ZIP6 and ZIP14 zinc transporter expression in normozoospermic and asthenozoospermic human spermatozoa - relationship to nuclear chromatin accessibility

Abstract

Introduction/Objective. Male infertility accounts for approximately 40% of infertility cases, with asthenozoospermia affecting nearly one in five infertile men. Beyond conventional semen parameters, nuclear DNA organization and chromatin accessibility provide deeper insight into sperm quality. Zinc transporters ZIP6 and ZIP14 have been implicated in chromatin stabilization and zinc homeostasis, potentially essential to sperm maturation and fertilization competence. Methods. Semen samples from normozoospermic and asthenozoospermic men undergoing intrauterine insemination were purified by density gradient. ZIP6 and ZIP14 were detected by immunofluorescence, nuclear DNA visualized by using Sytox Orange. Expression levels, nuclear area, and staining intensity were quantified in ImageJ, and analyzed using Kruskal-Wallis test with Dunn's post-hoc correction (p ≤ 0.05). Results. ZIP6 immunopositivity was significantly reduced in asthenozoospermic compared to normozoospermic samples, while ZIP14 expression remained stable. Nuclear area and overall Sytox Orange intensity did not differ significantly between transporter-positive and -negative sperm, though ZIP6-positive asthenozoospermic cells showed a modest reduction in nuclear surface area. Normozoospermic ZIP14-negative spermatozoa displayed lower Sytox Orange fluorescence than ZIP6-negative cells, suggesting focal differences in chromatin accessibility between transporter-defined subpopulations. Conclusion. ZIP6 expression reflects sperm maturation and functional competence, while ZIP14 remains constitutively stable. Both transporters may serve as molecular markers for selecting high-quality sperm in assisted reproduction, particularly in populations exposed to occupational stressors that compromise DNA integrity.