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

T. Soda, Y. Miyagawa, N. Ueda, K. Takezawa, H. Okuda, S. Fukuhara, K. Fujita, H. Kiuchi, M. Uemura, Y. Okamoto, A. Tsujimura, H. Tanaka, N. Nonomura

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Abstract

STUDY QUESTION: Is actin capping protein (CP) β3 involved in human spermatogenesis and male infertility? SUMMARY ANSWER: Human CPβ3 (hCPβ3) is expressed in testis, changes its localization dynamically during spermatogenesis, and has some association with male infertility. WHAT IS KNOWN ALREADY: The testis-specific α subunit of CP (CPα3) was previously identified in human, and mutations in the cpα3 gene in mouse were shown to induce malformation of the sperm head and male infertility. However, CPβ3, which is considered to be a heterodimeric counterpart of CPα3, has been neither characterized in human nor reported in association with male infertility. STUDY DESIGN, SIZE, DURATION: To confirm the existence of CPβ3 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 CPα3 (hCPα3) and hCPβ3 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 hCPβ3 was investigated by RT-PCR and Western blot analysis. To investigate whether hCPα3 and hCPβ3 form a heterodimer, a tandem expression vector containing hcpα3 tagged with monomeric red fluorescent protein 1 and hcpβ3 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 hCPα3 and hCPβ3 during spermatogenesis were examined by immunohistochemical analysis using human spermatogenic cells. The protein expressions of hCPα3 and hCPβ3 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 hcpβ3 was expressed exclusively in testis. Western blot analysis detected hCPβ3 with anti-bovine CPβ3 antibody. Co-IP assay with recombinant protein showed that hCPα3 and hCPβ3 form a protein complex. At each step during spermatogenesis, the cellular localization of hCPβ3 changed dynamically. In spermatogonia, hCPβ3 showed a slight signal in cytoplasm. hCPβ3 expression was conspicuous mainly from spermatocytes, and hCPβ3 localization dynamically migrated from cytoplasm to the acrosomal cap and acrosome. In mature spermatozoa, hCPβ3 accumulated in the postacrosomal region and less so at the midpiece of the tail. Double-staining analysis revealed that hCPα3 localization was identical to hCPβ3 at every step in the spermatogenic cells. Most spermatozoa from the Normo group were stained homogenously by both hCPα3 and hCPβ3. In contrast, significantly more spermatozoa in the O + A versus Normo group showed heterogeneous or lack of staining for either hCPα3 or hCPβ3 (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). hCPβ3 in conjunction with hCPα3 seemed to play an important role in spermatogenesis and may be associated with male infertility.
Original languageEnglish
Pages (from-to)514-522
Number of pages9
JournalHuman Reproduction
Volume32
Issue number3
DOIs
Publication statusPublished - 2017

Keywords

  • actin capping protein
  • male infertility
  • spermatogenesis
  • testis specific
  • sperm morphology

Cite this

Soda, T., Miyagawa, Y., Ueda, N., Takezawa, K., Okuda, H., Fukuhara, S., Fujita, K., Kiuchi, H., Uemura, M., Okamoto, Y., Tsujimura, A., Tanaka, H., & Nonomura, N. (2017). Systematic characterization of human testis-specific actin capping protein β3 as a possible biomarker for male infertility. Human Reproduction, 32(3), 514-522. https://doi.org/10.1093/humrep/dew353