Statistics and quantification dataset of Haspin and its related proteins analysis in mouse spermatocytes

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and therefore must be precisely regulated. One of the main centromeric regulatory signaling pathways is the Haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. In mitosis, Haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, whose catalytic component is Aurora B kinase. However, the centromeric Haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed Haspin functions by either its chemical inhibition in cultured spermatocytes using LDN-192960, or the ablation of Haspin gene in Haspin-/-. Our studies suggest that Haspin kinase activity is required for proper chromosome congression during both meiotic divisions and for the recruitment of Aurora B and kinesin MCAK to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter Borealin and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the Haspin-H3T3ph-CPC pathway and centromere function during meiosis.

Methods
Quantification of the immunofluorescence intensity was estimated by measuring the integrated fluorescence density in individual nuclei using ImageJ by creating a binary mask with the DAPI staining. The acquisition time was fixed for all acquired images, and the quantification was only performed using the original unmodified images. A minimum of 10 metaphase spermatocytes per condition were analyzed in each experiment. All graphics and statistical tests were performed with GraphPad Prism 9.0 software. Mean differences for each group were evaluated by an independent sample unpaired t-test. Values were expressed as mean ± SEM and p-values below 0.05 were considered statistically significant.