Obstet Gynecol Sci.  2015 May;58(3):232-238. 10.5468/ogs.2015.58.3.232.

Relationship between phospholipase C zeta immunoreactivity and DNA fragmentation and oxidation in human sperm

• Park JH
• Kim SK
• Kim J
• Kim JH
• Chang JH
• Jee BC
• Kim SH

• KMID: 2148936
• DOI: http://doi.org/10.5468/ogs.2015.58.3.232

Abstract

OBJECTIVE
The study aimed to evaluate the feasibility and reproducibility of measuring phospholipase C zeta (PLCzeta) using immunostaining in human sperm and to investigate the relationship between PLCzeta immunoreactivity and DNA fragmentation and oxidation in human sperm.
METHODS
Semen samples were obtained from participants (n=44) and processed by the conventional swim-up method. Sperm concentration, motility, normal form by strict morphology, DNA fragmentation index assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling method and immunofluorescent expression for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and PLCzeta were assessed.
RESULTS
When duplicate PLCzeta tests were performed on two sperm samples from each of the 44 participants, similar results were obtained (74.1+/-9.4% vs. 75.4+/-9.7%). Two measurements of PLCzeta were found to be highly correlated with each other (r=0.759, P<0.001). Immunoreactivity of PLCzeta was not associated with donor's age, sperm concentration, motility, and the percentage of normal form as well as DNA fragmentation index. However, immunoreactivity of PLCzeta showed a significant negative relationship with 8-OHdG immunoreactivity (r=-0.404, P=0.009).
CONCLUSION
Measurement of PLCzeta by immunostaining is feasible and reproducible. Lower expression of PLCzeta in human sperm may be associated with higher sperm DNA oxidation status.

Keyword

DNA oxidation; 8-Hydroxy-2'-deoxyguanosine; Phospholipase C zeta; Spermatozoa

MeSH Terms

DNA
DNA Fragmentation*
DNA Nucleotidylexotransferase
Humans
Semen
Spermatozoa*
Type C Phospholipases*
DNA
DNA Nucleotidylexotransferase
Type C Phospholipases

Figure

• 

  Fig. 1 A representative microphotograph showing sperm stained by immunofluorescent antibody for 8-hydroxy-2'-deoxyguanosine (×1,000); (A) 4,6-diamidino-2-phenylindole, (B) fluorescein isothiocyanate, and (C) merged capture.

• 

  Fig. 2 A representative microphotograph showing sperm stained by immunofluorescent antibody for anti-phospholipase C zeta (×400). (A) Hoechst 33258, (B) fluorescein isothiocyanate, and (C) merged capture. Four 'green' sperm were seen in (B). Non-specific binding in the tail has previously been reported by Grasa et al. [1].

• 

  Fig. 3 Two-times measurements of phospholipase C zeta immunoreactivity (round 1 and round 2) were highly correlated each other (r=0.759, P<0.001, by the Pearson correlation test).

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