de Pauli Larissa Fiamengui, Santos Elisa Gomes, Daher Arcangelo Fernanda Pazotti, Orcini Wilson Aparecido, Peruquetti Rita Luiza
Health Science Center, Universidade do Sagrado Coração (USC), Bauru, São Paulo, Brazil.
Laboratory of Molecular Biology and Citogenetics, Universidade do Sagrado Coração (USC), Bauru, São Paulo, Brazil.
Micron. 2017 Mar;94:37-45. doi: 10.1016/j.micron.2016.12.004. Epub 2016 Dec 14.
Chromatoid body (CB) is a cytoplasmic structure of male germ cells that has been indicated as having a role in the RNA and protein storage for the final differentiation of spermatozoa. Recent studies have indicated that some of these macromolecular complex components have nucleolar origin. The aims of the present study were to monitor the expression of fibrillarin nucleolar protein in mammalian seminiferous tubules at different stages of the spermatogenic cycle; to check fibrillarin distribution during the CB assembly; and also its interaction with two well-known CB markers (MIWI and HSP70). Seminiferous tubules were isolated by transilluminating microscope from testis of adult mice. Fibrillarin expression and also co-localization between fibrillarin and MIWI/HSP70 were performed by Western blot (WB) and by immunofluorescence (IF), respectively. Total proteins from testis of adult mice were also used to perform co-immunoprecipitation (Co-IP) experiments. Our results demonstrated higher fibrillarin expression in seminiferous tubules in stages IV-VI, and a close localization of fibrillarin with MIWI (a protein that plays a role in RNA metabolism in the CB), as well as with HSP70 (a protein that plays a role in the proteasome folding in the CB). We also performed Co-IP between fibrillarin/MIWI and between fibrillarin/HSP70 in order to determine whether MIWI or HSP70 interacts with this nucleolar protein. We found MIWI in the Co-IP precipitate, but not HSP70. In conclusion, our results show that fibrillarin may participate in the physiological activities performed by the CB by interacting with CB components that play a role in RNA metabolism.
拟染色体（CB）是雄性生殖细胞的一种细胞质结构，已被表明在精子最终分化的RNA和蛋白质储存中发挥作用。最近的研究表明，这些大分子复合物的一些成分起源于核仁。本研究的目的是监测生精周期不同阶段哺乳动物生精小管中核仁蛋白纤维蛋白原的表达；检查纤维蛋白原在CB组装过程中的分布；以及它与两种著名的CB标记物（MIWI和HSP70）的相互作用。通过透射显微镜从成年小鼠的睾丸中分离出生精小管。分别通过蛋白质免疫印迹（WB）和免疫荧光（IF）检测纤维蛋白原的表达以及纤维蛋白原与MIWI/HSP70之间的共定位。成年小鼠睾丸的总蛋白也用于进行免疫共沉淀（Co-IP）实验。我们的结果表明，在IV-VI期生精小管中纤维蛋白原表达较高，并且纤维蛋白原与MIWI（一种在CB的RNA代谢中起作用的蛋白质）以及与HSP70（一种在CB的蛋白酶体折叠中起作用的蛋白质）紧密定位。我们还进行了纤维蛋白原/MIWI之间以及纤维蛋白原/HSP70之间的免疫共沉淀实验，以确定MIWI或HSP70是否与这种核仁蛋白相互作用。我们在免疫共沉淀沉淀物中发现了MIWI，但没有发现HSP70。总之，我们的结果表明，纤维蛋白原可能通过与在RNA代谢中起作用的CB成分相互作用，参与CB所执行的生理活动。
