Sperm Mitochondrial Membrane Potential Research Articles

Factors affecting staining to discriminate between bull sperm with greater and lesser mitochondrial membrane potential

The sperm mitochondrial membrane potential (MMP) is usually evaluated using the JC-1 dye. This study aimed to verify the effect of incubation temperature (25 °C or 38 °C), incubation time (10, 30, and 45 min), JC-1 stain concentration (0.2 μM, 2 μM, 8 μM, 12 μM), and the presence of glycerol (6.6% compared with 0%), on the capacity of the stain to discriminate between sperm with high mitochondrial membrane potential (hMMP) and low mitochondrial membrane potential (lMMP) in fresh and frozen bull sample by both flow cytometry and epifluorescence microscopy. The temperature (38 °C for 10 min) and the dye concentration (8 μM and 12 μM) resulted in a greater proportion of hMMP (P < .05). The incubation for 45 min at 38 °C resulted in a significant reduction of hMMP in samples stained with JC-1 dye at 8 μM and 12 μM (P < .01). A longer incubation time (45 min) and greater dye concentration (8 μM and 12 μM) resulted in an increased proportion of hMMP sperm in cryopreserved samples. Fresh sperm incubated with glycerol had a hMMP (P < .05). Data for the present study indicate that the optimal incubation temperature was 38 °C, with an incubation time differing between fresh (10–30 min) and cryopreserved sperm (at least 45 min). Furthermore, the JC-1 dye concentration used that could reliably detect the proportion of hMMP sperm was 2 μM in fresh samples, and at least 8 μM in cryopreserved sperm.

HSP90 maintains boar spermatozoa motility and mitochondrial membrane potential during heat stress

Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were incubated in non-capacitating conditions (Tyrode’s basal medium or TBM) for 3, 8 and 24 h or in capacitating conditions (Tyrode’s complete medium or TCM) for 4 h at 38.5 °C or 40 °C (Heat stress) in the presence or absence of 5 or 20 μM of 17-AAG, a specific HSP90 inhibitor. Sperm viability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7 ± 4.1 vs. 1.9 ± 1.1; % mean ± SEM), PM (3.1 ± 0.9 vs. 0) and high mitochondrial membrane potential (19.5 ± 2.2 vs. 11.8 ± 0.8) decreased significantly in sperm incubated at 40 °C for 24 h in the presence of 20 μM 17-AAG (control vs. 20 μM 17-AAG, respectively; p < 0.05). In sperm incubated at 38.5 °C only a mild decrease in TM was observed (48.7 ± 3.1 vs. 32.1 ± 4.8; control vs. 20 μM 17-AAG, respectively; p < 0.05). However, under capacitating conditions none of the sperm parameters studied were affected by 17-AAG after 4 h of incubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non-capacitating conditions.

Protective effects of apigenin against acrylonitrile-induced subchronic sperm injury in rats

This study aimed to investigate the protective effects of apigenin (AP), a flavonoid found in plants, against acrylonitrile (ACN)-induced subchronic sperm and testes injury in rats. Male Sprague-Dawley rats were randomly divided into four groups: a control group (corn oil), an ACN group (ACN 50 mg kg−1), an ACN + AP1 group (ACN + AP 234 mg kg−1), and an ACN + AP2 group (ACN + AP 468 mg kg−1). The ACN + AP group received AP by gavage after treatment with 50 mg kg−1 ACN for 30 min, whereas the rats in the control group were given an equivalent volume of corn oil. The gavage was conducted 6 days per week for 12 weeks. The results showed that AP increased the sperm concentration, motility, and mitochondrial membrane potential (MMP) (P < 0.05), which were reduced by ACN. Conversely, reactive oxygen species (ROS) and malondialdehyde (MDA) were significantly decreased by AP (P < 0.05). AP improved the damage of the ultrastructure of sperm caused by ACN. AP reduced the pathological injuries and spermatogenic cell apoptosis caused by ACN in rat testes. AP also increased glutathione peroxidase activity and decreased MDA content. In conclusion, AP reduces ACN-induced decreasing sperm quality by inhibition of inflammation and oxidative stress.

Boar variability in sperm cryo-tolerance after cooling of semen in different long-term extenders at various temperatures

This study investigated individual boar variability in the quality of pre-freeze (PF) and post-thaw (PT) semen cooled in different long-term (LT) extenders and for different holding times (HT). Sperm rich fractions were diluted with Androhep® Plus (AHP), Androstar® Plus (ASP), Safecell® Plus (SCP) and TRIXcell® Plus (TCP) extenders, stored for 2h at 17°C (HT 1) and additionally for 24h at 10°C (HT 2) and the samples were subsequently evaluated and frozen. Besides the analysis of CASA sperm variables, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), normal apical ridge (NAR) acrosome integrity, and viability (YO-PRO-1−/PI−) of sperm were assessed in the PF and PT semen. Results indicated that boar, extender and HT group affected the sperm quality characteristics. There were great variations in PMOT and the sperm motion patterns of the PF semen among the boars. Differences in the HT groups of the PF semen, with respect to the sperm membrane integrity, were less marked among the boars. Consistent variations in TMOT and PMOT in the PT semen were observed among the boars, being greater in the HT 2 group. Most of the CASA-analyzed sperm motion patterns were greater in the HT 2 group of the PT semen. Furthermore, sperm MMP, PMI and viability were greater in the HT 2 group of the PT semen in most of the boars, while consistent differences were observed among the boars for sperm NAR acrosome integrity in either HT group. The significant effect of the cryopreservation process on the sperm membrane proteome was evident from the number of protein bands, detected in the electrophoretic profiles of sperm of the HT 1 and HT 2 groups. The electrophoretic profiles of the PF and PT semen among boars with poor and good semen freezability, however, differed with respect to the abundance and types of sperm membrane-associated proteins. The overall results of this study provided evidence that there are differences among boars in response to the different cooling regimens, and that cooling of extended semen for a 24-h period at 10°C modulated the functions of sperm in an extender-dependent manner, rendering the cells less susceptible to cryo-induced damage. It is suggested that the findings of this study have the potential to improve the technology of boar semen cryopreservation.

Osmotic tolerance of feline epididymal spermatozoa

During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezing-thawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P<0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P>0.01) and a difference between caput and corpus after return from 1200 mOsm (P<0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P<0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to non-returning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.

Cryopreservation of cynomolgus macaque (Macaca fascicularis) sperm with glycerol and ethylene glycol, and its effect on sperm-specific ion channels — CatSper and Hv1

The cryoprotective agent (CPA) is one of the most important factors that affects the cryosurvival of sperm. The aim of the present study was to compare two different CPAs, glycerol (Gly) and ethylene glycol (EG), on the cryopreservation of cynomolgus macaques sperm and evaluate the effects of cryopreservation on sperm motility, acrosomal integrity, DNA integrity, mitochondrial function and the sperm membrane ion channels CatSper and Hv1. Compared to fresh sperm, cryopreservation with either 0.7 M Gly or EG decreased the sperm motility (79.8 ± 1.5% Vs. 47.3 ± 1.8% and 47.6 ± 1.4%), acrosomal integrity (89.6 ± 1.2% Vs. 80.1 ± 1.8% and 79.6 ± 1.7%), DNA integrity (91.9 ± 0.7% Vs. 82.9 ± 1.0% and 82.3 ± 1.0%) and mitochondrial membrane potential (87.9 ± 1.8% Vs. 70.6 ± 2.7% and 67.9 ± 2.5%) and the quantity of the CatSper and Hv1 channels determined by Western Blot (p < 0.05), and EG showed equal cryoprotection to cynomolgus sperm in all of the sperm parameters. Our results indicated, for the first time, that cryopreservation decreases the quantity of sperm membrane ion channels (CatSper and Hv1), which might be one of the reasons that frozen sperm have a low fertilizing ability. The study will be beneficial to understand the biological process involved in sperm cryopreservation of nonhuman primates and contribute to improving cryopreservation protocols than can maintain sperm function and fertilizing ability.

Bovine serum albumin and skim-milk improve boar sperm motility by enhancing energy metabolism and protein modifications during liquid storage at 17 °C

Both bovine serum albumin (BSA) and skim-milk have been reported to improve sperm quality, primarily by enhancing sperm motility, but the underlying molecular mechanism remains unknown. In this study, boar semen samples were collected and diluted with Androstar® Plus extender containing different concentrations (0, 2, 4 g/l) of BSA and skim-milk. On days 0, 3, 5 and 7, the sperm motility parameters were determined using computer-assisted sperm analysis (CASA), and the ATP concentrations, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity and mitochondrial membrane potential were evaluated using commercial kits. The levels of protein phosphorylation, acylation and ubiquitination were analyzed by western blot. The results showed that supplementation with BSA and skim-milk provided higher sperm motility parameters, ATP levels, GAPDH activity and mitochondrial membrane potential than the control group (P < 0.05). Interestingly, we found that the levels of protein phosphorylation, acetylation and succinylation of the spermatozoa in the treated groups were dramatically higher than those in the control group (P < 0.05). Though the protein ubiquitination level had a decreasing trend, the change in ubiquitination modification was not significantly different between the control group and treated groups. Moreover, the changes in protein modifications between the BSA treated group and skim-milk treated group were not distinctly dissimilar. Taken together, these results suggest that BSA and skim-milk had a positive role in the regulation of boar sperm motility by influencing sperm protein modifications changes as well as increasing the GAPDH activity, mitochondrial membrane potential, and intracellular ATP content. This research provides novel insights into the molecular mechanisms underlying BSA and skim-milk protective effects on boar sperm in the male reproductive system and suggests the feasibility of using skim-milk instead of BSA as a boar semen extender supplement.

Comparación de 2 métodos de selección espermática sobre la fragmentación del ADN y el potencial de membrana mitocondrial de los espermatozoides humanos: en busca del gameto de oro

IntroductionTo select good-quality sperm for assisted reproductive technology procedures, the best spermatozoa should be elicited. The aim of this study is to compare the impact of sperm selection on human sperm DNA fragmentation and mitochondrial membrane potential by means of density gradient and swim-up techniques. Materials and methodsConventional and functional semen parameters (mitochondrial membrane potential and plasma membrane integrity) in sperm selected by density gradient and swim-up techniques in 14 semen samples were evaluated. ResultsIn the selected neat semen samples, the number of sperm recovered was lower using swim-up as compared to density gradients. By contrast, an increase in progressive motility was observed compared with the initial sample, with no statistically significant difference between both techniques. On the other hand, the swim-up technique was shown to decrease the amount of non-progressive motile sperm recovered, whereas the density gradient technique allowed for a better recovery of the total motile sperm selected. Finally, an increase in the proportion of cells with higher mitochondrial membrane potential and lower DNA fragmentation index was observed. ConclusionThe results of this study demonstrate that sperm selection using the swim-up technique allows for better-quality sperm to be recovered, with a higher sperm cell membrane potential and lower DNA fragmentation. The use of this method is therefore recommended prior to artificial insemination or in vitro fertilisation procedures.

Cytoplasmic droplet acting as a mitochondrial modulator during sperm maturation in dogs

Motility acquisition during sperm maturation and passage through the epididymis is closely related to mitochondrial function and appears to occur in parallel with cytoplasmic droplet (CD) migration. However, such mechanism remains unclear in dogs. Thus, the aim of this study was to characterize the influence of sperm CD in the mitochondrial functionality during epididymal sperm maturation in dogs. Twenty-one adult dogs were submitted to elective bilateral orchiectomy. Testicles were stored for 18–24h at 5°C and epididymal sperm samples were then collected from different segments of the epididymis (caput, corpus and cauda). Samples were evaluated for computer-assisted motility analysis (CASA), presence of CD (eosin/nigrosin stain), ultrastructural CD analysis and sperm mitochondrial activity (3,3' diaminobenzidine technique) and membrane potential (JC-1 probe). Samples collected from the corpus epididymis showed higher motility and mitochondrial activity in comparison to the caput sperm. Moreover, corpus sperm had lower percentage of proximal droplets compared to caput samples, while mitochondrial membrane potential remained unchanged. Cauda samples showed higher motility, mitochondrial activity and potential, however, lower presence of sperm droplets (proximal and distal). In conclusion, the CD is essential for epididymal sperm maturation in dogs, showing important functions along the transit in the epididymis. In the corpus segment, the migration of the CD along the sperm midpiece provides a high mitochondrial activity and the onset of sperm motility. On the other hand, sperm from cauda epididymis lack CD but suffered lipid membrane changes which allow a maximum mitochondrial membrane potential and motility.

Metabolic Activity of Boar Semen Stored in Different Extenders Supplemented with Ostrich Egg Yolk Lipoproteins.

IntroductionThe aim of this study was to evaluate the effect of lipoprotein fraction isolated from ostrich egg yolk (LPFo) on the metabolic activity of boar spermatozoa following liquid semen storage in different extenders and temperatures.Material and MethodsBoar ejaculates were extended in Androhep, Beltsville thawing solution (BTS), and Martín-Rillo and Alias (MR-A) without (control) or with the addition of LPFo and stored for three days at either 5°C or 16°C. The analysed sperm parameters included total motility (TMOT), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), oxygen consumption, and adenosine triphosphate (ATP) production.ResultsThe sperm metabolic activity seemed to be higher in the LPFo-based extenders following storage for three days, irrespective of the storage temperature. Compared with the LPFo-free extenders, significantly higher (P < 0.05) sperm PMI and MMP were observed in BTS and MR-A extenders supplemented with LPFo during storage for three days at 5°C. Spermatozoa stored in the BTS-LPFo extender exhibited higher (P < 0.05) TMOT and oxygen consumption, whereas higher (P < 0.05) PMI was observed in spermatozoa stored in Androhep-LPFo and MR-A-LPFo for three days at 16°C. No significant differences (P > 0.05) in ATP content were observed between the LPFo-free and LPFo-based extenders during storage.ConclusionsSupplementation of LPFo to semen extenders had varying effects on the metabolic activity of boar spermatozoa stored at different temperatures. It can be suggested that the interactions of various components of the extenders and seminal plasma with LPFo exert beneficial effects on the sperm metabolic activity during liquid storage of boar semen.

A New Method for Evaluating Stallion Sperm Viability and Mitochondrial Membrane Potential in Fixed Semen Samples.

Multiparametric assessment of stallion sperm quality using flow cytometry can be a useful adjunct in semen evaluation; however, the availability of flow cytometers in veterinary practice is limited. The ability to preserve and transport sperm samples for later flow cytometric analysis using fixable probes would potentially facilitate this process. In the current study, we validated the combination of live/dead Zombie Green® (a fixable dye used to assess live and dead sperm) and MitoTracker Deep Red® (used to assess mitochondrial membrane potential). The assay was validated against classic, non-fixable, membrane assays (SYBR-14/PI). Our results demonstrated the feasibility of the assay. In conclusion, stained and fixed semen samples stored for 72 h obtained equivalent results to the exam on the same day; this new protocol shall facilitate the wider use of flow cytometry in stallion andrology in the future. © 2017 International Clinical Cytometry Society.

Mitochondrial Biomarkers Reflect Semen Quality: Results from the MARCHS Study in Chongqing, China.

Unexplained infertility requires that more sensitive and mechanism-based biomarkers should be developed and used independently of or in addition to conventional semen parameters for an infertility diagnosis. In the present study, semen samples were collected from young men participating in the Male Reproductive Health in Chongqing College students (MARCHS) cohort study in the follow-up stage in 2014. Conventional semen parameters were measured in all 656 participants, whereas sperm mitochondrial membrane potential (MMP), mitochondrial DNA copy number (mtDNAcn), mtDNA integrity and apoptotic parameters were measured among 627, 386, 362, and 628 participants, respectively. We found that sperm MMP was significantly positively correlated with all of conventional semen parameters including semen volume (r = 0.090, p = 0.025), sperm concentration (r = 0.301, p<0.01), total sperm count (r = 0.324, p<0.01), and progressive motility (r = 0.399, p<0.01); sperm MMP was also negatively correlated with Annexin V+ sperm (r = -0.553, p<0.01); mtDNAcn was significantly negatively correlated with sperm concentration (r = -0.214, p<0.01), total sperm count (r = -0.232, p<0.01), and progressive motility (r = -0.164, p = 0.01); mtDNA integrity was also significantly positively correlated with sperm concentration (r = 0.195, p<0.01), total sperm count (r = 0.185, p<0.01), and progressive motility (r = 0.106, p = 0.043). After adjusting for potential confounders, these relationships remained significant. Furthermore, we explored the potential effects of lifestyles on such mitochondrial biomarkers and found that the current drinkers displayed a higher level of sperm MMP; additionally, mt DNAcn was increased with age. The results indicated that certain mitochondrial biomarkers could serve as predictors of semen quality in a general population, and the study provides a baseline for the effects of population characteristics and lifestyles on such mitochondrial markers.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer.

Sperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

The effect of aminoguanidine on sperm motility and mitochondrial membrane potential in varicocelized rats.

Objective(s):Increased levels of nitric oxide (NO) in the testicular veins of people suffering from varicocele have already been reported. However, the role of NO-synthase (NOS) isozymes and their inhibitors have not been extensively studied. We aimed to evaluate the inhibitory effects of aminoguanidine (AG), on sperm motility, vitality, and mitochondrial membrane potential (MMP) in varicocelized rats.Materials and Methods:Twenty fore male Wister rats were divided into control, sham, varicocele, and treatment groups. Varicocele and treatment groups underwent partial ligation of left renal vein. Rats in the sham group underwent the same procedures as the varicocele group with the exception of vein ligation. 10 weeks after varicocele induction, sperm parameters were evaluated in all groups. The treatment group received 50 mg/kg AG injection daily for 10 weeks after which they were sacrificed prior to assessment of the parameters. Sperm viability and MMP were assessed by flow cytometry using propidium iodide (PI) and rhodamine 123 (Rh123), respectively.Results:The results of this study show a decrease in sperm viability, motility and MMP in the varicocele group compared with the other groups. After AG injection, we observed that all the parameters were significantly enhanced in the treatment group compared with the other groups. Rh123 staining revealed a positive relation between MMP and sperm motility, whereas PI staining showed a positive relation between sperm motility and viability.Conclusion:The findings of our study show that AG improves sperm motility and MMP, and thus, might be useful in the management of varicocele-related infertility.

Beneficial effect of extracellular adenosine 5'-triphosphate treatment on the Indochinese leopard (Panthera pardus delacouri) sperm quality after cryopreservation.

The Indochinese leopard (Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization (IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5'-triphosphate (ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10×106 cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30min and 3hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process (p≤.05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ (p>.05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced (p≤.05) by ATPe treatment (1.0 and 2.5 mM; 10min to 3hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) (p≤.05). The percentage of pronuclear formation and cleaved embryos was increased (p≤.05) after 1.0 ATPe treatment (49.8±2.8; 45.9±1.5) compared to 0 mM (41.4±3.3; 38.9±0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe activated the velocity of Indochinese leopard sperm motility that may lead to faster sperm/oocyte binding and sperm penetration (factors of successful embryo development).

Polycyclic aromatic hydrocarbons exposure decreased sperm mitochondrial DNA copy number: A cross-sectional study (MARHCS) in Chongqing, China

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that have adverse effects on the male reproductive function. Many studies have confirmed that PAHs preferentially accumulate in mitochondria DNA relative to nuclear DNA and disrupt mitochondrial functions. However, it is rare whether exposure to PAHs is associated with mitochondrial damage and dysfunction in sperm. To evaluate the effects of PAHs on sperm mitochondria, we measured mitochondrial membrane potential (MMP), mitochondrial DNA copy number (mtDNAcn) and mtDNA integrity in 666 individuals from the Male Reproductive Health in Chongqing College Students (MARHCS) study. PAHs exposure was estimated by measuring eight urinary PAH metabolites (1-OHNap, 2-OHNap, 1-OHPhe, 2-OHPhe, 3-OHPhe, 4-OHPhe, 2-OHFlu and 1-OHPyr). The subjects were divided into low, median and high exposure groups using the tertile levels of urinary PAH metabolites. In univariate analyses, the results showed that increased levels of 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu were found to be associated with decreased sperm mtDNAcn. After adjusting for potential confounders, significantly negative associations of these metabolites remained (p = 0.039, 0.012, 0.01, 0.035, respectively). Each 1 μg/g creatinine increase in 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu was associated with a decrease in sperm mtDNAcn of 9.427%, 11.488%, 9.635% and 11.692%, respectively. There were no significant associations between urinary PAH metabolites and sperm MMP or mtDNA integrity. The results indicated that the low exposure levels of PAHs can cause abnormities in sperm mitochondria.

Transient scrotal hyperthermia affects human sperm DNA integrity, sperm apoptosis, and sperm protein expression

This prospective randomized clinical study is aimed to evidence the reproductive impairment of frequent scrotal heat exposure. A total of 20 normozoospermic subjects were randomly divided into two groups to undergo testicular warming in a 43°C water bath 10 times, for 30min each time; the subjects in group 1 underwent testicular warming for 10 consecutive days and those in group 2 once every 3days. Sperm chromatin structure assay (SCSA), sperm mitochondrial membrane potential (MMP), apoptosis, and seminal plasma-soluble Fas (sFas) were analyzed before treatment and every 2weeks after, for a total of 10 times. In group 1, some critical proteins involved in heat stress, hypoxia, structure, and function of sperm mitochondria and flagella were evaluated before hyperthermia and 2, 6, 10, and 16weeks after hyperthermia. Both groups showed a reversible increase in the proportion of spermatozoa with a disrupted MMP (both p<0.05 when the minimums were compared with baseline levels, the same below), sperm apoptosis (both p<0.01) and high DNA stainability (both p<0.05). The sFas concentration in both groups showed no obvious changes except one: the value at week 2 was significantly increased over baseline in group 1 (p=0.036). The level of Bcl-2 decreased significantly at weeks 6 and 10 (p=0.017 and 0.05, respectively) and recovered to baseline at week 16. Proteins involved in heat stress and mitochondria functions were up-regulated, whereas in flagella structure and function was down-regulated (all p<0.05). This study demonstrated that transient and frequent scrotal hyperthermia severely and reversibly damaged spermatogenesis, consecutive heat exposure had more serious effects than intermittent exposure, whereas intermittent exposure led to a later recovery of sperm damage.

Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion), scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot) and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342). The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

Protective effect of silymarin on viability, motility and mitochondrial membrane potential of ram sperm treated with sodium arsenite

Sodium arsenite can impair male reproductive function by inducing oxidative stress. Silymarin is known as a potent antioxidant. This study was performed to investigate if silymarin can prevent the adverse effect of sodium arsenite on ram sperm viability, motility and mitochondrial membrane potential. Epidydimal spermatozoa obtained from ram were divided into five groups: 1) Spermatozoa at 0 hr, 2) spermatozoa at 180 min (control), 3) spermatozoa treated with sodium arsenite (10 μM) for 180 min, 4) spermatozoa treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 min and 5) spermatozoa treated with silymarin (20 μM) for 180 min. MTT assay and Rhodamine 123 staining were used to assess sperm viability and mitochondrial membrane potential respectively. Sperm motility was performed according to World Health Organization (WHO) guidelines. Viability (p<0.01), nonprogressive motility (p<0.001) and intact mitochondrial membrane potential (p<0.001) of the spermatozoa were significantly decreased in sodium arsenite treated group compared to control group. In silymarin + sodium arsenite group, silymarin could significantly reverse the adverse effect of sodium arsenite on these sperm parameters compared to sodium arsenite group (p<0.001). In addition, the application of silymarin alone for 180 minutes could significantly increase progressively motile sperm (p<0.001) and decrease non motile sperm (p<0.01) compared to the control. Silymarin could compensate the adverse effect of sodium arsenite on viability, nonprogressive motility and mitochondrial membrane potential of ram sperm.

Sperm mitochondrial membrane potential and motility pattern in the Holstein bull semen positive for heparin binding proteins

Present study was aimed to evaluate sperm motility and mitochondrial membrane potential (MMP) in relation to the presence of heparin binding proteins (HBPs) in semen. Semen was collected from 17 Holstein Friesian breeding bulls. Seminal plasma and sperm membrane proteins were isolated by precipitation method; HBPs were isolated using heparin-sepharose affinity chromatography. SDS-PAGE was performed and bulls were categorized into 3 groups based on presence/absence of 24 and 30 kDa HBPs. Computer-assisted semen analysis revealed that fresh ejaculates of bulls positive for 30 kDa HBP had 4.24 and 14.18% of higher total motile sperm than semen with 24 kDa and negative for HBPs. Semen with 30 and 24 kDa had 18.72 and 12.34% of higher number of sperm with progressive motility and velocity than semen devoid of HBPs. Simultaneous assessment of plasma membrane integrity (PMI) and MMP by fluorescence staining revealed that post-thaw sperm of semen with 30 kDa HBP had 6.28 and 12.79% of higher intact PMI. Further, sperm of semen with 30 kDa HBP had 4.61 and 10.50% of higher MMP, which had travelled an extra distance of 9.71 and 4.95 mm through cervical mucus than semen with 24 kDa and devoid of HBPs. The mucus penetration distance showed a strong positive correlation with high MMP, PMI, sperm motility and velocity. It is to be concluded that HBPs were able to sustain higher percentage of motile sperm with progressive motility and MMP which was depicted by more mucus penetration distance.