Effect of Different Dilution Methods and Ratios of Ram Semen on Sperm Parameters after Cryopreservation.

The dilution method and ratio were tested to assess their effects on the Hu ram semen after cryopreservation. Experiment I aimed to explore the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I (Tris-based and egg yolk) under the condition of 1:1 dilution of diluent II (diluent I and glycerol) on the Hu ram semen preserved in liquid nitrogen regarding spermatozoa motility and kinetic parameters. Experiment II aimed to investigate the effect of various dilution ratios (1:1, 1:2, 1:3, 1:4) of diluent I under the condition of 1:2 dilution of diluent II to the Hu ram semen for cryopreservation on spermatozoa motility and kinetic parameters. The purpose of experiment III is to assess the effect of various dilution methods and ratios on the cryopreservation of Hu ram semen by detecting spermatozoa motility, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level. Experiment III includes four groups: one-step dilution method and two-step dilution method. The two-step dilution method includes two groups: 1:2, 1:1 and 1:3, 1:2, and the one-step dilution method includes two groups: 1:5 and 1:11. The results indicated that the post-thawed spermatozoa total motility (TM), progressive motility (PM) and average motion degree (MAD) were highest in the 1:2 group and significantly higher (p < 0.05) than those in the 1:1 and 1:4 groups under the condition of 1:1 dilution of diluent II. The post-thawed spermatozoa TM and PM of the 1:3 group were significantly higher (p < 0.05) than those of the other groups under the condition of 1:2 dilution of diluent II. The post-thawed spermatozoa TM, PM, plasma membrane integrity and acrosome integrity of the two-step group (1:3, 1:2) were the highest and significantly higher (p < 0.05) than those in the other groups. Additionally, the post-thawed spermatozoa ROS level of the two-step group (1:3, 1:2) was significantly lower (p < 0.05) than that in the one-step groups (1:5 and 1:11). Therefore, a two-step dilution (1:3, 1:2) was found to be the most suitable method and ratio for diluting the Hu ram semen after cryopreservation.

Sorted Bulls' X-Chromosome-Bearing Spermatozoa Show Increased GAPDHS Activity Correlating with Motility.

Sperm sexing is a technique for spermatozoa sorting into populations enriched with X- or Y-chromosome-bearing cells and is widely used in the dairy industry. Investigation of the characteristics of sorted semen is of practical interest, because it could contribute to the enhancement of sexed semen fertility characteristics, which are currently lower than those of conventional semen. Comparison of a spermatozoa population enriched with X-chromosome-bearing cells to a mixed population is also intriguing in the context of potential differences that drive the mechanisms of primary sex-ratio determination. In this work, sexed (X spermatozoa) and conventional spermatozoa of Holstein bulls were analyzed for the content and enzymatic activity of GAPDHS, a sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase that plays a significant role in the regulation of flagellar activity. No difference in the amount of this glycolysis enzyme per cell was revealed, but, notably, GAPDHS enzymatic activity in the sexed samples was significantly higher. Enzymatic activity among the group of sexed but not conventional sperm samples positively correlated with spermatozoa motility, which indicates the significant role of this enzyme for the sorted cells population.

Effect of chronic sleep deprivation on acrosomal integrity and functional parameters of murine sperm.

OBJECTIVE: To evaluate the effect of chronic sleep deprivation on sperm function quality in mice. DESIGN: Experimental study. SETTING: Not applicable. ANIMALS: Spermatozoa from twenty-four 10-week-old C57BL/6J male mice. INTERVENTION(S): The sleep deprivation group underwent gentle handling for 6 hours for 5 consecutive days. The mice in the sleep recovery group were allowed to sleep during the 24-hour period after the sleep deprivation protocol. MAIN OUTCOME MEASURE(S): After euthanasia, the spermatozoa were collected for analysis. Sperm motility was evaluated using computer-assisted sperm analyzer. Intracellular superoxide anion (O2-) activity, acrosome integrity, mitochondrial activity, and DNA fragmentation assays were conducted afterward. RESULT(S): Sleep deprivation and sleep recovery groups presented a lower percentage of spermatozoa with an intact acrosome, compared with the respective control groups. Regarding DNA fragmentation, a decreased proportion of spermatozoa with Comet I class intact DNA was observed in the sleep recovery group, compared with the recovery control group. Beat cross frequency was increased in the sleep recovery group. CONCLUSION(S): Sleep deprivation can reduce sperm quality, impairing acrosome integrity. Sleep recovery decreased DNA integrity and increased beat cross frequency.

Pikeperch (Sander lucioperca) spermatozoa motility and volume regulation under different osmotic and ionic conditions.

Pikeperch (Sander lucioperca) is a highly profitable commercial species whose economic value has greatly increased in the last decade. As in other species, the quality of spermatozoa in this species is a principal feature inherent in fertilization success and efficient natural and artificial reproduction. The capacity of fish spermatozoa to be activated and tolerate environmental changes (in osmolality, ion composition, external pH, temperature, etc.) during the motility period contributes to fertilization success. In this study, we investigated the effects of environmental osmolality and ion composition on spermatozoa motility. To determine if the activation mechanism is affected by sperm quality parameters, we measured semen characteristics such as semen volume, spermatozoa concentration, seminal fluid osmolality and ion composition, and spermatozoa lipid composition. An additional parameter of sperm quality reflecting spermatozoa osmoresistance, the swelling rate, was measured by the nephelometry method. We detected that sperm samples with the highest content of palmitic (C16:0) and palmitoleic (C16:1) acids showed the lowest motility activation under the studied conditions, suggesting that these fatty acids are possible markers for the determination of spermatozoa quality in fish. Our results show that pikeperch spermatozoa can be activated under different osmotic conditions and that cell swelling always accompanies motility. However, spermatozoa sustain their volume under hypotonic conditions when motility is not initiated, suggesting that pikeperch spermatozoa activation is mainly controlled by ion composition rather than the osmolarity of the surrounding medium.

Defining Reference Ranges for Serum Anti-Müllerian Hormone on a Large Cohort of Normozoospermic Adult Men Highlights New Potential Physiological Functions of AMH on FSH Secretion and Sperm Motility.

BACKGROUND: Few studies to date have attempted to measure serum anti-Müllerian hormone (AMH) levels in adult men, and solid references ranges have not yet been defined in a large cohort. OBJECTIVE: In this study, we aimed, first, to establish the reference ranges for serum AMH and AMH-to-total testosterone ratio (AMH/tT) in adult males. Second, we investigated the relationship between serum AMH and both reproductive hormones and semen parameters. METHODS: This single-center retrospective study included 578 normozoospermic adult men. Serum AMH concentrations were determined with an automated sandwich chemiluminescent immunoassay. RESULTS: The median serum AMH was 43.5 pmol/L. The 2.5th and 97.5th percentile values for serum AMH and AMH/tT were 16.4 and 90.3 pmol/L and 0.45 and 3.43, respectively. AMH was positively correlated with inhibin B and sperm concentration and negatively correlated with age, follicle-stimulating hormone (FSH), and progressive sperm motility. Interestingly, using immunofluorescence, we documented for the first time that AMH type II receptor (AMH-R2) is expressed in ejaculated human spermatozoa and gonadotrophic cells in the postmortem pituitary gland. CONCLUSIONS: We establish a new age-specific reference range for serum AMH and AMH/tT. Moreover, AMH-R2 expression in human spermatozoa and gonadotrophic cells, together with the relationship between serum AMH levels and sperm motility or mean FSH levels, highlight new potential functions of AMH in regulating sperm motility or FSH secretion in adult men.

Lysine acetylation participates in boar spermatozoa motility and acrosome status regulation under different glucose conditions.

Efficient artificial insemination (AI) with liquid preserved boar semen is essential for the swine industry. Glucose is the most widely utilized energy source in refrigerated boar semen extenders. However, the relationship between glucose concentration and spermatozoa quality is not fully understood. In the present study, boar spermatozoa were used as a model to investigate the impact of different glucose concentrations on spermatozoa motility, mitochondrial activity, and acrosome integrity at physiological temperature (37 °C) and during refrigeration (17 °C). The proportion of progressively motile spermatozoa and mitochondrial activity in the high glucose group were significantly lower than in the low glucose group when incubated at 37 °C for 3 h or 17 °C for 3 d, but not at 17 °C for 7 d. Lysine acetylation is a reversible post-translational modification that plays a crucial role in spermatozoa function. Our results show that spermatozoa protein acetylation levels were higher in the high glucose group than in the low glucose group. The proportions of progressively motile and acrosome-intact spermatozoa were higher in acetyltransferase inhibitor (WM-1119)-treated spermatozoa than in the control. Spermatozoa acetyl-CoA concentration, which is directly linked to acetylation, was significantly higher in the high glucose group than in the low glucose group. Taken together, spermatozoa motility and acrosome integrity can be altered by changing the concentration of glucose in the extender. High glucose concentration-induced lysine acetylation participates in the regulation of boar spermatozoa motility and acrosome integrity during preservation. These results can provide insights into spermatozoa preservation and AI in the swine industry.

Vitamin C ameliorates tetrahydrocannabinol-induced spermatotoxicity in-vitro.

BACKGROUND: We investigated the in-vitro effects of vitamin C on delta-9-tetrahydrocannabinol (THC) -induced reduction in spermatozoa motility and kinematics. METHODS: Six rats were used for the study. Semen from each of the 6 rats was randomly divided into 6 groups such that each rat's semen was in all of the groups. Groups I-III received placebo, THC (1 mM), and vitamin C (5 mM) respectively. Group IV was pre-treated with cannabinoid receptors' blockers (CBs-) 1 and 2, followed by THC. Groups V and VI received THC and vitamin C, but group VI was additionally pre-treated with CBs-. RESULTS: The spermatozoa progressive motility, average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), amplitude of lateral head (ALH) and beat cross frequency (BCF) were reduced by THC (6.08 ± 1.16%; 5.64 ± 0.82 µm/s; 6.96 ± 0.74 µm/s; 2.75 ± 0.23 µm/s; 0.31 ± 0.02 µm; and 0.78 ± 0.08 Hz respectively) but increased by vitamin C (51.20 ± 1.32%; 17.90 ± 0.21 µm/s; 25.11 ± 0.96 µm/s; 8.80 ± 0.27 µm/s; 0.75 ± 0.01 µm; and 3.15 ± 0.03 Hz respectively) when compared to control (39.72 ± 0.38%; 13.70 ± 0.29 µm/s; 18.04 ± 0.58 µm/s; 7.54 ± 0.34 µm/s; 0.65 ± 0.02 µm; and 2.79 ± 0.01 Hz respectively). Vitamin C inhibited the THC-induced reduction in these parameters (37.36 ± 0.73%; 10.98 ± 0.45 µm/s; 13.58 ± 0.30 µm/s; 7.11 ± 0.22 µm/s; 0.58 ± 0.01 µm; and 2.60 ± 0.01 Hz respectively) in the absence of CBs- 1 and 2, and even caused additional increases in progressive motility (49.54 ± 1.01%), VAP (15.70 ± 0.38 µm/s) and VCL (22.53 ± 0.29 µm/s) above the control levels with CBs-. CONCLUSION: Vitamin C ameliorates the THC-induced reduction in spermatozoa motility in-vitro by modulation of their kinematics.

Inhibiting the influence of ovarian fluid on spermatozoa activation and spermatozoa kinetic characteristics in the common barbel Barbus barbus.

As a result of evolution, various finfish species have developed different breeding strategies. However, there are some similarities, and one of them is the positive effect of ovarian fluid on spermatozoa. The opposite of this phenomenon was found in the common barbel (Barbus barbus). The present study analyzed the effect of ovarian fluid (OF), distilled water (DW) and Woynarovich solution (WS) on the motility, longevity and kinetics of barbel spermatozoa. These spermatozoa parameters were also evaluated with various dilutions of ovarian fluid (OF) in relation to distilled water [0:4 (Group OF 0%), 1:3 (Group OF 25%), 1:1 (Group OF 50%), 3:1 (Group OF 75%), 4:0 (Group OF 100%)] and spermatozoa reactivation after a 30 s (Group OFR30s 100%) treatment in ovarian fluid. The motility analysis was carried out using computer-assisted semen analysis (CASA). The negative interaction of ovarian fluid with spermatozoa motility in the same fish species was recorded for the first time. In pure ovarian fluid, the average spermatozoa motility (MOT) decreased significantly (1.40 ± 0.94%). The negative effect of ovarian fluid-to-spermatozoa motility was reversible, and after a 30 s treatment in ovarian fluid and later dilution with water, spermatozoa motility was reactivated (from 2.25 ± 0.53% vs 69.78 ± 6.02%). The use of Woynarovich solution as an activator of spermatozoa movement had a positive effect (P < 0.05) on spermatozoa movement longevity (motility up to 90 s) and the percentage of motile spermatozoa compared to distilled water (up to 45 s) and ovarian fluid (P < 0.05).

Chemerin Impairs In Vitro Testosterone Production, Sperm Motility, and Fertility in Chicken: Possible Involvement of Its Receptor CMKLR1.

The chemokine chemerin is a novel adipokine involved in the regulation of energy metabolism but also female reproductive functions in mammals. Its effects on male fertility are less studied. Here, we investigated the involvement of chemerin in chicken male reproduction. Indeed, the improvement of the sperm of roosters is a challenge for the breeders since the sperm quantity and quality have largely decreased for several years. By using specific chicken antibodies, here we show that chemerin and its main receptor CMKLR1 (chemokine-like receptor 1) are expressed within the chicken testis with the lowest expression in adults as compared to the embryo or postnatal stages. Chemerin and CMKLR1 are present in all testicular cells, including Leydig, Sertoli, and germinal cells. Using in vitro testis explants, we observed that recombinant chicken chemerin through CMKLR1 inhibits hCG (human chorionic gonadotropin) stimulated testosterone production and this was associated to lower 3ßHSD (3beta-hydroxysteroid dehydrogenase) and StAR (steroidogenic acute regulatory protein) expression and MAPK ERK2 (Mitogen-Activated Protein Kinase Extracellular signal-regulated kinase 2) phosphorylation. Furthermore, we demonstrate that chemerin in seminal plasma is lower than in blood plasma, but it is negatively correlated with the percentage of motility and the spermatozoa concentration in vivo in roosters. In vitro, we show that recombinant chicken chemerin reduces sperm mass and individual motility in roosters, and this effect is abolished when sperm is pre-incubated with an anti-CMKLR1 antibody. Moreover, we demonstrate that fresh chicken sperm treated with chemerin and used for artificial insemination (AI) in hen presented a lower efficiency in terms of eggs fertility for the four first days after AI. Taken together, seminal chemerin levels are negatively associated with the rooster fertility, and chemerin produced locally by the testis or male tract could negatively affect in vivo sperm quality and testosterone production through CMKLR1.

Potential of coconut water to enhance fresh semen quality and fertility in rabbits.

Coconut water as a natural diluent was evaluated on rabbit semen quality, conception and litter size in artificially inseminated does. A total of 10 bucks and 90 does, all crossbred New Zealand White × chinchilla, were used for the trial. Pooled semen was obtained from bucks using artificial vagina. Semen diluents were prepared at 0%, 20%, 40%, 60% and 80% and control 50% normal saline and designated as T1, T2, T3, T4, T5 and T6 respectively, in a completely randomised design. Semen analysis was conducted on fresh semen and the various diluted semen immediately at 37 °C. Fifteen does each were randomly allotted to the different treatment and were inseminated with their respective treatment diluted semen using the standard procedure, and conception rate, litter size and productivity index at birth were assessed at kindling. Result obtained revealed that spermatozoa motility and structural membrane integrity of the diluted semen were significantly (p < 0.05) affected by the percentage of dilution. The range of values obtained for spermatozoa motility, structural membrane integrity and acrosome integrity is within the accepted values for good-quality semen. Conception rate, litter size and productivity index at birth were highest in does inseminate with T4 (60%), and the least values were obtained in T5 (80%). In conclusion, coconut water is a suitable diluent for rabbit semen for on-farm artificial insemination, the optimal dilution rate of coconut water in rabbit semen is 60%, which guarantees high conception, litter size and productivity index at birth.

Spermatozoa motility in bivalves: Signaling, flagellar beating behavior, and energetics.

Though bivalve mollusks are keystone species and major species groups in aquaculture production worldwide, gamete biology is still largely unknown. This review aims to provide a synthesis of current knowledge in the field of sperm biology, including spermatozoa motility, flagellar beating, and energy metabolism; and to illustrate cellular signaling controlling spermatozoa motility initiation in bivalves. Serotonin (5-HT) induces hyper-motility in spermatozoa via a 5-HT receptor, suggesting a serotoninergic system in the male reproductive tract that might regulate sperm physiology. Acidic pH and high concentration of K+ are inhibitory factors of spermatozoa motility in the testis. Motility is initiated at spawning by a Na+-dependent alkalization of intracellular pH mediated by a Na+/H+ exchanger. Increase of 5-HT in the testis and decrease of extracellular K+ when sperm is released in seawater induce hyperpolarization of spermatozoa membrane potential mediated by K+ efflux and associated with an increase in intracellular Ca2+ via opening of voltage-dependent Ca2+ channels under alkaline conditions. These events activate dynein ATPases and Ca2+/calmodulin-dependent proteins resulting in flagellar beating. It may be possible that 5-HT is also involved in intracellular cAMP rise controlling cAMP-dependent protein kinase phosphorylation in the flagellum. Once motility is triggered, flagellum beats in asymmetric wave pattern leading to circular trajectories of spermatozoa. Three different flagellar wave characteristics are reported, including "full", "twitching", and "declining" propagation of wave, which are described and illustrated in the present review. Mitochondrial respiration, ATP content, and metabolic pathways producing ATP in bivalve spermatozoa are discussed. Energy metabolism of Pacific oyster spermatozoa differs from previously studied marine species since oxidative phosphorylation synthetizes a stable level of ATP throughout 24-h motility period and the end of movement is not explained by a low intracellular ATP content, revealing different strategy to improve oocyte fertilization success. Finally, our review highlights physiological mechanisms that require further researches and points out some advantages of bivalve spermatozoa to extend knowledge on mechanisms of motility.

Eel sperm cryopreservation: An overview.

The eels are teleost fishes from the order Anguilliformes that includes several species with high commercial value. Due to the high interest for aquaculture production of some eel species and for the need to restore eel species that are endangered, several research groups have directed their research toward developing protocols to cryopreserve the spermatozoa of Japanese eel (Anguilla japonica) and European eel (Anguilla anguilla). In this review, we provide an overview on the different protocols that have been developed so far. The first developed protocols used DMSO as cryoprotectant in both species with good success, obtaining sperm motilities of over 45% in Japanese eel and over 35% in European eel. Moreover, sperm cryopreserved using DMSO was successfully used in fertilization trials, although with low fertilization rates. However, recent studies show that DMSO produce epigenetic changes in eel sperm and therefore, the last developed protocols used methanol as cryoprotectant instead. Cryopreservation protocols using methanol as cryoprotectant, showed improved motility values in both Japanese and European eel. In addition, the latest protocols have been adapted to cryopreserve larger volumes of sperm of up to 5 mL, which is useful for larger scale fertilization trials. The present study introduces the state of the art and future perspectives of the eel sperm cryopreservation to be applied in aquaculture and biological conservation programs.

Seminal plasma transferrin effects on cryopreserved common carp Cyprinus carpio sperm and comparison with bovine serum albumin and antifreeze proteins.

In the current study, there was evaluation of the cryopreservation effectiveness of common carp Cyprinus carpio sperm when cryopreservation medium was supplemented with proteins. Semen was diluted with Kurokura's extender composing 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, 2.38 mM NaHCO3, and 10% dimethylsulfoxide (DMSO). Cryopreservation medium was supplemented with purified seminal plasma transferrin (Tf), bovine serum albumin (BSA) or antifreeze protein (AFP) Types I and III. Concentration of proteins evaluated was 0.1 µg/ml, 1 µg/ml, and 10 µg/ml. Motility and curvilinear velocity of spermatozoa was evaluated by the Computer Assisted Semen Analyzer (CASA). The percent of motile cells and spermatozoa curvilinear velocity of frozen-thawed sperm with supplementation of Tf and AFP III at all concentrations were greater compared to samples with no added proteins. The protective effect of BSA and AFP I was less and dose-dependent. Thus, it is concluded that incorporation of Tf in the extender before freezing improves crypreservation of common carp spermatozoa whereas supplementation with AFP III in greater concentrations was more effective.

Fish sperm biology in relation to urogenital system structure.

Morphology of the urogenital system has evolved during fish speciation. Chondrostei (sturgeons and paddlefishes) possess an excretory system which is called "primitive" in that the sperm ducts enter the kidneys and share the excretory ducts where sperm is mixed with urine before it is released into the spawning environment. Further, in this group of fishes there are also physiological characteristics which are associated with these anatomical features where the mixing of sperm and urine is a prerequisite for the final sperm maturation rather than contamination. In the Holostei (gars and bowfins) which are closely related to the Chondrostei, sperm also naturally mixed with urine, but the physiological role of such mixing for sperm biology has not been described. In contrast, urinary and sperm ducts in the more evolved Teleostei are completely separate, and sperm and urine are not mixed before being released during spawning. Thus, urine constitutes an inappropriate environment which can be a source of problems when sperm is collected during fisheries practices. In this review, the consequences of such divergent conditions in the urogenital anatomy will be considered in relation to general features of fish sperm biology and in relation to aquaculture and fisheries practices.

Evaluation of seminal plasma composition and spermatozoa quality parameters of silver barb, Barbonymus gonionotus Bleeker, 1850.

Seminal composition and semen quality are the important determinants in assessing the reproductive performance of different fishes. This study was carried out to evaluate the seminal composition and sperm quality of Barbonymus gonionotus. The seminal plasma contained 17.2 ± 0.34 mmol/l, 20.9 ± 0.48 mmol/l, 0.72 ± 0.04 mmol/l, 3.8 ± 0.2 mmol/l, and 1.49 ± 0.02 g/dl of Na+, K+, Ca++, Mg++, and total protein, respectively. The physical spermatological parameters, such as sperm volume, sperm motility, motility duration, sperm density, osmolality, and pH values were 1.55 ± 0.15 ml, 89 ± 2%, 391.9 ± 8.5 s, 2.8 ± 0.2 × 1010 /ml, 400.6 ± 5.1 mmol/kg, and 8.75 ± 0.10, respectively. In correlation matrix, the K+ (R2 = 0.39, P < 0.01) and Ca++ (R2 = 0.27, P < 0.05) ions and osmolality (R2 = 0.29, P < 0.05) showed significant positive correlations with sperm motility. Similarly, fertilization rate significantly influenced by sperm motility (R2 = 0.26, P < 0.05) and K+ (R2 = 0.30, P < 0.05) and Ca++ (R2 = 0.26, P < 0.05) ions. Also, osmolality significantly and negatively correlated with Mg++ (R2 = 0.33, P < 0.05) and sperm motility duration (R2 = 0.28, P < 0.05). Therefore, based on this results, it can be concluded that seminal plasma ions, K+ and Ca++ and osmolality are the key factors for the determination of sperm quality of silver barb, and these parameters could be considered during standardization of artificial fertilization or cryopreservation technique of silver barb spermatozoa.

UCP2 Mitigates the Loss of Human Spermatozoa Motility by Promoting mROS Elimination.

BACKGROUND/AIMS: To demonstrate the function of uncoupling protein 2 (UCP2) in the regulation of human spermatozoa motility. METHODS: Semen samples were collected from donors with either normal spermatozoa motility (normospermia [NS]) or poor spermatozoa motility (asthenospermia [AS]). UCP2 protein in spermatozoawas quantified by Western blotting. The level of mitochondrial reactive oxygen species (mROS) was evaluated by MitoSOX Red. The activity of mitochondrial membrane potential (MMP) in spermatozoa was evaluated by a JC-1 assay and the ATP level was monitored by a luciferin-luciferase assay. RESULTS: UCP2 was expressed in both NS and AS groups, with the former exhibiting a higher level than the latter. Immunofluorescence analysis shows that UCP2 is mainly located at the mid-region of human spermatozoa. The inhibition of UCP2 by a highly selective inhibitor, Genipin, results in not only impaired spermatozoa mobility (P<.05) but also an elevated level of mROS (P<.05), suggesting that UCP2 is involved in the maintenance of the spermatozoa mobility, which probably is achieved by promoting mROS elimination. Furthermore, H2O2 treatment of spermatozoa increases the mROS level coupled with the loss of spermatozoa mobility. Unexpectedly, this treatment also has a positive impact on the expression of UCP2 within a certain range of supplemental H2O2, indicating the moderate mROS level possibly serves as a feedback signal to stimulate the expression of UCP2. Finally, the treatment of spermatozoa by an ROS scavenger, N-acetyl-l-cysteine (NAC),decreases the level of mROS and increases the curvilinear velocity (VCL) of spermatozoa, but the UCP2 level is not affected. CONCLUSION: These results suggest an UCP2-mROS-motility regulatory system exists for maintaining spermatozoa mobility in humans. In such a system, UCP2 fulfills its function by promoting mROS elimination, and slightly over-produced mROS in turn serves as a signal to stimulates the expression of UCP2. This regulatory system represents a new potential target for the discovery of novel pharmaceuticals for the treatment of patients with low spermatozoa motility.

Effects of tetrabrombisphenol A on DNA integrity, oxidative stress, and sterlet (Acipenser ruthenus) spermatozoa quality variables.

The sperm of sterlet (Acispenser ruthenus) was used to investigate the effect of the xenobiotic tetrabrombisphenol A (TBBPA) on sperm quality variables (ATP content, spermatozoa motility, and velocity), DNA integrity, and oxidative stress indices. Sperm was diluted to obtain a spermatozoa density of 5 × 10(8) cells/mL and exposed for 2 h to final concentrations of TBBPA (0.5, 1.75, 2.5, 5, and 10 µg/L). The oxidative stress indices, including lipid peroxidation, carbonyl derivatives of proteins, and antioxidant activity were significantly higher with increased concentrations of TBBPA. There was significantly less intracellular ATP in sperm samples at TBBPA concentrations of 2.5 µg/L and above. Spermatozoa velocity and percent motile sperm were significantly lower at each sampling time post-activation compared to controls. DNA damage expressed as percent DNA in Tail and Olive Tail moment was significantly higher with exposures ≥2.5 µg/L TBBPA. The results demonstrated that TBBPA and other xenobiotics can induce reactive oxygen species stress in fish spermatozoa, which could impair the sperm quality, DNA integrity, ATP content, and the antioxidant defense system. This study confirmed that fish spermatozoa can be used in in vitro assays for monitoring residual pollution in aquatic environments.

Toxicity of environmental contaminants to fish spermatozoa function in vitro--a review.

In vitro techniques for investigating the toxic effects of environmental contaminants (EC) on fish spermatozoa motility kinetics and fertilizing ability are valuable tools to understand toxicity mechanisms and sites of action. In vitro techniques may also be well-suited to studies of endocrine disruption in male fertility in vivo. This review shows ECs to decrease or suppress spermatozoa motility kinetics and fertilizing ability in a dose-dependent manner, with toxic concentrations being much higher than those reported in the aquatic environment. Sites of action depend on EC concentration and duration of exposure. Both instant (immediate) and incubated exposure of spermatozoa to ECs results in damage to the plasma membrane and the axoneme, while disruption of energy metabolism appears only during incubated exposure. Spermatozoa lose fertilizing ability following exposure to ECs in vitro, not only due to inhibition or suppression of the initiation of motility, but also through damage to DNA. This review highlights the significant lack of information about disruption of spermatozoa function associated with exposure to water from polluted areas as well as combined effects of ECs. Specifics of alterations in intracellular signaling cascades involved in the initiation of spermatozoa motility following exposure to sublethal concentrations of ECs remain unknown. Further studies are also needed to elucidate in vitro EC effects during spermatozoa maturation, when spermatozoa acquire the potential for motility.

The in vitro effect of duroquinone on functional competence, genomic integrity, and oxidative stress indices of sterlet (Acipenser ruthenus) spermatozoa.

The sturgeon is a highly endangered fish species mostly due to over-fishing, habitat destruction, and water pollution. Duroquinone (derivative of 1,4-benzoquinone) is a xenobiotic compound widespread in the environment. The effect of duroquinone on motility, DNA integrity, and oxidative stress indices in sterlet, Acispenser ruthenus, spermatozoa was investigated. Sterlet sperm was exposed for 2h to duroquinone at concentrations of 25, 50, 100, and 150 µM. Spermatozoa motility, velocity, and ATP content were significantly decreased with exposure to duroquinone. The level of DNA damage significantly increased at concentrations of 50 µM and above. Oxidative stress indices (lipid peroxidation and content of carbonyl proteins) and superoxide dismutase (SOD) activity increased significantly with increasing concentrations of duroquinone. Oxidative stress in sterlet spermatozoa induced by duroquinone was shown to impair spermatozoa DNA integrity, motility parameters, and the antioxidant defense system. Spermatozoa motility, content of carbonyl proteins, and SOD activity were shown to be sensitive biomarkers, exhibiting strong responses to low concentrations of the xenobiotic. Results also suggested that fish spermatozoa in vitro assays may provide a simple and efficient means of monitoring residual pollutants in the aquatic environment.