Systematic characterization of human testis-specific actin capping protein β3 as a possible biomarker for male infertility

STUDY QUESTION: Is actin capping protein (CP) beta 3 involved in human spermatogenesis and male infertility? SUMMARY ANSWER: Human CP beta 3 (hCP beta 3) is expressed in testis, changes its localization dynamically during spermatogenesis, and has some association with male infertility. WHAT IS KNOWN ALREADY: The testis-specific a subunit of CP (CP alpha 3) was previously identified in human, and mutations in the cp alpha 3 gene in mouse were shown to induce malformation of the sperm head and male infertility. However, CP beta 3, which is considered to be a heterodimeric counterpart of CP alpha 3, has been neither characterized in human nor reported in association with male infertility. STUDY DESIGN, SIZE, DURATION: To confirm the existence of CP beta 3 in human testis, fresh semen samples from proven fertile men were analyzed. To investigate protein expression during spermatogenesis, cryopreserved testis obtained from men with obstructive azoospermia were examined by immunofluorescent analysis. To assess the association of CP with male infertility, we compared protein expression of human CPa3 (hCP alpha 3) and hCP beta 3 using immunofluorescent analysis of cryopreserved sperm between men with normozoospermia (volunteers: Normo group, n = 20) and infertile men with oligozoospermia and/or asthenozoospermia (O + A group, n = 21). PARTICIPANTS/MATERIALS, SETTING, METHODS: The tissue-specific expression of hCP beta 3 was investigated by RT-PCR and Western blot analysis. To investigate whether hCP alpha 3 and hCP beta 3 form a heterodimer, a tandem expression vector containing hcp alpha 3 tagged with monomeric red fluorescent protein 1 and hcp beta 3 tagged with enhanced green fluorescent protein in a single plasmid was constructed and analyzed by co-immunoprecipitation (Co-IP) assay. The protein expression profiles of hCP alpha 3 and hCP beta 3 during spermatogenesis were examined by immunohistochemical analysis using human spermatogenic cells. The protein expressions of hCP alpha 3 and hCP beta 3 in sperm were compared between the Normo and O + A groups by immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: RT-PCR showed that mRNA of hcp beta 3 was expressed exclusively in testis. Western blot analysis detected hCP beta 3 with anti-bovine CP beta 3 antibody. Co-IP assay with recombinant protein showed that hCPa alpha 3 and hCPa beta 3 form a protein complex. At each step during spermatogenesis, the cellular localization of hCP alpha 3 changed dynamically. In spermatogonia, hCP beta 3 showed a slight signal in cytoplasm. hCP alpha 3 expression was conspicuous mainly from spermatocytes, and hCP beta 3 localization dynamically migrated from cytoplasm to the acrosomal cap and acrosome. In mature spermatozoa, hCP alpha 3 accumulated in the postacrosomal region and less so at the midpiece of the tail. Double-staining analysis revealed that hCP alpha 3 localization was identical to hCP beta 3 at every step in the spermatogenic cells. Most spermatozoa from the Normo group were stained homogenously by both hCP alpha 3 and hCP beta 3. In contrast, significantly more spermatozoa in the O + A versus Normo group showed heterogeneous or lack of staining for either hCP alpha 3 or hCP beta 3 (abnormal staining) (P < 0.001). The percentage of abnormal staining was higher in the O + A group (52.4 +/- 3.0%) than in the Normo group (31.2 +/- 2.5%). Even by confining the observations to morphologically normal spermatozoa selected in accordance with David's criteria, the percentage of abnormal staining was still higher in the O + A group (39.9 +/- 2.9%) versus the Normo group (22.5 +/- 2.1%) (P < 0.001). hCP beta 3 in conjunction with hCP alpha 3 seemed to play an important role in spermatogenesis and may be associated with male infertility. LARGE SCALE DATA: Not applicable. LIMITATIONS REASONS FOR CAUTION: Owing to the difficulty of collecting fresh samples of human testis, we used cryopreserved samples from testicular sperm extraction. To examine the interaction of spermatogenic cells or localization in seminiferous tubules, fresh testis sample of healthy males are ideal. WIDER IMPLICATIONS OF THE FINDINGS: The altered expression of hCP alpha 3 and hCP beta 3 may not only be a cause of male infertility but also a prognostic factor for the results of ART. They may be useful biomarkers to determine the fertilization ability of human sperm in ART.