Abstract
Spermiogenesis is a postmeiotic process that drives development of round spermatids into fully elongated spermatozoa. Spermatid elongation is largely controlled post-transcriptionally after global silencing of mRNA synthesis from the haploid genome. Here, rats that differentially express EGFP from a lentiviral transgene during early and late steps of spermiogenesis were used to flow sort fractions of round and elongating spermatids. Mass-spectral analysis of 2D gel protein spots enriched >3-fold in each fraction revealed a heterogeneous RNA binding proteome (hnRNPA2/b1, hnRNPA3, hnRPDL, hnRNPK, hnRNPL, hnRNPM, PABPC1, PABPC4, PCBP1, PCBP3, PTBP2, PSIP1, RGSL1, RUVBL2, SARNP2, TDRD6, TDRD7) abundantly expressed in round spermatids prior to their elongation. Notably, each protein within this ontology cluster regulates alternative splicing, sub-cellular transport, degradation and/or translational repression of mRNAs. In contrast, elongating spermatid fractions were enriched with glycolytic enzymes, redox enzymes and protein synthesis factors. Retrogene-encoded proteins were over-represented among the most abundant elongating spermatid factors identified. Consistent with these biochemical activities, plus corresponding histological profiles, the identified RNA processing factors are predicted to collectively drive post-transcriptional expression of an alternative exome that fuels finishing steps of sperm maturation and fitness.
Highlights
Post-transcriptional regulation of gene expression is essential for cells to transition into and out of distinct developmental, physiological, and pathological states (1– 4)
It should be stressed that linear “steps” in germ cell development are different from the “stages” of the seminiferous epithelium cycle (24)
Spermiogenic Expression of EGFP in Rats—Novel strains of transgenic rats were generated by transducing donor spermatogonial stem cells with a self-inactivating, lentiviral vector (33), and evaluated as animal models for studying sperm development
Summary
Post-transcriptional regulation of gene expression is essential for cells to transition into and out of distinct developmental, physiological, and pathological states (1– 4). Newly formed round spermatids undergo up to 19 well-defined steps of spermiogenesis before being shed into the lumen of the seminiferous epithelium as fully elongated, yet functionally immature spermatozoa (reviewed by Yves Clermont) (24). It should be stressed that linear “steps” in germ cell development are different from the “stages” of the seminiferous epithelium cycle (24) This is because the epithelial stages are defined by physical associations formed between different testis cell types during an epithelial cycle (see Supplemental Fig. 1 for review). Developmental gaps between each generation of germ cells comprising a given stage is defined by the time taken to complete one cycle of the seminiferous epithelium (i.e. ϳ12.9 days/cycle in rats) (24). Each epithelial stage merely represents subsequent snap-shots in cycle time within the same seminiferous tubule segment (Supplemental Fig. 1)
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