E-Poster Presentation ESA-SRB-ANZBMS 2021

Importins: Diverse roles in male fertility explored using new and old tools (#582)

Benedict Nathaniel 1 , Penny Whiley 1 2 , Yoichi Miyamoto 3 , Kate Loveland 1 2
  1. Centre for Reproductive Health, Hudson Institute of Medical Research, Clayton, VIC, Australia
  2. Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia
  3. Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan

Regulated nucleocytoplasmic transport is central to gene expression changes that underpin cellular development and homeostasis, including in the testis, and proteins in the importin family are the predominant facilitators of cargo transport through the nuclear envelope. Earlier reports by us and others that documented cell-specific profiles of importin transcripts and proteins during spermatogenesis led us to hypothesize that importins facilitate developmental switches in the testis. Importins serve additional functions, both inside and outside the nucleus that include acting as subcellular scaffolding, mediating cellular stress responses, and controlling transcription. The importance of importin functions is further supported by multispecies studies in which importin gene knockouts showed they are variously essential for life and fertility. To expand knowledge of importin roles and their potential transport and non-transport functions in the testis, we combined new and old tools. This involved side-by-side analysis of published single cell RNAseq (scRNA-seq) data and immunohistochemistry on sections of developing and adult mouse testes. The single cell transcriptomes were delineated and compared to the intracellular and intercellular locations of importin proteins, in both germ and somatic cell populations.  The importin beta 1 transcript and its encoded protein, IMPb1, required for classical alpha/beta transport of cargo proteins into the nucleus, are abundant in maturing somatic cells after they cease mitosis, whilst in maturing germ cells, levels decline in post-mitotic spermatocytes and are absent in elongated spermatids. This highlights differential reliance on classical transport in distinct cell types. The importin alpha proteins, IMPa2 and IMPa4, display distinct nuclear and cytoplasmic localization profiles as spermatogenesis progresses. These changes in transcript expression profiles and importin protein subcellular distribution spotlight changing roles during testis development. Prospective analyses of existing human testis scRNAseq data can also identify additional avenues for impactful investigations of these conserved and highly important proteins.