Up-regulation of heme oxygenase-1 expression modulates reactive oxygen species level during the cryopreservation of human seminiferous tubules

Abstract

Objective: To study the effect of freezing techniques and to optimize a method for trace amounts of testicular spermatozoa from biopsed seminiferous tubules. The level of reactive oxygen species (ROS) and the gene expression of heme oxygenase-1 was evaluated.

Design: Prospective experimental study.

Setting: University-based laboratory.

Patient(s): Eighteen adults with male fator infertility underwent testicular biopsy surgery.

Intervention(s): Seminiferous tubular fragments from each man were evenly allocated to three groups: fresh control, slow freezing, and vitrifiaction groups. The morphology and ROS levels before and after freezing were evaluated for seminiferous tubular fragments. The expression of heme oxygenase-1 (HO-1) at both the transcriptional and protein levels was determined.

Main outcome measure(s): The morphology was analyzed by light microscopy. The ROS levels were measured with ELISA. The proliferation and differentiation were evaluated by immunohistochemistry, and the expression of HO-1 was evaluated using a real-time polymerase chain reaction (PCR) and Western blotting.

Result(s): Decreased ROS levels and increased HO-1 expression at the transcriptional and protein levels were observed after thawing the human seminiferous tubules. The ROS level was negatively correlated with HO-1 expression. Slow freezing was more effective than vitrification in terms of HO-1 up-regulation and ROS alteration.

Conclusion(s): Based on our study, the slow freezing technique was more effective compared with the vitrification method.

Keywords: Oxidative stress; cryopreservation; testicular spermatozoa; trace amounts.