomic approach, we have previously identified two C. elegans Glc7/PP1 (Protein Phosphatase 1) phosphatases specifically associated to sperm chromatin. Glc7/PP1 family members play a role in many cellular functions. In oocyte mitosis and meiosis, Glc7/PP1's act antagonistically to the
lpl1/aurora kinases, which phosphorylate histone H3 at serine10 to function in chromosome segregation. The two sperm chromatin associated Glc7 phosphatases that were identified are
gsp-3 (W09C3.6) and
gsp-4 (T03F1.5), and share a 90% nucleotide sequence identity. We have found that GSP-3 and GSP-4 are necessary for chromosome segregation in meiosis in sperm. Due to the sequence similarity, antibodies against GSP-3 and GSP-4 cross-react. Antibody staining in wildtype revealed localization around sperm DNA during and after meiosis in both hermaphrodites and male animals. In order to assess the loss of function phenotype of
gsp-3 and
gsp-4 mutants, we utilized RNA-mediated interference via microinjection. RNAi of either
gsp-3 or
gsp-4 decreased the fertility of injected animals. When wildtype males were mated with
gsp-3(RNAi) or
gsp-4(RNAi) hermaphrodites, the fertility defect of the hermaphrodites was rescued. Therefore, the RNAi experiments determined that
gsp-3 and
gsp-4 function in a sperm specific manner. When gsp(RNAi) sperm chromatin was visualized by DAPI staining, we observed chromosomal bridging during sperm meiosis, suggesting that the fertility defect was a caused by chromosome segregation problems. Therefore, we have identified two sperm specific Glc7 phosphatases required for sperm chromosome segregation. One of our goals is to distinguish the seperate functions of
gsp-3 and
gsp-4. To do this we have generated a
gsp-4 mutant strain which is null for
gsp-4. This animal shows no visible defects and stains for -GSP-3/GSP-4 antibody similarly to wildtype. We will examine a
gsp3 mutant strain similarly to determine if
gsp-3 and
gsp-4 act redundantly. When RNAi animals are stained with -GSP-3/GSP-4 antibody there is still visible staining, indicating a partial loss of function. We are generating a
gsp-3gsp-4 double mutant strain to analyze the null phenotype. This strain will provide us with further insight on the role of these proteins in DNA condensation and segregation.