Z. Gibb

Sperm Motility Is Lost In Vitro As a Consequence of Mitochondrial Free Radical Production and the Generation of Electrophilic Aldehydes but Can Be Significantly Rescued by the Presence of Nucleophilic Thiols

ABSTRACT The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2′-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.

The Paradoxical Relationship Between Stallion Fertility and Oxidative Stress

ABSTRACT The relationship between stallion fertility and oxidative stress remains poorly understood. The purpose of this study was to identify criteria for thoroughbred fertility assessment by performing a logistical regression analysis using “dismount” sperm parameters as predictors and weekly per-cycle conception rate as the dependent variable. Paradoxically, positive relationships between fertility and oxidative stress were revealed, such that samples that produced pregnancies exhibited higher rates of 8-hydroxy-2′-deoxyguanosine release (1490.2% vs. 705.5 pg/ml/24 h) and lower vitality (60.5% vs. 69.6%) and acrosome integrity (40.2% vs. 50.1%) than those that did not. We hypothesized that the most fertile spermatozoa exhibited the highest levels of oxidative phosphorylation (OXPHOS), with oxidative stress simply being a by-product of intense mitochondrial activity. Accordingly, an experiment to investigate the relationship between oxidative stress and motility was conducted and revealed positive correlations between mitochondrial ROS and total motility (R2 = 0.90), rapid motility (R2 = 0.89), average path velocity (VAP; R2 = 0.59), and curvilinear velocity (VCL; R2 = 0.66). Similarly, lipid peroxidation was positively correlated with total motility (R2 = 0.46), rapid motility (R2 = 0.51), average path velocity (R2 = 0.62), and VCL (R2 = 0.56), supporting the aforementioned hypothesis. The relative importance of OXPHOS in supporting the motility of equine spermatozoa was contrasted with human spermatozoa, which primarily utilize glycolysis. In this study, mitochondrial inhibition significantly reduced the velocity (P < 0.01) and ATP (P < 0.05) content of equine, but not human, spermatozoa, emphasizing the formers relative dependence on OXPHOS. The equine is the first mammal in which such a positive relationship between oxidative stress and functionality has been observed, with implications for the management of stallion fertility in vitro and in vivo.

Proteomic characterization and cross species comparison of mammalian seminal plasma

UNLABELLED Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammalian species. Nucleobindin 1 and RSVP14, a member of the BSP (binder of sperm protein) family, were identified in all species. Beta nerve growth factor (bNGF), previously identified as an ovulation inducing factor in alpacas and llamas, was identified in this study in alpaca and camel (induced ovulators), cattle, sheep and horse (spontaneous ovulators) seminal plasma. These findings indicate that while the mammalian species studied have common ancestry as ungulates, their seminal plasma is divergent in protein composition, which may explain variation in reproductive capacity and function. The identification of major specific proteins within seminal plasma facilitates future investigation of the role of each protein in mammalian reproduction. BIOLOGICAL SIGNIFICANCE This proteomic study is the first study to compare the protein composition of seminal plasma from seven mammalian species including two camelid species. Beta nerve growth factor, previously described as the ovulation inducing factor in camelids is shown to be the major protein in alpaca and camel seminal plasma and also present in small amounts in bull, ram, and horse seminal plasma.

Causes and consequences of oxidative stress in spermatozoa.

Spermatozoa are highly vulnerable to oxidative attack because they lack significant antioxidant protection due to the limited volume and restricted distribution of cytoplasmic space in which to house an appropriate armoury of defensive enzymes. In particular, sperm membrane lipids are susceptible to oxidative stress because they abound in significant amounts of polyunsaturated fatty acids. Susceptibility to oxidative attack is further exacerbated by the fact that these cells actively generate reactive oxygen species (ROS) in order to drive the increase in tyrosine phosphorylation associated with sperm capacitation. However, this positive role for ROS is reversed when spermatozoa are stressed. Under these conditions, they default to an intrinsic apoptotic pathway characterised by mitochondrial ROS generation, loss of mitochondrial membrane potential, caspase activation, phosphatidylserine exposure and oxidative DNA damage. In responding to oxidative stress, spermatozoa only possess the first enzyme in the base excision repair pathway, 8-oxoguanine DNA glycosylase. This enzyme catalyses the formation of abasic sites, thereby destabilising the DNA backbone and generating strand breaks. Because oxidative damage to sperm DNA is associated with both miscarriage and developmental abnormalities in the offspring, strategies for the amelioration of such stress, including the development of effective antioxidant formulations, are becoming increasingly urgent.

The Simmet Lecture: New Horizons on an Old Landscape – Oxidative Stress, DNA Damage and Apoptosis in the Male Germ Line

Our ability to diagnose and treat male infertility is gradually improving in concert with advances in our understanding of the molecular mechanisms underpinning defective sperm function. In this context, one of the factors to emerge as a major causative agent in male infertility is oxidative stress. Spermatozoa are particularly susceptible to such stress because they are exceptionally rich in vulnerable substrates such as polyunsaturated fatty acids, proteins and DNA. The lack of sperm cytoplasm also provides these cells with little capacity to protect themselves from oxidative attack or to effect any repair, should damage occur. Similarly, sperm chromatin is in a quasi-crystalline state and has very little capacity to respond to any DNA damage induced by oxidative attack. When the latter does occur, it appears to be initiated by reactive oxygen species (ROS) generated by the sperm mitochondria. These free radicals attack the lipids present in the sperm mitochondria generating electrophilic aldehydes, which bind to components of the mitochondrial electron transport chain stimulating yet more ROS production. The oxidative stress created via this self-propagating mechanism initiates an apoptotic cascade as a result of which the spermatozoa loose their capacity for fertilization and suffer damage to their DNA. Phosphatidylserine externalization is a late event in sperm apoptosis and may facilitate the silent phagocytosis of moribund cells in the female reproductive tract, that is, the phagocytosis of senescent spermatozoa without the accompanying generation of an inflammatory response. Encouragingly, the involvement of oxidative stress in the aetiology of male infertility has opened up new opportunities for therapeutic interventions involving the judicious administration of nucleophiles and other forms of antioxidants.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.

Investigation of the stallion sperm proteome by mass spectrometry

Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell-cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC-MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim of furthering our understanding of this cells complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to β-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of β-oxidation, reduced motility parameters, indicating that β-oxidation contributes to maintenance of motility in stallion spermatozoa.

Use of a defined diluent increases the sex-sorting efficiency of stallion sperm.

The low efficiency of flow cytometric sex-sorting of stallion sperm has been attributed to the use of an opaque skim milk-based diluent during Hoechst 33342 (H33342) staining. Three experiments were conducted to formulate an optically clear stallion semen diluent for use during H33342 staining, and to determine whether a clear diluent improved resolution during sorting. For Experiment 1, sperm were incubated at 34 °C in each of five diluents containing either no protein, skim milk, 0.25% Cohns Fraction V BSA, 0.5% BSA, or 1% BSA, following an 18 h storage (15 °C) period, or shortly after collection. Sperm incubated in both skim milk and 1% BSA-supplemented diluents had equivalent total (47 and 49.5%, respectively) and progressive (4.73 and 5.67%, respectively) sperm motilities after 45 min, and comparable acrosome integrity (65.9 and 67.9%, respectively). For Experiment 2, the protein source was optimised by comparing the characteristics of sperm stored and incubated in five diluents supplemented with skim milk, BSA, fatty acid and endotoxin free BSA (I-BSA), KnockOut™ Serum Replacement, and β-lactoglobulin, respectively. The I-BSA diluent was superior to skim milk for motility maintenance during incubation (74.0 vs 63.7%). The effect of diluent on sorting was investigated in Experiment 3 using a range of H33342 concentrations and incubation durations. The clear (1% BSA) diluent improved the split ratio compared with the opaque (skim milk) diluent (0.17 vs 0.08), with an optimum staining time of 45 min using 0.09 mM H33342. In conclusion, a diluent containing 1% fatty acid free, low endotoxin BSA in lieu of skim milk improved the sorting efficiency and motility characteristics of stallion sperm after storage for 18 h.

Stallion fertility: a focus on the spermatozoon.

Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermatozoal motility, the acrosome reaction and spermatozoon-oocyte interactions. Closing comments address the need for development and standardisation of molecular-based assays for use with spermatozoa of stallions whose subfertility cannot be explained with conventional tests.

Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa

ABSTRACT This study demonstrates for the first time the presence of an L-amino acid oxidase (LAAO) enzyme in equine spermatozoa that is able to generate significant amounts of reactive oxygen species (ROS) and create a state of oxidative stress. RT-PCR analysis revealed that the mRNA for this enzyme was present in the equine testis and spermatozoa, while immunocytochemical studies demonstrated that the mature LAAO protein was located in the sperm head, particularly in the acrosomal and postacrosomal domains. Experimental studies demonstrated that the aromatic amino acids (L-phenylalanine > L-tryptophan > L-tyrosine) were substrates for this enzyme, eliciting the dose- and time-dependent generation of ROS via mechanisms that were enhanced by cell death. This unexpected result was confirmed by analyses of ROS generation in subcellular sperm fractions, which again located a majority of LAAO activity to the sperm head. Equine cryopreservation medium was shown to contain sufficient quantities of aromatic amino acids to activate the LAAO system and generate ROS. The biological significance of this activity was established in an experiment in which physiological concentrations of aromatic amino acids were found to suppress sperm motility but only if dead spermatozoa were present in the same suspension. The combination of aromatic amino acids and nonviable cells was also found to enhance the levels of lipid peroxidation in live spermatozoa. These results suggest the potential significance of LAAO activity in generating the oxidative stress associated with the cryopreservation of equine spermatozoa. It is possible that inhibitors of this enzyme system may facilitate the development of modified cryostorage regimes for clinical validation in vivo.