Testicular heat stress, a historical perspective and two postulates for why male germ cells are heat sensitive.

Herein, we compare the different experimental regimes used to induce testicular heat stress and summarise their impact on sperm production and male fertility. Irrespective of the protocol used, scrotal heat stress causes loss of sperm production. This is first seen 1-2 weeks post heat stress, peaking 4-5 weeks thereafter. The higher the temperature, or the longer the duration of heat, the more pronounced germ cell loss becomes, within extreme cases this leads to azoospermia. The second, and often underappreciated impact of testicular hyperthermia is the production of poor-quality spermatozoa. Typically, those cells that survive hyperthermia develop into morphologically abnormal and poorly motile spermatozoa. While both apoptotic and non-apoptotic pathways are known to contribute to hyperthermic germ cell loss, the mechanisms leading to formation of poor-quality sperm remain unclear. Mechanistically, it is unlikely that testicular hyperthermia affects messenger RNA (mRNA) abundance, as a comparison of four different mammalian studies shows no consistent single gene changes. Using available evidence, we propose two novel models to explain how testicular hyperthermia impairs sperm formation. Our first model suggests aberrant alternative splicing, while the second model proposes a loss of RNA repression. Importantly, neither model requires consistent changes in RNA species.

Seasonal variation in bull semen quality demonstrates there are heat-sensitive and heat-tolerant bulls.

Using semen data from 1271 ejaculates (79 different bulls, 11 different breeds) we have investigated the variability of semen quality in cattle living in sub-tropical conditions. Modelling shows definitive evidence of seasonal variation. Semen quality from the same bulls had a 90% "pass rate" for cryopreservation purposes in winter, dropping to less than 50% in summer. Notably, individual bulls could be classified as either "heat-tolerant" (produce good quality spermatozoa all year regardless of temperature) or "heat-sensitive" (only produce good quality sperm in summer). Nominal logistic regression demonstrated when temperatures reach 30.5 °C, 40% of heat-sensitive bulls fail a semen analysis 17 days later. At 34 °C, the proportion of bulls failing reached 63%. Ratifying this, the purposeful heating of bulls to 40 °C for 12 h showed that individual animals had different degrees of heat-sensitivity. Using historical temperature data, we then modelled how many days/decade bulls would be subject to heat-events. Beginning from 1939 to 1949, on average, the area in which bulls were kept recorded 19, 7 and 1 day over 38 °C, 39 °C and 40 °C respectively. This number steadily increases and of last decade (2010-2010), the numbers of days per decade over 38 °C, 39 °C and 40 °C jumped to a staggering 75, 39 and 15 respectively. These data show the urgent need to identify heat-tolerant bulls as future sires. Such variation likely explains why the veterinary bull breeding test often fails to accurately predict bull breeding potential.

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 MS coupled with liquid chromatography (LC-MS/MS), we found 142 nonreductant 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), a TCA cycle enzyme, was the only protein to show an increase in Sia content during capacitation. Interestingly, although the loss of Sia within EL (N62) was accompanied by a reduction in its phospholipase A1 activity, a decrease in the activity of ACO2 (i.e. stereospecific isomerization of citrate to isocitrate) occurred when sialylation increased (N612). The latter was confirmed by N612D recombinant protein tagged with both His and GFP. The replacement of Sia for the negatively charged Aspartic acid in the N612D mutant caused complete loss of aconitase activity compared with the WT. Computer modeling show that N612 sits atop the catalytic site of ACO2. The introduction of Sia causes a large conformational 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.

Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology.

Male infertility is widespread and estimated to affect 1 in 20 men. Although in some cases the etiology of the condition is well understood, for at least 50% of men, the underlying cause is yet to be classified. Male infertility, or subfertility, is often diagnosed by looking at total sperm produced, motility of the cells and overall morphology. Although counting spermatozoa and their associated motility is routine, morphology assessment is highly subjective, mainly because of the procedure being based on microscopic examination. A failure to diagnose male-infertility or sub-fertility has led to a situation where assisted conception is often used unnecessarily. As such, biomarkers of male infertility are needed to help establish a more consistent diagnosis. In the present study, we compared nuclear extracts from both high- and low-quality spermatozoa by LC-MS/MS based proteomic analysis. Our data shows that nuclear retention of specific proteins is a common facet among low-quality sperm cells. We demonstrate that the presence of Topoisomerase 2A in the sperm head is highly correlated to poor head morphology. Topoisomerase 2A is therefore a potential new biomarker for confirming male infertility in clinical practice.

Proteomic analysis of good- and poor-quality human sperm demonstrates that several proteins are routinely aberrantly regulated.

Male infertility is a complex condition, and for the most part, all men produce defective spermatozoa, but infertile men have a tendency to produce more. Despite attempts to classify infertility, there is no definitive test. One approach would be to use protein biomarkers; however as yet, we still do not understand proteins that are differentially expressed within defective spermatozoa. As such, we took nine men (fertility status unknown) and used Percoll density gradients to isolate a population of good- and poor-quality sperm. For four of these men, we also obtained multiple ejaculations. The most noticeable differences between the Percoll-isolated fractions were motility and CMA3 staining. While the good sperm fraction produced cells with at least 80% forward progressive motility and low levels of CMA3 staining, the poor-quality sperm demonstrated less than 10% forward progressive motility and higher levels CMA3 staining. Using the technique of sequential window activation of all theoretical mass spectra, we quantified 2774 proteins and found 171 proteins to be significantly more abundant in the good sperm fraction, while 104 proteins were significantly more abundant in the lower sperm fraction (adjusted Benjamini-Hochberg significance of P < 0.018, minimum 2-fold difference).