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).

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.

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STUDY QUESTION What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. SUMMARY ANSWER The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. WHAT IS KNOWN ALREADY Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. STUDY DESIGN, SIZE, DURATION We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. PARTICIPANTS/MATERIALS, SETTING, METHODS We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. LIMITATIONS, REASONS FOR CAUTION The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. WIDER IMPLICATIONS OF THE FINDINGS CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. STUDY FUNDING/COMPETING INTEREST(S) The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

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.

The free-radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid-peroxidation product 4-hydroxynonenal (4-HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4-HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4-HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics-based findings propose a revised understanding of oxidative stress within the male reproductive tract.

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.

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), a TCA cycle enzyme, 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, 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 to the WT. Computer modelling 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.