Characterisation of the Transcriptome and Proteome of Residual Bodies and Uncovering of a Non-Canonical Role of the Histone Variant H2A.B3 During Spermatogenesis

Abstract

Spermatogenesis is a process where male germ cells undergo morphological changes to become motile and fertile spermatozoa. Key changes include the shrinkage of the nucleus, development of the flagellum, and extrusion of most cytoplasmic contents into the lumen of the seminiferous tubules in the form of Residual Bodies (RBs). Conventionally, RBs were thought to be phagocytosed by Sertoli cells (SCs). However, recent evidence challenges this view, highlighting a more intricate utilisation of RB contents. Specifically, H2A.B3, a spermatid-specific histone variant known for its positive regulation of germ cell transcription, is preserved in a full-length non-chromatin-bound form within RBs. Remarkably, upon RB absorption, H2A.B3 is detected in the nucleus of SCs. Intriguingly, the H2A.B3 KO mouse exhibits tubules congested with the cytoplasmic content of maturing spermatids and defective SC phenotypes. This suggests a novel epigenetic information transfer mechanism between germ and Sertoli cells mediated by RBs, where H2A.B3 potentially acts as a chaperone to transport germ cell-specific factors to SCs.

The first aim of the thesis was to establish an improved purification method for RBs from mouse seminiferous tubules. Chapter 3 introduces a continuous sucrose gradient ultracentrifugation-based method, enabling the isolation of up to 98% pure RBs from only 1 g of testis tissue. Morphological characterisation of RBs using RB-specific EMA staining and transmission electron microscopy (TEM) revealed that the tubules of H2A.B3 KO mice contain a more dispersed form of RBs with a different size distribution, suggesting delayed RB absorption by SCs and slower tubule differentiation. Furthermore, H2A.B3 KO RBs demonstrated more filled vacuoles of lysosomal nature compared to WT, implying defects in lysosomal clearance.

The second aim, explored in Chapters 4 and 5, was to comprehensively characterise the transcriptome and proteome of RBs, respectively. Using Nanopore RNA-sequencing (RNA-seq), diverse classes of biologically important RNA were identified in RBs, with a significant overrepresentation of replication-dependent histone genes in H2A.B3 KO RBs. This implies a defective chromatin compaction process, as evidenced by a higher sperm chromatin density in H2A.B3 KO mice. Additionally, H2A.B3 was found to be involved in splicing, showing a preference for first and last exon inclusion. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic study of RBs detected 3,226 proteins, which are mainly involved in protein transport and metabolism. Our investigation also reported the presence several histone proteins in RBs for the first time. Moreover, RBs contain nine unique H2A.B3-interacting proteins, the majority of which are associated with splicing.

The third aim of the thesis, explored in Chapter 6, was to investigate the role of H2A.B3 in RNA degradation. A newly developed bioinformatic pipeline was used to measure and compare the 3'- and 5'-end degradation levels of replication-dependent histone mRNA transcripts between WT and H2A.B3 KO across various spermatid stages and RBs. The 3'-end of replication-dependent histone mRNAs was the most degraded in early-round spermatids of H2A.B3 KO mice, whereas the 5'-end remained mostly unaffected, which was also validated by overexpressing the H2A.B3 protein in Neuro2a cells.

In summary, this thesis presents a modernised RB purification method, aiding in understanding RB biology and the crucial roles played by H2A.B3. The findings lay the groundwork for investigating broader hypothesis regarding epigenetic information transfer between germ cells and SCs via RBs. Additionally, a novel non-canonical role of H2A.B3 in the degradation process of replication-dependent histone mRNA is uncovered, further expanding our understanding of spermatogenesis. Show more