Analysis and significance of mRNA in human ejaculated sperm from normozoospermic donors: relationship to sperm motility and capacitation.
The existence of a complex population of mRNA in human sperm is well documented but their role is not yet elucidated. Using discontinuous density gradients, we have isolated high and low motile sperm from the same semen sample. The levels of different transcripts coding for molecules either involved in nuclear condensation (protamines 1 and 2) or in capacitation [endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and c-myc] were then assessed in the two populations using semi-quantitative RT-PCR. Sperm viability was estimated by eosin-nigrosin staining and by hypo-osmotic swelling test; apoptosis percentage was measured by the TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling technique. The contamination by somatic and germ cells was assessed by looking for specific molecular markers of these cells, respectively CD-45 and E-cadherin for somatic cells and c-kit for germ cells. The viability of sperm was unchanged in high and low motile fractions, as well as DNA fragmentation percentage. The amount of Prm-1 mRNA was significantly higher in low density motile than in the high motile fraction. In most of high motile sperm samples eNOS and nNOS transcripts were undetectable whereas they were present in the low motile sperm. In contrast, no significant variation was found in the c-myc/Prm-2 mRNA ratio between the two populations. Moreover, a partial or complete disappearance of c-myc transcripts was observed after capacitation. Thus analysing mRNA profiles could be helpful as a diagnostic tool and prognosis value for fertilization.
Lambard
S
S
UPRES EA 2608-USC INRA, University of Caen, France.
Galeraud-Denis
I
I
Martin
G
G
Levy
R
R
Chocat
A
A
Carreau
S
S
D000328:Adult; D002469:Cell Separation; D002470:Cell Survival; D004542:Ejaculation; D006801:Humans; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D011479:Protamines / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D013075:Sperm Capacitation / Q000235:genetics*; D013081:Sperm Motility / Q000235:genetics*; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*
C489427:MYC protein, human; D009419:Nerve Tissue Proteins; C082073:PRM1 protein, human; D011479:Protamines; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; C068621:protamine 2; C496320:NOS1 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III