L-type voltage-dependent calcium channel alpha-1C subunit mRNA is present in ejaculated human spermatozoa

The current study adds to the growing body of evidence that RNA is present in mature ejaculated human spermatozoa. We report that a sodium dodecyl sul phate (SDS)/citric acid extraction method is superior to guanidinium isothi ocyanate in terms of reproducibility of RNA recovery from motile sperm popu lations from individual ejaculates. Using the SDS/citric acid method, RNA w as recovered from both fresh and frozen-thawed motile spermatozoa. Sperm RN A were used as templates in reverse transcription-polymerase chain reaction (RT-PCR), in an attempt to identify partial RNA transcripts of a highly co nserved region within the alpha-1C (pore-forming) subunit of L-type voltage -dependent calcium channels from 11 individual donors. Control reactions em ployed primers derived from the human glyceraldehyde-3-phosphate dehydrogen ase (GAPDH) sequence. In nine of the 11 specimens, gene-specific PCR produc ts were obtained with both the GAPDH and alpha-1C primer pairs. DNA sequenc ing analysis confirmed that the respective spliced transcripts were amplifi ed. The two cases in which no amplification was obtained were attributed to reduced RNA yield. These data are consistent with results from in-situ RT- PCR of rat testis sections indicating that the testis-specific calcium chan nel of that species was expressed uniformly in all stages of the germinal e pithelium, including mature spermatozoa.