Determination of Poly (ADP-ribose) polymerase (PARP) homologues in human ejaculated sperm and its correlation with sperm maturation.
OBJECTIVE
To investigate the presence of Poly (ADP-ribose) polymerase (PARP) and evaluate its function in ejaculated spermatozoa.


METHODS
Experimental study.


METHODS
Reproductive Research Center.


METHODS
Semen specimens from 18 healthy donors and 12 infertile males.


METHODS
Ejaculated spermatozoa were subjected to sperm fractionation with a double-layer density gradient, protein extraction, detection, immunoblotting, and mass spectrometry. Semen samples were exposed to PARP inducer staurosporine, or hydrogen peroxide with or without PARP inhibitor 3-aminobenzimide. Annexin V assay was examined for apoptosis by flow cytometry.


RESULTS
We detected approximately 75 kDa, approximately 63 kDa, and approximately 60 kDa PARP homologues on the immunoblot of mature and immature sperm fraction isolated from human ejaculate, which were identified as PARP-1 ( approximately 75 kDa), PARP-9 ( approximately 63 kDa), and PARP-2 ( approximately 60 kDa), respectively. Western blot analysis showed a positive correlation between the amount of PARP protein and sperm maturity. PARP proteins (75 kDa, 63 kDa, and 60 kDa) were evaluated after inducing apoptosis by hydrogen peroxide and staurosporine exposure.


CONCLUSIONS
PARP-2 may have a role in prevention of oxygen species/oxidative stress and chemical (staurosporine)-induced sperm cell apoptosis. The presence of the PARP homologue, that is, PARP-1 ( approximately 75 kDa), suggests its role in preventing damage of mature sperm. Additional studies are needed to delineate the role of PARP-9 in sperm physiology. The results from our study indicate an active role for PARPs in sperm cell physiology in preventing apoptosis.
Jha
Rajesh
R
Reproductive Research Center, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio, USA.
Agarwal
Ashok
A
Mahfouz
Reda
R
Paasch
Uwe
U
Grunewald
Sonja
S
Sabanegh
Edmund
E
Yadav
Satya P
SP
Sharma
Rakesh
R
D017209:Apoptosis; D001549:Benzamides / Q000494:pharmacology; D015153:Blotting, Western; D016022:Case-Control Studies; D002499:Centrifugation, Density Gradient; D004542:Ejaculation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007150:Immunohistochemistry; D007527:Isoenzymes; D008297:Male; D008970:Molecular Weight; D010449:Peptide Mapping; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013091:Spermatogenesis / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D019311:Staurosporine / Q000494:pharmacology
D001549:Benzamides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C025160:3-aminobenzamide; D006861:Hydrogen Peroxide; C499320:PARP1 protein, human; C483041:PARP2 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019311:Staurosporine