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 sulphate (SDS)/citric acid extraction method is superior to guanidinium isothiocyanate in terms of reproducibility of RNA recovery from motile sperm populations from individual ejaculates. Using the SDS/citric acid method, RNA was recovered from both fresh and frozen-thawed motile spermatozoa. Sperm RNA were used as templates in reverse transcription-polymerase chain reaction (RT-PCR), in an attempt to identify partial RNA transcripts of a highly conserved region within the alpha-1C (pore-forming) subunit of L-type voltage-dependent calcium channels from 11 individual donors. Control reactions employed primers derived from the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequence. In nine of the 11 specimens, gene-specific PCR products were obtained with both the GAPDH and alpha-1C primer pairs. DNA sequencing analysis confirmed that the respective spliced transcripts were amplified. 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 channel of that species was expressed uniformly in all stages of the germinal epithelium, including mature spermatozoa.
Goodwin
L O
LO
Division of Molecular Genetics, Department of Research, North Shore University Hospital-New York University School of Medicine, 300 Community Drive, Manhasset, New York 11030, USA.
Karabinus
D S
DS
Pergolizzi
R G
RG
Benoff
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics*; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000166:cytology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism
D020746:Calcium Channels, L-Type; D017931:DNA Primers; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases