#338 : Insights into the Zona-Pellucida Mediated Selection of Fertilization-Competent Spermatozoa

Abstract

Background and Aims: Human zona pellucida (ZP) selectively interacts with high-quality spermatozoa characterized by superior morphology and normal chromatin structure to achieve fertilization. Human ejaculate contains millions of spermatozoa with varying degrees of fertilization potential and genetic quality, of which only thousands of motile spermatozoa can bind to the ZP at the fertilization site. However, direct evidence for ZP-mediated sperm selection process is lacking. This study aims to demonstrate that spermatozoa-ZP interaction represents a crucial step in selecting fertilization-competent spermatozoa in humans. Method: Human spermatozoa and oocytes were obtained with written consents from the donors attending our assisted reproduction program. Acrosome intact, ZP-bound and unbound spermatozoa were recovered using the modified spermatozoa-ZP coincubation assays. ZP-binding protein expression on ZP-bound and unbound spermatozoa were examined by immunofluorescence staining. And the genetic integrity and epigenetic landscapes were evaluated by TUNEL assay, COMET assay, acridine orange staining, chromomycin A3, and immunofluorescence staining for 5-methylcytosine. Results: The time course data demonstrated that ZP interacted with a small proportion of motile spermatozoa. Heat shock 70 kDa protein 2 (HSPA2) and sperm acrosome associated 3 (SPACA 3) are two protein markers associated with the sperm ZP-binding ability. ZP-bound spermatozoa had a higher percentage of positive HSPA2 (85.8% vs. 45.9%, p<0.05) and SPACA3 signals (86.3% vs. 30.5%, p<0.05) than the unbound ones. The signal intensities of both markers were stronger in the ZP-bound spermatozoa than the unbound ones. ZP-bound spermatozoa had a significantly higher level of normal morphology, DNA integrity, chromatin integrity, protamination and global methylation when compared to the unbound ones. Conclusion: Our results validated the functional role of spermatozoa-ZP interaction in providing diagnostic information regarding the fertilization potential and genetic qualities of spermatozoa independent of those derived from the standard semen analysis.