Rachel A. Ogle

Objective: To understand the immediate impact that testicular heat stress has on isolated populations of precursor male germ cells including Spermatocytes and Spermatids. Design: Mice were given testicular heat stress and pre-cursor male germ cells were immediately isolated. RNA sequencing was performed and validated using qPCR. Subjects: This work was carried out in adult male CD1 mice. Results: Using 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. Conclusion: Testicular 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.

Testicular heat stress is a well described phenomenon that occurs in mammals that possess a scrotum. Different models to induce testicular hyperthermia, such as surgical cryptorchidism, hot water bath, scrotal insulation or increased environmental temperature have all shown that spermatocytes and spermatids are unambiguously affected by high temperature, resulting in poor sperm production weeks later. Furthermore, a body of evidence suggest the involvement of oxidative stress is either a major or contributory pathway, which gives rise to the potential to overcome this condition. Whilst experimental models conclusively show the deleterious effect of testicular heat on sperm quality, the physiological relevance of the work is still debated. Herein we summarise a cohort of studies that report the effect of “season” on sperm quality. The data show season can affect sperm production, motility and morphology depending on where the work was performed. In countries where temperatures drop below zero, there is evidence showing summer conditions tend to improve semen quality. However, in sub-tropical countries, some studies show a decrease in summer, whilst others show no change. Herein we offer a reasonable explanation for this apparent controversy and present a range of antioxidant supplements that may offer some protection against testicular hyperthermia.

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 A ₁ 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.