250. Importin α2-recognised testis cargoes; relevance to spermatogenesis

Abstract

Spermatogenesis, the morphogenetic process of generating haploid sperm capable of fertilising the female gamete, requires transport into the nucleus of transcription and chromatin remodelling factors to implement genome compaction. This transport is mediated by members of the importin (IMP) superfamily, which exhibit distinct expression patterns during male germ cell development, consistent with the idea that each IMP carries specific cargo(es) at discrete stages of spermatogenesis. To identify IMP cargoes in the testis to help identify potential developmental switches critical to the spermatogenic process, we performed a yeast two-hybrid screen using full length IMPα2 as bait and an adult mouse testis library. Binding partners identified included coilin, Hop2, Chrp (cysteine and histidine rich protein), TAF9 (TATA Binding Protein Associated Factor 9) and Cdyl (Chromodomain Y chromosome-like). These proteins may be important in cell cycle regulation, homologous chromosome pairing and recombination, transcriptional regulation, splicing, mRNA storage or histone-protamine exchange during spermatogenesis. IMPα2 interaction with these cargoes was verified by binding assays, coimmunoprecipation and cotransfection approaches, while immunohistochemical staining of rodent testis sections indicated their co-expression with IMPα2 in specific testicular cell types. A key cargo in this context is the chromatin remodelling component, Cdyl where we could show that interaction with IMPα2 (and not IMPα4 or IMPα6 which are known to be expressed in the same testicular cell types as IMPα2) is necessary for its efficient nuclear import and function in facilitating histone H4 acetylation, the prerequisite step for incorporation of protamines during the final stages of spermatogenesis. A similar approach is currently in progress to demonstrate the importance of IMPα2 for the efficient nuclear transport of the other cargoes identified for their function(s) during spermatogenesis. IMPα2-mediated nuclear localisation of specific cargoes would thus seem to represent a critical developmental switch during spermatogenesis.