title: Systematic characterization of human testis-specific actin capping protein β3 as a possible biomarker for male infertility.
abstract: Is actin capping protein (CP) β3 involved in human spermatogenesis and male infertility?
  
  
  
  Human CPβ3 (hCPβ3) is expressed in testis, changes its localization dynamically during spermatogenesis, and has some association with male infertility.
  
  
  
  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.
  
  
  
  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).
  
  
  
  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.
  
  
  
  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.
  
  
  
  Not applicable.
  
  
  
  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.
  
  
  
  The altered expression of hCPα3 and hCPβ3 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.
  
  
  
  This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JP16K20133). The authors declare no competing interests.
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Soda
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Miyagawa
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Ueda
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Takezawa
authors.affiliation: Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: H
authors.initials: H
authors.lastname: Okuda
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Fukuhara
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Fujita
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kiuchi
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Uemura
authors.affiliation: Okamoto Clinic, Osaka 558-0004, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Okamoto
authors.affiliation: Department of Urology, Juntendo University Urayasu Hospital, Urayasu 279-0021, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Tsujimura
authors.affiliation: Molecular Biology laboratory, Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Nagasaki 859-3298, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Nonomura
mesh_terms: D051344:Actin Capping Proteins / Q000378:metabolism*; D000328:Adult; D053627:Asthenozoospermia / Q000378:metabolism; D053713:Azoospermia / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis* / Q000378:metabolism; D008297:Male; D013091:Spermatogenesis / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*
chemical_list: D051344:Actin Capping Proteins; D015415:Biomarkers
keywords: actin capping protein; male infertility; sperm morphology; spermatogenesis; testis specific