During the process of spermatogenesis, male germ cells must undergo morphological changes in order to become motile and fertile spermatozoa. As a part of this process nucleus shrinks, flagellum develops, and majority of cytoplasmic contents is extruded into the lumen of the seminiferous tubules in the form of residual bodies (RBs). It is believed that the RBs are subsequently phagocytosed by Sertoli cells (SCs), and fully degraded generating ATP. Contrasting evidence, however, points out to a more delicate utilization of RBs’ contents. In particular, H2A.B3, a spermatid-specific histone variant that positively regulates germ cell transcription, is well preserved in a non-chromatin-bound form within RBs and is detected in the SC nucleus after RB absorption. Intriguingly, the H2A.B3 knock-out mouse model developed in our laboratory shows tubules that are clogged with cytoplasmic content of maturing spermatids, and SCs exhibit a defective phenotype. Therefore, we set out to investigate how RB contents are used by the SCs and the role of H2A.B3 throughout the process.
As a first step, we decided to identify the full proteome and transcriptome of the RBs. For that, a sucrose gradient-based ultracentrifugation method was developed in our laboratory to isolate extra pure fractions of RBs from wild type and H2A.B3 KO mouse testes. To characterize the RNA and protein contents of RBs, Oxford nanopore technology (ONT)-based long read RNA sequencing and liquid chromatography-based mass spectroscopy (LC-MS/MS) techniques were applied, respectively.
Our findings revealed several new aspects of the RB biology. Amongst these, we have characterised the bio-types and origin of RNA species that reside in the RB, and have found a correlation between RNA and corresponding protein species that could reflect dynamics of RNA translation by the maturing germ cells. These and other aspects of Residual Body biology will be presented.