In a blinded study of couples undergoing in vitro fertilization, (IVF) more than 40% of the males had serum levels of lead above the action limit for occupationally exposed workers. Both serum and seminal plasma Pb correlated inversely with rates of oocyte fertilization in IVF. Seminal plasma Pb also correlated inversely with the ability of the sperm to undergo a progesterone-stimulated acrosome reaction (PSAR) and the aggregation/translocation of NNPR to the equatorial region of sperm which precedes exocytosis. In preliminary experiments, we demonstrated that the human PSAR was blocked by specific inhibitors of voltage-gated K+ channels, and that PSAR was augmented by increasing K+, suggesting that a K+ channel opened early in the PSAR. To show that the channels are present in male germ cells, the investigator used PCR primers based on a rat neural cortex voltage-dependent K+ channel to amplify a product from rat testes mRNA. North analysis using this sequence as a probe identified 2.4 and 4.2kB transcripts in pooled rat testes mRNA, but not in any other tissue tested. In situ RT/PCR found these transcripts in the cytoplasm of stage IX and X rat spermatocytes as well as elongating spermatids. The goal of this proposal is to characterize the K+ channel associated with the NNPR. The investigator will perform dose-response studies with specific ion channel inhibitors to further characterize the K+ channel of human sperm, with the aim of distinguishing among possible subtypes (e.g. delayed-rectifier voltage-gated, Ca++ activated). The endpoint will be PSAR as assay with rhodamine-labeled pes agglutinin. The investigator will prepare biotinylated charybdotoxin by standard protocols and attempt to visualize K+ channels on intact human sperm using our product in conjunction with anti-biotin antibodies. The investigator will clone and sequence the cDNA of the human sperm channel K+ from pooled human sperm RNA, starting from primers based on the human voltage-gated K+ channel. They will determine the stage-specific expression of the human sperm K+ channel by in situ RT/PCR on human testes biopsy sections.
| Benoff, S; Jacob, A; Hurley, I R (2000) Male infertility and environmental exposure to lead and cadmium. Hum Reprod Update 6:107-21 |
