Nguyen Edward B, Westmuckett Andrew D, Moore Kevin L
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.
Biol Reprod. 2014 Jan 30;90(1):16. doi: 10.1095/biolreprod.113.111831. Print 2014 Jan.
Sperm acrosome associated 7 (SPACA7) is a novel protein of unknown function with no homology to any known protein. Spaca7 transcripts are detected only in testis and predict a 158-residue mature polypeptide with one potential N-glycosylation site and no cysteines. Orthologs are present in various species, including mice and humans. We developed a polyclonal antibody to mouse SPACA7 to study its expression and function. Western blotting and immunofluorescence microscopy detected SPACA7 only in testis, and it was detected in testis starting at Postnatal Day 21 and into adulthood. Immunofluorescence staining of testicular germ cells detected weak SPACA7 expression as early as zygotene spermatocytes. Higher expression was observed in round spermatids, where SPACA7 was localized to a perinuclear spot adjacent to the Golgi and to the acrosome of elongating spermatids and spermatozoa. Immunogold electron microscopy demonstrated that SPACA7 is localized within the proacrosomal granule of round spermatids and the acrosome of spermatozoa. Finally, we showed that SPACA7 was retained within the acrosome of epididymal sperm and was released upon the acrosome reaction. To assess if SPACA7 was involved in fertilization, in vitro fertilization assays in the presence of anti-SPACA7 IgG were performed. Anti-SPACA7 inhibited fertilization of cumulus-intact eggs and prominently delayed cumulus dispersal. However, anti-SPACA7 did not inhibit fertilization of cumulus-free eggs. Our findings indicate that release of SPACA7 from the acrosome accelerates cumulus dispersal and facilitates fertilization via unknown mechanisms. This study is the first to document the expression of endogenous SPACA7 and a function for this novel acrosomal protein.
精子顶体相关蛋白7（SPACA7）是一种功能未知的新型蛋白质，与任何已知蛋白质均无同源性。仅在睾丸中检测到Spaca7转录本，预测其成熟多肽有158个氨基酸残基，有一个潜在的N-糖基化位点，且无半胱氨酸。在包括小鼠和人类在内的各种物种中都存在直系同源物。我们制备了针对小鼠SPACA7的多克隆抗体，以研究其表达和功能。蛋白质免疫印迹法和免疫荧光显微镜检查仅在睾丸中检测到SPACA7，且在出生后第21天直至成年期的睾丸中均可检测到。对睾丸生殖细胞进行免疫荧光染色，最早在偶线期精母细胞中检测到较弱的SPACA7表达。在圆形精子细胞中观察到较高的表达，其中SPACA7定位于高尔基体附近的核周斑点以及伸长的精子细胞和精子的顶体。免疫金电子显微镜显示，SPACA7定位于圆形精子细胞的前顶体颗粒和精子的顶体内。最后，我们发现SPACA7保留在附睾精子的顶体内，并在顶体反应时释放。为了评估SPACA7是否参与受精，我们进行了在抗SPACA7 IgG存在下的体外受精试验。抗SPACA7抑制了完整卵丘卵母细胞的受精，并显著延迟了卵丘扩散。然而，抗SPACA7并未抑制无卵丘卵母细胞的受精。我们的研究结果表明，SPACA7从顶体释放可加速卵丘扩散，并通过未知机制促进受精。本研究首次记录了内源性SPACA7的表达以及这种新型顶体蛋白的功能。
