Investigating Sperm-Specific Gene Expression Required For Embryogenesis

Zygotic gene expression in the nematode C. elegans starts at the four-cell stage of embryogenesis. Therefore, the gene products necessary to regulate the first two ordered and asymmetric cell divisions are maternally or paternally derived. Though a number of maternal factors have been identified, paternally derived factors necessary for the early development of a zygote into an embryo are little known or understood.

We isolated RNA from highly purified sperm cells and used 17 putative paternal effect genes. My research focuses on the further analysis of one of these genes, K07A12.5. I used cDNA clones of K07A12.5 to generate DIG labeled sense and anti-sense probes using asymmetric PCR. The probes were used for in-situ hybridization in dissected male and hermaphrodite gonads. Individual dissected worms were fixed in paraformaldehyde, permeablized with a detergent and protenase K treatment, and allowed to hybridize for 36 hours at 48˚C. After washing, bound probes were detected using anti-DIG antibodies and the signal was amplified using Tryamide. The samples were mounted on agarose pads in Prolong-Gold mounting medium with DAPI for imaging and examination.

Preliminary data show that the gene K07A12.5 is sperm-specific given that it was isolated from purified sperm cells. In-situ hybridization was inconclusive and I am currently troubleshooting before conducting the protocol once again.