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.

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.

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4-Hydroxynonenal Adducted Proteins in Human Sperm.

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.

Quantitative Glycopeptide Changes in Rat Sperm During Epididymal Transit.

Mammalian spermatozoa acquire fertilizing potential as they undergo a series of changes during epididymal transit. One major facet of such is the alterations in the sperm glycome. Modifications of the sialic acid content within glycan moieties are known to regulate epitope presentation and cellular adhesion and signaling, all of which may be critical for sperm to successfully reach and fertilize the egg. To date, there is paucity of information regarding the sialic acid changes that occur on spermatozoa during epididymal transit. Therefore, the aim of this study was to identify N-linked sialylated glycoproteins in rat epididymal sperm and investigate whether they are regulated during epididymal transit. Sialylated glycopeptides from caput, corpus, and cauda spermatozoa were enriched using titanium dioxide beads. Bound N-linked glycopeptides were released by enzymatic deglycosylation using PNGase F and then analyzed by liquid chromatography tandem-mass spectrometry. A total of 92 unique N-linked sialylated glycopeptides were identified from 65 different proteins. These included members of the disintegrin and metalloproteinase domain-containing protein family (ADAM), Basigin, and Testis-expressed protein 101 (TEX101). Remarkably, label-free quantification showed that more than half of these peptides (48/92) were regulated during epididymal transit. Of interest, the protein TEX101 exhibited PNGase F-resistant deglycosylation under the conditions used in this study. The results from this study showed that changes in the N-linked sialoglycoprotein profile is a major hallmark of sperm maturation in rats.

Analysis of protein thiol changes occurring during rat sperm epididymal maturation.

The maturation of spermatozoa throughout the epididymal environment occurs in the complete absence of nuclear protein biosynthesis. As such, these cells rely heavily on posttranslational modifications of existing proteins in order to obtain the potential for fertilization. We have used an OxiCat approach to label both free and oxidized cysteine residues in rat sperm proteins and compared the ratio of reduced:oxidized peptides as these cells undergo epididymal transit. In all, 20 peptides, corresponding to 15 proteins, underwent a change in their redox status. Included in this list were A-kinase anchoring protein 4 and fatty acid-binding protein 9. Both of these proteins undergo intradisulfide bonding, leading to reduced solubility and, in the case of the latter, is likely to cause a loss of protein function. Interestingly, two glycolytic enzymes, hexokinase-1 and lactate dehydrogenase, also display increased cysteine oxidation during epididymal transit, which may be involved in the regulation of the enzyme activities.

Defining the mechanisms by which the reactive oxygen species by-product, 4-hydroxynonenal, affects human sperm cell function.

Lipid peroxidation products such as the naturally occurring aldehyde 4-hydroxynonenal (4-HNE) are known to be cytotoxic toward different cell types, including spermatozoa. In order to understand this at the molecular level, we have employed a proteomic approach to characterize direct 4-HNE adducts on human spermatozoa. Several proteins were identified to be of particular interest, including aldehyde labeling of histone methyltransferase and dynein heavy chain. In addition, we found that 4-HNE bound to part of the activation segment, cysteine residue 199, of protein kinase A (PKA). Interestingly, at low levels, addition of 4-HNE had a stimulatory effect on PKA. However, this did not correlate to increased phosphotyrosine levels during capacitation. This data explains the link between reactive oxygen species and sperm toxicity. Given that epigenetic regulation is likely affected in oxidative-stressed spermatozoa, this data show that spermatozoa appear to shut down under these conditions before reaching the egg.

Effects of high-fat diet-induced diabetes on autophagy in the murine liver: A systematic review and meta-analysis.

AIMS: We conducted a meta-analysis to investigate whether diabetes induced by a high-fat diet (HFD) has the potential to alter the process of autophagy in the murine liver. METHODS: A systematic literature search was performed with electronic databases (PubMed, EMBASE, Web of Science). Study design, population, intervention, outcome, and risk of bias were analyzed. Given the availability of studies, a quantitative meta-analysis including 23 studies was performed. KEY FINDINGS: The search found 5754 articles, with 48 matching the eligibility criteria, comprising of 1033 animals. The meta-analysis showed that diabetic murines fed with HFD presented an absence of p62 degradation (SMD 4.63, 95 % CI 2.02 to 7.24, p = 0.0005; I2 = 77 %), higher expression of p-mTOR/mTOR (SMD 5.20, 95 % CI 1.00 to 9.39, p = 0.01; I2 = 78 %), and a decreased p-AMPK/AMPK ratio (SMD -2.02, 95 % CI -3.96 to -0.09, p = 0.04; I2 = 85 %) when compared to nondiabetic murines. When associated with streptozotocin, the animals presented decreased ATG-7 and LC3-II. The meta-regression results showed a decrease in autophagy responses due to increased glycemic levels, fat content, and long-term exposure to HFD, and advanced animal age. The common and species-specific protein responses were also consistent with the inhibition of autophagy. SIGNIFICANCE: The normal process of autophagy mechanisms in the liver is less competent after HFD consumption. The destabilization of (auto)phagolysosomes contributes to the perpetuation of diabetes, metabolic dysfunction-associated fatty liver disease, and cell death.

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.

Nonpregnant and pregnant adult female rats affected by maternal diabetes environment.

Maternal diabetes-mediated fetal programming is widely discussed, however, it is important to define the extent to which intrauterine hyperglycemia interferes with the health of female pups, along with determining whether these changes can be perpetuated across generations. This study aimed to evaluate the effects of maternal diabetes on fetal programming and the repercussions on the metabolism of pregnant and nonpregnant female pups. Diabetes status was induced (diabetic group-D) using streptozotocin (a beta cell cytotoxic drug) on the fifth postnatal day of female rats, while controls received a citrate buffer (Control-C). In adulthood, the rats were mated to obtain their female pups. At 90 days of age, half of the female pups were mated (preg) and the other half continued virgin (Npreg). Furthermore, they were distributed into four groups: OC/Npreg and OC/preg-female pups from control mothers; OD/Npreg and OD/preg-female pups from diabetic mothers. At 115 days of life and/or 17 days of pregnancy, the oral glucose tolerance test (OGTT) was performed with blood collection for insulin measurement. At 120 days of life and/or 21 days of pregnancy, the rats were anesthetized and euthanized to determine their blood oxidative stress status. The OD/Npreg group showed glucose intolerance during OGTT (p < 0.0001), while the OD/preg group showed increased insulin secretion during OGTT (p < 0.0001) and insulin resistance (IR; p = 0.0027). An increase in homeostatic model assessment ß was shown in the pregnant groups, regardless of maternal diabetes (p < 0.0001). The OD/preg group presented increased thiobarbituric acid reactive substances (p < 0.0001) and -SH levels (p = 0.0005) and decreased superoxide dismutase activity (p = 0.0063). Additionally, small fetuses for gestational age (p < 0.0001) were found in these rats. In conclusion, exposure to maternal hyperglycemia compromises the glycemic metabolism of female pups before and during pregnancy and causes oxidative stress, IR, and impaired fetal growth during pregnancy.

From Past to Present: The Link Between Reactive Oxygen Species in Sperm and Male Infertility.

Reactive oxygen species (ROS) can be generated in mammalian cells via both enzymatic and non-enzymatic mechanisms. In sperm cells, while ROS may function as signalling molecules for some physiological pathways, the oxidative stress arising from the ubiquitous production of these compounds has been implicated in the pathogenesis of male infertility. In vitro studies have undoubtedly shown that spermatozoa are indeed susceptible to free radicals. However, many reports correlating ROS with sperm function impairment are based on an oxidative stress scenario created in vitro, lacking a more concrete observation of the real capacity of sperm in the production of ROS. Furthermore, sample contamination by leukocytes and the drawbacks of many dyes and techniques used to measure ROS also greatly impact the reliability of most studies in this field. Therefore, in addition to a careful scrutiny of the data already available, many aspects of the relationship between ROS and sperm physiopathology are still in need of further controlled and solid experiments before any definitive conclusions are drawn.

Blood and seminal plasma concentrations of selenium, zinc and testosterone and their relationship to sperm quality and testicular biometry in domestic cats.

The aim of this study was to assess seminal plasma (SP) and serum concentrations of zinc (Zn), selenium (Se) and testosterone (T) in domestic cats and determine whether these are related to sperm quality and testicular biometry. Six tomcats were collected using an artificial vagina and sperm analysis included motility by CASA, morphology, plasma membrane integrity, and sperm count. Serum and SP were submitted to total T concentration determination using a solid-phase radioimmunoassay technique while Zn and Se were measured by atomic absorption spectroscopy. Serum T concentrations were greater compared to SP concentrations, but both values were significantly correlated. Se concentrations were higher in serum, whereas SP had greater Zn values. Concentrations of Se, Zn and T were not correlated with each other either in serum or SP. Negative correlations were detected between Se concentrations in SP and total sperm head defects, and between Se concentrations in serum and VAP, VSL, STR, and LIN. Serum concentrations of Zn were negatively correlated with total abnormal sperm and midpiece defects and positively related to progressive motility. Both serum and SP concentrations of T had no relationship with sperm quality. Concentrations of Se exhibited a negative correlation with total testicular weight, whereas T concentrations in SP and serum were correlated with total testicular volume and weight. In conclusion, both Se and Zn concentrations in serum were correlated to sperm quality variables in the domestic cat, thus, making these potential candidates for fertility markers.

Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa.

Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk-based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate-conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration.

High incidence of 'Dag-like' sperm defect in the domestic cat.

The occurrence of a high incidence of sperm tail defects in a male domestic cat resembling the known 'Dag-like' defect is reported. Sperm analyses were performed in ejaculated samples collected by an artificial vagina and in testicular and epididymal sperm cells after castration. The following alterations were observed using transmission electron microscope: heavily coiled sperm tails containing several axonemal units enclosed in the same common cell membrane; aberrations in the axonemal main structure; and swollen and unevenly distributed mitochondria in the midpiece. Abnormal modifications in the mitochondrial sheath were also found in sperm cells retrieved from testes and epididymides. Considering these findings, we can conclude that this is the Dag-like defect, described previously in other domestic species and a testicular origin may be involved.

Identification of phospholipase C zeta in normospermic and teratospermic domestic cat sperm.

In mammalian species, oocyte activation is initiated by oscillations in the intracellular concentration of free calcium ([Ca(2+)]i), which are also essential to allow embryonic development. To date, evidence supporting the hypothesis that a sperm factor is responsible for initiating oocyte activation has been presented in various mammalian species. Among the possible candidates to be the active sperm factor is the novel sperm-specific phospholipase C ζ (PLCζ), which besides its testis-specific expression is capable of initiating [Ca(2+)]i oscillations. In this study, we investigated the presence of PLCζ in the sperm of the domestic cat and whether normospermic and teratospermic cats differ in their PLCζ expression. Immunoblotting with anti-PLCζ antibodies confirmed the presence of an immunoreactive band of ∼70 kDa in whole sperm lysates of domestic cat as well as in both soluble and "insoluble" fractions from this sperm. Additional immunoreactive bands, probably C- and N-terminal truncated versions of PLCζ, were also visualized in the soluble sperm fractions. Interestingly, immunoreactivity of PLCζ was detectable in teratospermic sperm, although with slightly less intensity than in normospermic sperm. In conclusion, domestic cat sperm express PLCζ in both cytosolic and high-pH fractions, which is consistent with data in other mammals. Sperm from teratospermic cats also express PLCζ, albeit at reduced concentrations, which may affect the fertility of these males.

Comparison of efficiency between two artificial insemination methods using frozen-thawed semen in domestic cat (Felis catus): artificial insemination in domestic cats.

The aim of this study was to compare the efficiency of the intravaginal (IVAI) vs. intrauterine artificial insemination (IUAI) using frozen-thawed sperm in the domestic cat. Semen was collected from two tom cats using an artificial vagina and samples were assessed for motility (computer-assisted sperm analysis (CASA)), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP/YOLK (4% of glycerol), sperm samples were loaded into 0.25 mL straws (25 x 10(6)motile sperm/straw), incubated at 5 degrees C for 20 min and cryopreserved over liquid nitrogen (LN(2)) vapor for 15 min and then immersed in LN(2). For each AI, four straws from the same male were thawed (12s at 46 degrees C) and centrifuged at 250 x g for 8 min to pellet the sperm. The supernatant was discarded and sperm pellet resuspended with the remaining liquid, approximately 100 microL, and analyzed as described above. Queens were treated with a single im injection of 100 IU eCG to induce ovarian follicular development. Final oocyte maturation and ovulation was induced with 100 IU hCG given im at 82-84 h after eCG administration. Thirty hours after hCG administration, females were inseminated either intrauterine (n=8 queens) or intravaginally (n=8 queens), using thawed sperm from a single male. Although a pronounced decrease in sperm motility, acrosome and plasma membrane integrity was observed in sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method compared with 0% pregnancy when inseminated intravaginally.