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
Department of Urology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria 3800, Australia.
Hum Reprod. 2017 Mar 1;32(3):514-522. doi: 10.1093/humrep/dew353.
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.
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.
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.
STUDY FUNDING/COMPETING INTEREST(S): 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.
肌动蛋白封端蛋白（CP）β3是否参与人类精子发生及男性不育？
人类CPβ3（hCPβ3）在睾丸中表达，在精子发生过程中其定位动态变化，并且与男性不育存在一定关联。
先前已在人类中鉴定出睾丸特异性CPα亚基（CPα3），并且小鼠cpα3基因中的突变可导致精子头部畸形及男性不育。然而，被认为是CPα3异二聚体对应物的CPβ3，在人类中尚未被表征，也未报道其与男性不育相关。
研究设计、规模、持续时间：为证实CPβ3在人类睾丸中的存在，对来自已证实可育男性的新鲜精液样本进行分析。为研究精子发生过程中的蛋白质表达，通过免疫荧光分析检测从梗阻性无精子症男性获取的冷冻保存睾丸。为评估CP与男性不育的关联，我们使用免疫荧光分析比较了正常精子症男性（志愿者：正常组，n = 20）与少精子症和/或弱精子症不育男性（O + A组，n = 21）冷冻保存精子中人类CPα3（hCPα3）和hCPβ3的蛋白质表达。
参与者/材料、环境、方法：通过逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹分析研究hCPβ3的组织特异性表达。为研究hCPα3和hCPβ3是否形成异二聚体，构建了在单个质粒中包含标记有单体红色荧光蛋白1的hcpα3和标记有增强型绿色荧光蛋白的hcpβ3的串联表达载体，并通过免疫共沉淀（Co-IP）分析进行检测。使用人类生精细胞通过免疫组织化学分析检查精子发生过程中hCPα3和hCPβ3的蛋白质表达谱。通过免疫组织化学分析比较正常组和O + A组精子中hCPα3和hCPβ3的蛋白质表达。
RT-PCR显示hcpβ3的mRNA仅在睾丸中表达。蛋白质免疫印迹分析用抗牛CPβ3抗体检测到hCPβ3。重组蛋白的Co-IP分析表明hCPα3和hCPβ3形成蛋白质复合物。在精子发生的每个阶段，hCPβ3的细胞定位动态变化。在精原细胞中，hCPβ3在细胞质中显示轻微信号。hCPβ3的表达主要在精母细胞中明显，并且hCPβ3的定位从细胞质动态迁移至顶体帽和顶体。在成熟精子中.hCPβ3积聚在顶体后区域，在尾部的中段较少。双重染色分析显示在生精细胞的每个阶段hCPα3的定位与hCPβ3相同。正常组的大多数精子被hCPα3和hCPβ3均匀染色。相比之下，O + A组中与正常组相比，明显更多的精子对hCPα3或hCPβ3显示出异质性或缺乏染色（异常染色）（P < 0.001）。O + A组的异常染色百分比（52.4±3.0%）高于正常组（31.2±2.5%）。即使将观察限于根据大卫标准选择的形态学正常的精子，O + A组的异常染色百分比（39.9±2.9%）仍高于正常组（22.5±2.1%）（P < 0.001）。hCPβ3与hCPα3一起似乎在精子发生中起重要作用，并且可能与男性不育有关。
不适用。
由于收集人类睾丸新鲜样本困难，我们使用了来自睾丸精子提取的冷冻保存样本。为检查生精细胞的相互作用或在生精小管中的定位，健康男性的新鲜睾丸样本是理想的。
hCPα3和hCPβ3表达的改变不仅可能是男性不育的原因，也可能是辅助生殖技术（ART）结果的预后因素。它们可能是确定ART中人类精子受精能力的有用生物标志物。
研究资金/竞争利益：这项工作得到了日本科学促进会青年科学家（B）资助（JP16K20133）。作者声明无竞争利益。
