Whelly Sandra, Muthusubramanian Archana, Powell Jonathan, Johnson Seethal, Hastert Mary Catherine, Cornwall Gail A
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430, USA.
College of Arts and Sciences Microscopy, Texas Tech University, Canton and Main, Lubbock, TX 79409, USA.
Mol Hum Reprod. 2016 Nov;22(11):729-744. doi: 10.1093/molehr/gaw049. Epub 2016 Jul 21.
Do the CRES (cystatin-related epididymal spermatogenic) subgroup members, including CRES2, CRES3 and cystatin E2, contribute to the formation of a nonpathological, functional amyloid matrix in the mouse epididymal lumen?
CRES2, CRES3 and cystatin E2 self-assemble with different aggregation properties into amyloids in vitro, are part of a common amyloid matrix in the mouse epididymal lumen and are present in extracellular vesicles.
Although previously thought only to be pathological, accumulating evidence has established that amyloids, which are highly ordered protein aggregates, can also carry out functional roles in the absence of pathology. We previously demonstrated that nonpathological amyloids are present in the epididymis; specifically, that the reproductive cystatin CRES forms amyloid and is present in the mouse epididymal lumen in a film-like amyloid matrix that is intimately associated with spermatozoa. Because the related proteins CRES2, CRES3 and cystatin E2 are also expressed in the epididymis, the present studies were carried out to determine if these proteins are also amyloidogenic in vitro and in vivo and thus may coordinately function with CRES as an amyloid structure.
STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The epididymides from CD1 and Cst8 (CRES)129SvEv/B6 gene knockout (KO) and wild-type mice and antibodies that specifically recognize each CRES subgroup member were used for immunohistochemical and biochemical analyzes of CRES subgroup proteins. Methods classically used to identify amyloid, including the conformation-dependent dyes thioflavin S (ThS) and thioflavin T (ThT), conformation-dependent antibodies, protein aggregation disease ligand (which binds any amyloid independent of sequence) and negative stain electron microscopy (EM) were carried out to examine the amyloidogenic properties of CRES subgroup members. Immunofluorescence analysis and confocal microscopy were used for colocalization studies.
Immunoblot and immunofluorescence analyzes showed that CRES2, CRES3 and cystatin E2 were primarily found in the initial segment and intermediate zone of the epididymis and were profoundly downregulated in epididymides from CRES KO mice, suggesting integrated functions. Except for CRES3, which was only detected in a particulate form, proteins were present in the epididymal lumen in both soluble and particulate forms including in a film-like matrix and in extracellular vesicles. The use of amyloid-specific reagents determined that all CRES subgroup members were present as amyloids and colocalized to a common amyloid matrix present in the epididymal lumen. Negative stain EM, dot blot analysis and ThT plate assays showed that recombinant CRES2, CRES3 and cystatin E2 formed amyloid in vitro, albeit with different aggregation properties. Together, our studies demonstrate that a unique amyloid matrix composed of the CRES family of reproductive-specific cystatins and cystatin C is a normal component of the mouse epididymal lumen and may play a functional role in sperm maturation by coordinating interactions between the luminal fluid and spermatozoa.
LIMITATIONS, REASONS FOR CAUTION: The structures examined in our studies were isolated from luminal fluid obtained by puncture of the epididymis and therefore we cannot rule out some contamination by epithelial cells. Although our studies show CRES family members are associated with extracellular vesicles, we have yet to determine if proteins are present on the surface or are within the vesicles. We also have not established if narrow/apical cells are the source of the CRES family extracellular vesicles. CRES and CRES2 have been previously found in the human epididymis and associated with spermatozoa; however, we have yet to determine if the human CRES subgroup proteins are amyloidogenic and if an amyloid matrix is present in the human epididymal lumen.
Understanding the regulation and biological roles of amyloids, such as the CRES subgroup amyloid matrix that functions without causing pathology, could have broad implications for understanding pathological amyloids including those associated with neurodegenerative diseases and prionopathies.
None.
This work was supported by NIH grants RO1HD033903 and RO1HD056182 to G.A.C. The authors declare there are no conflicts of interest.
包括CRES2、CRES3和胱抑素E2在内的CRES（胱抑素相关附睾生精蛋白）亚组成员是否有助于在小鼠附睾管腔中形成非病理性的功能性淀粉样蛋白基质？
CRES2、CRES3和胱抑素E2在体外以不同的聚集特性自组装成淀粉样蛋白，是小鼠附睾管腔中常见淀粉样蛋白基质的一部分，并存在于细胞外囊泡中。
尽管以前认为淀粉样蛋白仅是病理性的，但越来越多的证据表明，高度有序的蛋白质聚集体——淀粉样蛋白，在无病理状态下也能发挥功能作用。我们之前证明附睾中存在非病理性淀粉样蛋白；具体而言，生殖胱抑素CRES形成淀粉样蛋白，并以与精子密切相关的膜状淀粉样蛋白基质形式存在于小鼠附睾管腔中。由于相关蛋白CRES2、CRES3和胱抑素E2也在附睾中表达，因此开展本研究以确定这些蛋白在体外和体内是否也具有淀粉样变性，从而可能与CRES作为一种淀粉样结构协同发挥作用。
研究设计、样本/材料、方法：使用来自CD1和Cst8（CRES）129SvEv/B6基因敲除（KO）小鼠及野生型小鼠的附睾，以及特异性识别每个CRES亚组成员的抗体，对CRES亚组蛋白进行免疫组织化学和生化分析。采用经典的鉴定淀粉样蛋白的方法，包括构象依赖性染料硫黄素S（ThS）和硫黄素T（ThT）、构象依赖性抗体、蛋白聚集病配体（可结合任何序列无关的淀粉样蛋白）以及负染电子显微镜（EM），来检测CRES亚组成员的淀粉样变性特性。免疫荧光分析和共聚焦显微镜用于共定位研究。
免疫印迹和免疫荧光分析表明，CRES2、CRES3和胱抑素E2主要存在于附睾的起始段和中间区，在CRES基因敲除小鼠的附睾中显著下调，提示它们具有整合功能。除了仅以颗粒形式检测到的CRES3外，这些蛋白在附睾管腔中以可溶性和颗粒性形式存在，包括在膜状基质和细胞外囊泡中。使用淀粉样蛋白特异性试剂确定所有CRES亚组成员均以淀粉样蛋白形式存在，并共定位于附睾管腔中存在的共同淀粉样蛋白基质。负染EM、斑点印迹分析和ThT平板试验表明，重组CRES2、CRES3和胱抑素E2在体外形成淀粉样蛋白，尽管具有不同的聚集特性。总之，我们的研究表明，由生殖特异性胱抑素CRES家族和胱抑素C组成的独特淀粉样蛋白基质是小鼠附睾管腔的正常组成部分，可能通过协调管腔液与精子之间的相互作用在精子成熟中发挥功能作用。
局限性、注意事项：我们研究中检测的结构是从通过穿刺附睾获得的管腔液中分离出来的，因此我们不能排除上皮细胞的一些污染。尽管我们的研究表明CRES家族成员与细胞外囊泡相关，但我们尚未确定这些蛋白是存在于囊泡表面还是囊泡内部。我们也尚未确定窄/顶端细胞是否是CRES家族细胞外囊泡的来源。CRES和CRES2先前已在人类附睾中发现并与精子相关；然而，我们尚未确定人类CRES亚组蛋白是否具有淀粉样变性，以及人类附睾管腔中是否存在淀粉样蛋白基质。
了解淀粉样蛋白的调节和生物学作用，例如无病理状态下发挥功能的CRES亚组淀粉样蛋白基质，可能对理解包括与神经退行性疾病和朊病毒病相关的病理性淀粉样蛋白具有广泛意义。
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本研究得到美国国立卫生研究院（NIH）授予G.A.C.的RO1HD033903和RO1HD056182两项基金支持。作者声明无利益冲突。
