A Population of CRES Resides in the Outer Dense Fibers of Spermatozoa
A Population of CRES Resides in the Outer Dense Fibers of Spermatozoa
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DOI：
10.1095/biolreprod.112.104745
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发表时间：
2013-03-01
影响因子：
3.6
通讯作者：
Oko, Richard
中科院分区：
文献类型：
--
作者：
Ferrer, Marvin;Cornwall, Gail;Oko, Richard
胱氨酸蛋白酶抑制剂相关的附睾精子发生蛋白（克雷斯，也称为CST 8）在睾丸和附睾中表达，并发现与精子相关。它表现为非糖基化（14和12 kDa）和糖基化亚型（19和17 kDa）。克雷斯的作用仍然是谜，是依赖于其异构体的本地化，这是本研究的目的。我们最初的方法是通过免疫组织化学和免疫金电子显微镜来研究这些亚型的睾丸起源。我们证实了先前的报道，即克雷斯在第8步至第16步的伸长精子细胞的细胞质中表达。然而，我们注意到睾丸表达的模式让人联想到外致密纤维（ODF）和纤维鞘（FS）蛋白。附睾尾精子提取物的Western印迹分析显示非离子型，洗涤剂不溶性的14-kDa克雷斯亚型。为了进一步精确定位和确认克雷斯定位，我们分离了精子头部和尾部，并用逐渐更苛刻的蛋白质增溶剂提取尾部。这些连续提取物的Western印迹，旨在逐步去除线粒体鞘和ODF或FS，检测到CRES免疫反应性14 kDa的带与附属纤维的尾巴。免疫金标记主要集中在睾丸中生长的ODF上，并持续存在于精子中。这项研究发现了一个克雷斯异构体，组装的一部分，在精子发生的延伸和成熟阶段的ODF，并保留作为一个共价结合的组成部分，在精子中的ODF。
Cystatin-related epididymal spermatogenic protein (CRES, also CST8) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as nonglycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES remains enigmatic and is dependent on localization of its isoforms, which is the objective of this study. Our initial approach was to investigate testicular origins of these isoforms by immunohistochemistry and immunogold electron microscopy. We confirmed previous reports that CRES was expressed in the cytoplasm of elongating spermatids from step 8 to step 16. However, we noticed that the pattern of testicular expression was reminiscent of outer dense fiber (ODF) and fibrous sheath (FS) proteins. Western blot analysis of extracts of cauda epididymal sperm revealed a nonionic, detergent-insoluble 14-kDa CRES isoform. To further pinpoint and confirm CRES localization we separated sperm heads and tails and extracted the tails with progressively harsher protein solubilizing agents. Western blots of these sequential extracts, designed to progressively remove the mitochondrial sheath and the ODFs or FS, detected a CRES-immunoreactive 14-kDa band associated with the accessory fibers of the tail. Immunogold labeling was concentrated over growing ODFs in the testes and persisted in spermatozoa. This study discovers a CRES isoform that assembles as part of the ODFs during the elongation and maturation phases of spermiogenesis and is retained as a covalently bound component of the ODFs in spermatozoa.