Hum. Reprod. Advance Access originally published online on April 11, 2008
Human Reproduction 2008 23(6):1271-1279; doi:10.1093/humrep/den113
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Fas receptor is not present on ejaculated human sperm
1 Clinical Analysis Unit, Department of Laboratory Medicine, Institute of Child Health IRCCS Burlo Garofolo, Via dell’Istria 65/1, 34137 Trieste, Italy 2 Assisted Reproduction Unit, Department of Obstetrics and Gynaecology, Institute of Child Health IRCCS Burlo Garofolo and University of Trieste, Via dell’Istria 65/1, 34137 Trieste, Italy
3 Correspondence address. Tel: +39-40-3785-322; Fax: +39-40-76 12-66; E-mail: ricci{at}burlo.trieste.it
BACKGROUND: Apoptosis appears to have an essential role in the control of testis germ cell number and Fas expression has been reported in apoptotic spermatocytes and spermatids. We investigated if Fas (CD95) was present on ejaculated human sperm and any relationship between Fas on sperm and the apoptotic marker Syto16.
METHODS: Semen samples from 77 male partners of infertile couples were evaluated. Each sample was analysed both before and after semen preparation by conventional microscopical procedures and by flow cytometry (FC). A multiparameter FC analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, CD45 positive (leukocyte) and CD95 (Fas) positive cell concentration was carried out. A further 10 samples were studied by indirect immunofluorescence to confirm results.
RESULTS: The mean concentration of CD95 positive cells was very low (<1%), with no significant difference between normozoospermic and non-normozoospermic men. There was no correlation between apoptotic sperm and CD95 positive cell concentration. A linear correlation was found between CD95 positive cell and leukocyte (CD45 positive) concentration (r = 0.9946, P < 0.0001). CD95 mean fluorescence intensity of leukocytes was 10-fold greater than that of sperm and of isotypic control. Both incubation with activating anti-Fas antibody and betulinic acid induced apoptosis in leukocytes. Incubation with betulinic acid, but not with activating anti-Fas antibody, induced apoptosis in sperm. Pre-incubation with neutralizing anti-Fas antibody suppressed CD95 expression on leukocytes, whereas it did not change sperm CD95 peak fluorescence.
CONCLUSIONS: There is no detectable quantity of Fas on human ejaculated sperm.
Key words: apoptosis/flow cytometry/Fas/sperm/Syto 16
Submitted on September 23, 2007; resubmitted on February 18, 2008; accepted on March 12, 2008.
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