Biology subjects

Netherton, J. K.

Publications and source records attributed to Netherton, J. K..

3 recordsLinked to original sources

bioRxiv2025

Acute testicular hyperthermia leads to a rapid loss of global piRNA levels and a consequent increase in transcript abundance, including LINE1 activity within heat-sensitive male germ cells.

ObjectiveTo understand the immediate impact that testicular heat stress has on isolated populations of precursor male germ cells including Spermatocytes and Spermatids. DesignMice were given testicular heat stress and pre-cursor male germ cells were immediately isolated. RNA sequencing was performed and validated using qPCR. SubjectsThis work was carried out in adult male CD1 mice. ResultsUsing next-generation RNA sequencing 134 differentially expressed transcripts were found to be differentially expressed upon exposure to testicular hyperthermia, 93% of which were upregulated. In addition, testicular hyperthermia induced 395 differential splicing events and altered the usage of 61 polyadenylation sites. To explain these observations and understand why transcript abundance appears to favour upregulation following testicular hyperthermia, we assessed global piRNA levels and found an overall, rapid reduction. Concomitantly, we observed an increase in transposable element RNA (LINE1) and protein (ORF1p) abundance. Furthermore, increased LINE1 expression appeared to be correlated with DNA damage in the male germline. At 24 hours post heat stress, piRNA levels recovered close to control levels, coincident with a significant reduction in LINE1 transcript expression within spermatocytes. ConclusionTesticular hyperthermia needs to be considered in the context of all reproductive outcomes. Affected spermatozoa are likely to be genetically compromised, leading to adverse outcomes such as infertility or loss of embryo following fertilization.

molecular biology↗

bioRxiv2024

Comparison of the CDC2-like kinase family across eukaryotes highlights the functional conservation of these unique biological thermometers

The family of CDC2-like kinases (CLKs) play a crucial role in regulating alternative splicing (AS), a process fundamental to eukaryotic gene expression and adaptation. Of particular interest, these enzymes exhibit unique responsiveness to minor temperature shifts, enabling them to modulate AS accordingly. Dysregulated CLK expression is linked to a wide variety of human diseases, establishing them as promising therapeutic targets. Despite the importance of CLKs, limited research has explored the genetic and functional diversification of this gene family. This report investigates the evolutionary origins, diversification, and functional implications of CLKs across major eukaryotic lineages through phylogenetic and structural comparisons. Our data demonstrate these kinases are prevalent throughout eukaryotes, with the original gene (which shares orthology to human CLK2), dating back to the Last Eukaryotic Common Ancestor. We identified three key duplication events in vertebrates, highlighting how this gene family has expanded and diversified in complex metazoans. Despite two instances of CLK paralog loss in vertebrate lineages, CLKs remain prevalent throughout metazoans, suggesting they are essential for complex eukaryotic life. Structural comparisons across diverse eukaryotes demonstrate kinase domain conservation, which is in line with their maintained function in AS regulation. While their N-terminal regions vary significantly in amino acid sequence, the function of this domain to regulate phosphorylation of AS factors is conserved, albeit in a species-specific manner. CLKs exhibit unique thermo-sensitive properties across diverse species, challenging conventional enzymatic behaviour. This temperature regulation, mediated by their kinase activation segment, is characterised by increased activity at lower physiological temperatures. The conservation of this structure, and a thermo-sensitive amino acid motif within it, suggests this was an ancient adaptation for responding to environmental cues. Species-specific temperature profiles highlight the adaptive evolution of CLKs, enabling organisms to thrive in diverse environmental conditions including extreme temperatures. Our analysis expands the understanding of CLK biology across diverse eukaryotes and connects insights from model organisms to human biology.

evolutionary biology↗

bioRxiv2020

Sialylation of Asparagine 612 inhibits Aconitase activity during mouse sperm capacitation; A possible mechanism for the switch from oxidative phosphorylation to glycolysis

After ejaculation, mammalian spermatozoa must undergo a process known as capacitation in order to successfully fertilize the oocyte. Several post-translational modifications occur during capacitation, including sialylation, which despite being limited to a few proteins, seems to be essential for proper sperm-oocyte interaction. Regardless of its importance, to date, no single study has ever identified nor quantified which glycoproteins bearing terminal sialic acid (Sia) are altered during capacitation. Here we characterize sialylation during mouse sperm capacitation. Using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS), we found 142 non-reductant peptides, with 9 of them showing potential modifications on their sialylated oligosaccharides during capacitation. As such, N-linked sialoglycopeptides from C4b-binding protein, endothelial lipase (EL), serine proteases 39 and 52, testis-expressed protein 101 and zonadhesin were reduced following capacitation. In contrast, mitochondrial aconitate hydratase (aconitase; ACO2) was the only protein to show an increase in Sia content during capacitation. Interestingly, while the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, the increase of sialylation in the ACO2 (N612) also resulted in a decrease of the activity of this TCA cycle enzyme. The latter was confirmed by N612D recombinant protein with both His and GFP tag, in which the N612D mutant had no activity compared to WT when protein. Computer modelling show that N612 sits atop the catalytic site of ACO2. The introduction of sialic acid causes a large confirmation change in the alpha helix, essentially, distorting the active site, leading to complete loss of function. These findings suggest that the switch from oxidative phosphorylation, over to glycolysis that occurs during capacitation may come about through sialylation of ACO2.

developmental biology↗