Bernstein, I. R. | bioRxiv Science

bioRxiv2026

A proteomic and phosphoproteomic comparison of mouse spermatogonial stem cells and progenitor spermatogonia

In this manuscript, we used an Id4-eGfp mouse line to produce the first proteomic and phosphoproteomic database for mouse spermatogonial stem cells (SSCs) and progenitor spermatogonia. Our proteomic analyses identified 8,464 proteins in spermatogonia, superseding the depth of previously published spermatogonia datasets by >2000 proteins. While the comparison of SSCs and progenitors revealed few unique proteins (18 and 3, respectively), 532 proteins exhibited significantly different abundance (FC {+/-} 1.5, p-value [≤] 0.05) between these sub-populations. Interestingly, in overlaying the proteome with transcriptomic data, correlation was poor (R2 = 0.236), re-iterating discordance between transcript and protein abundance in the testis. In our phosphoproteomic analyses, phosphosites were identified in 19.5% of proteins (3,604 total phosphosites). Unique protein phosphorylation was substantially more common than unique protein expression when comparing SSCs and progenitors, with 38 and 191 unique phosphosites identified, respectively, in addition to significant differences in abundance at 60 and 257 phosphosites. In identifying a wave of phosphorylation that accompanies the progenitor transition, we performed predictive analyses to identify three potential master kinases for follow up validation studies (PAK1, BUB1, ABL2). The inhibition of these kinases resulted in a significant reduction in the capacity for progenitor spermatogonia to differentiate, and caused elevated apoptosis and DNA damage. Finally, we explored the testis phenotype of 42 knockout mouse lines for proteins that were either differentially expressed (26) or differentially phosphorylated (15) in our dataset. Of these lines, 21 exhibited a testis phenotype in at least one of two biological replicates observed (severity score [≥] 1, compared to 0.15 for controls), with increased numbers of Sertoli-only tubules being evident in four of these lines. This manuscript provides a comprehensive roadmap to understand the multifaceted layers of regulation over fate decisions in undifferentiated spermatogonia. These data have been provided in an accessible platform via our ShinySpermatogoniaCells app to encourage future progress in the field.

cell biology↗

bioRxiv2024

Sub-chronic elevation in ambient temperature drives alterations to the sperm epigenome and accelerates early embryonic development in mice

Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of humans and animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked negative effect on the male reproductive system, and particularly the production of spermatozoa, less is known about the immediate impact on male reproductive function following sub-chronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, we exposed unrestrained male mice to heat stress conditions that emulate a heat wave (daily cycle of 8_h at 35{degrees}C followed by 16 h at 25{degrees}C) for a period of seven days. Neither the testes or epididymides of heat exposed male mice exhibited evidence of gross histological change, and similarly, spermatozoa of exposed males retained their functionality and ability to support embryonic development. However, the embryos generated from heat exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching and increased fetal weight. Such changes were causally associated with an altered sperm small non-coding RNA (sncRNA) profile, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat exposed spermatozoa. Such data highlight that even a relatively modest excursion in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress. Significance StatementThe fidelity of sperm production underpins successful reproduction yet is highly vulnerable to various forms of environmental challenge, including heat stress. Despite this knowledge, we lack a complete understanding of the immediate impact on male reproduction of whole-body exposure to elevated ambient temperatures such as those encountered during a heatwave. By experimentally emulating heatwave conditions, we demonstrate that the spermatozoa of exposed male mice accumulate changes in their small RNA profile that are causally linked to pronounced changes in embryonic gene expression, accelerated pre-implantation development, aberrant blastocyst hatching, and increased fetal weight. Such data highlight that even a relatively modest alteration in ambient temperature can affect male reproductive function, demonstrating the acute sensitivity of sperm small RNAs to environmental stress.

cell biology↗