Influence of sampling factors on canine sperm motility parameters measured by the Sperm Class Analyzer

The aim of the present study was to investigate the effect of different technical settings and semen processing on sperm motility parameters measured by the Sperm Class Analyzer (SCA). Semen was collected from 3 dogs, pooled, and diluted in phosphate buffered saline and subsequently assessed by the SCA for the different sperm motility characteristics. The data were statistically analyzed by ANOVA and the repeatability was assessed by coefficient of variation (CV). After a principal component analysis, the reliability was determined with intra-class correlation coefficient (ICC). In experiment 1, the CV's were below 10% for all evaluated parameters. Significant differences (P??0.05) in SCA read-outs were found between the number of microscopic fields captured (1, 2, 3, 4, or 5 fields), yielding the highest ICC (0.83) when 3 fields were captured. No significant differences (P?>?0.05) in motility parameters were found between the number of cells analyzed in each field (20, 50, and 100 spermatozoa) with the exception of beat cross frequency. Reliability of the SCA was good (ICC?=?0.71 to 0.90) for all motility measurements when 20 (ICC?=?0.89) or 50 (ICC?=?0.77) cells were captured in each field, but only just acceptable (ICC?=?0.51 to 0.70) when 100 cells were counted (ICC?=?0.67). The frame settings significantly (P?相似文献   

Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility

We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n?=?81) and non-male factor (NMF, n?=?52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p?<?0.001). Sperm chromatin damage was correlated with percentage of CMA₃- positive sperm (r?=?0.64, p?<?0.001) and with sperm concentration (r?=??0.36, p?<?0.001), motility (r?=??0.21, p?<?0.05), and morphologically normal spermatozoa (r?=??0.29, p?<?0.001). Linear regression showed sperm chromatin damage was related to percentage of CMA₃- positive sperm (p?<?0.001) in ungrouped patients. It was related to both percentage of CMA₃- positive sperm and serum DHEA in the MF group (p?<?0.001 and p?<?0.05, respectively). Sperm chromatin maturity assessed by CMA₃ test was inversely related to sperm chromatin damage assessed by the toludine blue assay. Male factor infertility associated with sperm chromatin damage may be related to sperm protamination and to serum DHEA.     

Relationship of seminal plasma antioxidants and serum male hormones with sperm chromatin status in male factor infertility

We explored the relationship between sperm chromatin integrity, hormone levels, seminal plasma total antioxidant capacity (TAC), and routine sperm parameters in men with male factor (MF, n?=?81) and non-male factor (NMF, n?=?52) infertility. Semen and blood were collected and examined from men undergoing evaluation for infertility in the Avicenna Infertility Clinic. We have examined each patient for serum hormones (LH, FSH, E2, DHEA), sperm chromatin damage, level of protamination and seminal plasma TAC. Levels of FSH, LH, sperm chromatin damage, and abnormal protamination were significantly higher in MF vs. NMF groups (p?相似文献   

DNA fragmentation status in patients with necrozoospermia

The aim of this study was to determine if a relationship exists between the levels of sperm DNA fragmentation and necrospermia in infertile men. Semen samples obtained from 70 men consulting for infertility evaluation were analyzed according to World Health Organization (WHO) guidelines. Patients were subdivided into three groups according to the percentage of necrotic spermatozoa: normozoospermia (<30%; n?=?20), moderate necrozoospermia (50-80%; n?=?30), and severe necrozoospermia (>80%; n?=?20). DNA fragmentation was detected by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay. The sperm DNA fragmentation index (DFI) was 9.28?±?2.98% in patients with a normal level of necrotic spermatozoa, 20.25?±?3.21% in patients with moderate necrozoospermia, and 35.31?±?5.25% in patients with severe necrozoospermia. There was a statistically significant increase of DNA fragmentation in the necrozoospermic group (P?<?0.01). A strong correlation was found between the degree of necrozoospermia and sperm DNA fragmentation. We concluded that patients with necrozoospermia showed a high level of DNA fragmentation compared to normozoospermic men. Severe necrozoospermia (>80%) is a predictive factor for increased sperm DNA damage.     

RELATIONSHIP BETWEEN SOME REPRODUCTIVE PARAMETERS AND BIOCHEMICAL PROPERTIES OF BLOOD SERUM IN RAMS

The objective of the present study was to describe the relationship between some reproductive parameters, hormonal levels, and some biochemical properties of blood serum in rams under the Afyon province conditions. The total protein and globulin were positively (P?<?0.01) correlated with sperm motility and sperm concentration in all of rams. Total lipid was negatively (P?<?0.05) correlated with sperm motility and concentration in Daglic, and it was positively (P?<?0.01) correlated with percentage of abnormal spermatozoa in Chios rams. Total lipid and cholesterol was positively (P?<?0.01) correlated with triiodothyronine in all of rams and was negatively (P?<?0.05) correlated with testosterone in Daglic rams. Percentage of abnormal spermatozoa were negatively (P?<?0.01) correlated with alanine amino transaminase (ALT) level, but were positively (P?<?0.01) associated with aspartate amino transaminase (AST) level and AST/ALT ratio in all of rams.     

A comparison of ejaculated and testicular spermatozoa aneuploidy rates in patients with high sperm DNA damage

Testicular spermatozoa are utilized to achieve pregnancy in couples with severe male factor infertility. Several studies suggest that aneuploidy rates in spermatozoa are elevated at the testicular level in infertile patients compared to ejaculates of normal controls. However, essential data regarding aneuploidy rates between ejaculated and testicular spermatozoa in the same individuals is lacking. The purpose of our study was to compare aneuploidy rates at the testicular and post-testicular level from the same patients with persistently high sperm DNA damage. Ejaculates and testicular biopsies were obtained from eight patients with persistently high DNA damage (>30%). Both ejaculated and testicular samples were analyzed for sperm DNA damage and sperm aneuploidy for chromosomes 13, 18, 21, X, and Y. In addition, semen samples from ten normozoospermic men presenting for fertility evaluation served as a control group. A strong correlation between the alteration of spermatogenesis and chromatin deterioration was observed in our study. In the same individuals, testicular samples showed a significantly lower DNA damage compared to ejaculated spermatozoa (14.9%?±?5.0 vs. 40.6%?±?14.8, P?<?0.05), but significantly higher aneuploidy rates for the five analyzed chromosomes (12.41%?±?3.7 vs. 5.77%?±?1.2, P?<?0.05). While testicular spermatozoa appear favourable for ICSI in terms of lower DNA damage, this potential advantage could be offset by the higher aneuploidy rates in testicular spermatozoa.     

Oleuropein protects against ethanol-induced oxidative stress and modulates sperm quality in the rat testis

The aim of the present study was to evaluate the antioxidant properties of oleuropein on ethanol-induced oxidative stress in the rat testis. 32 adult male Sprague?CDawley rats were divided into four equal groups: the first group as a control, the second group of rats were given ethanol (4?g/kg), the third group received oleuropein (15?mg/kg), and the fourth group of rats were supplemented of oleuropein (15?mg/kg) and after 120?min were ingested via ethanol (4?g/kg) orally. Oleuropein could prevent the reduction of motility and plasma membrane integrity of the spermatozoa against ethanol-induced oxidative stress in treated rats (P?<?0.05). While, thiobarbituric acid reactive substances (as a lipid peroxidation marker) concentration was decreased significantly in the oleuropein plus ethanol group compared to the ethanol group (P?<?0.05). Notably, glutathione peroxidase and superoxide dismutase activity increased significantly in the ethanol-treated animals compared to controls and total glutathione (GSH-content) increased significantly in ethanol-treated rats compared to the oleuropein group. Our findings suggest that oleuropein possesses beneficial antioxidant effects on ethanol-induced sperm toxicity, subsequently enhancing sperm motility and plasma membrane integrity.     

DHR123: an alternative probe for assessment of ROS in human spermatozoa

The aim of this study was to assess the potential of dihydrorhodamine 123 (DHR123) to measure oxidative stress produced by human spermatozoa. The results were compared with 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) that is routinely used for assessment of H₂O₂ produced by spermatozoa. Fluorescence intensity and percentage R123 and DCF positive sperm were measured by flow cytometry. The optimal condition for assessment of reactive oxygen species (ROS) produced by sperm with DHR123 was 0.05 µM for 1 million sperm per ml for 20 minutes. The results of ROS measurement by DHR123 showed a significant correlation (r?=?+0.818, P?<?0.001) with DCFH-DA staining. Immunofluorescence of sperm stained with DHR123 revealed ROS production in the sperm mid-piece. In addition a significant correlation was observed between oxidant production assessed by DHR123 and semen parameters. Therefore, DHR123 may be considered a suitable probe for estimating oxidants produced by human spermatozoa, and can present heterogeneity in oxidant production between different samples.     

Inhibition of the metabolism and motility of human spermatozoa by various alkaloids

The ability of several alkaloids to inhibit the metabolism and motility of human spermatozoa has been investigated.Of the agents tested, chloroquine was the most effective in inhibiting sperm metabolism (production of carbon dioxide and lactic acid) and motility. It was active at a concentration of 3.6 × 10^?5M. Quinine and Quinacrine were active at concentrations of 5 × 10^?4M and emetine required concentrations as high as 3.6 × 10^?3M to achieve an inhibitory effect. Detailed studies with emetine showed that the time needed for inhibition of sperm motility was inversely proportional to the drug concentration and directly related to the sperm density. In addition, the inhibition was shown not to be reversible.     

Profiling of sperm proteins and association of sperm PDC-109 with bull fertility

Abstract

The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n?=?12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n?=?3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1–5.6, 37%) and basic (pI; 7.9–10.0, 27%) proteins and their pattern of expression varied significantly (p?<?0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4?kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p?<?0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.     

Comparative study of sperm washing and selection methods after cryopreservation and its influence on sperm subpopulational structure in a bovine model

Abstract

The effect of different sperm washing-selection methods on sperm morphometric characteristics as a study to detect differences in the subpopulational structure has been carried out in detail in a bovine model. Cryopreserved sperm samples from 5 bulls were thawed, pooled, and processed by TALP-washing centrifugation method (TWCM), selective Percoll discontinuous density-gradient centrifugation method (PDGM), and self-migration swim-up separation method (SUMM). Live-dead assay (SYBR-14/ethidium homodimer-1), chlortetracycline assay (CTC), and sperm motility were assessed, and aliquots of sperm were processed for automated sperm morphometry analysis (ASMA) simultaneously before (raw thawed sperm used as control, RTS) and after different sperm washing-selection techniques. Deleterious effects of different methods were evident, particularly on sperm membrane integrity (p?<?0.05) and capacitation status (p?<?0.05). Moreover, each cell was measured for four primary dimensional parameters, and three shape parameters. All sperm morphometric parameters evaluated were analyzed by principal component analysis (PCA) and multivariate clustering analyses. PCA revealed two principal components for each sperm washing or separation method explaining more than the 91% of the variance. The number of subpopulations found was the same for all methods (four) except for PDGM (three). However, irrespective of the number of subpopulations defined by PCA and clustering analyses, the sperm subpopulational structure was found to be different and strongly influenced by the sperm selection procedure due to statistical differences found regarding the sperm biophysical changes induced by each method used (p?<?0.001). It is concluded that different sperm washing-selection methods commonly used during IVF process, may lead to alterations in sperm morphometric characteristics, which might explain the different results seen after IVF, since an important influence of these methods on sperm subpopulational structure has been demonstrated.     

Does sperm DNA fragmentation affect the developmental potential and the incidence of apoptosis following blastomere biopsy?

Common methods employed in assisted reproduction technology (ART) include intracytoplasmic sperm injection (ICSI) with an unspecified level of sperm DNA fragmentation (SDF) and preimplantation genetic diagnosis (PGD). The aim of this study was to investigate the impact of SDF on human preimplantation embryo development and the incidence of apoptosis following a single blastomere biopsy. Using sperm chromatin dispersion (SCD) to assess SDF, a total of 20 processed semen samples were categorized into two groups; group I: SDF ≤30% and group II: SDF >30%. After ICSI, fertilization, cleavage, and embryo quality score were assessed. A single blastomere was biopsied from day 3 embryos and development was monitored on day 4. The frequency of apoptosis in biopsied embryos was assayed by TUNEL and the level of BCL-2, BAX, hsa-mir-15a, and hsa-mir-16-1 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). SCD was found to be negatively correlated with sperm motility and normal form spermatozoa (p?<?0.05). The rate of fertilization, cleavage, and embryo quality score were not significantly different between the two groups (all p?>?0.05). SDF >30% had no negative effect on potential development and did not increase the proportion of apoptotic cells and the level of apoptosis-related genes and microRNAs (miRNAs) in group II vs. group I (p?>?0.05). It appears that at the levels assessed paternal genome damage had little if any negative effect on preimplantaton embryo development and apoptosis following single blastomere biopsy. This may reflect the selection of morphologically normal sperm for ICSI and the repair capacity of the oocyte.     

The effects of probiotics on mental health and hypothalamic–pituitary–adrenal axis: A randomized,double-blind,placebo-controlled trial in petrochemical workers

Objective: The aim of this study was to determine effects of probiotic yogurt and multispecies probiotic capsule supplementation on mental health and hypothalamic–pituitary–adrenal axis in petrochemical workers.

Methods: The present randomized double-blind, placebo-controlled trial was conducted on 70 petrochemical workers. Subjects were randomly divided into three groups to receive 100?g/day probiotic yogurt?+?one placebo capsule (n?=?25) or one probiotic capsule daily?+?100?g/day conventional yogurt (n?=?25) or 100?g/day conventional yogurt?+?one placebo capsule (n?=?20) for 6 weeks. Mental health parameters including general health questionnaire (GHQ) and depression anxiety and stress scale (DASS) scores were measured. Fasting blood samples were obtained at the beginning and 6 weeks after the intervention to quantify hypothalamic–pituitary–adrenal axis.

Results: After 6 weeks of intervention, a significant improvement of GHQ was observed in the probiotic yogurt (18.0?±?1.5 vs. 13.5?±?1.9, P?=?0.007) and in the probiotic capsule group (16.9?±?1.8 vs. 9.8?±?1.9, P?=?0.001), as well as a significant improvement in DASS scores in the probiotic yogurt (23.3?±?3.7 vs. 13.0?±?3.7, P?=?0.02) and the probiotic capsule group (18.9?±?3.2 vs. 9.4?±?4.0, P?=?0.006). However, there was no significant improvement in the conventional yogurt group (P?=?0.05 for GHQ and P?=?0.08 for DASS).

Discussion: The consumption of probiotic yogurt or a multispecies probiotic capsule had beneficial effects on mental health parameters in petrochemical workers.     

Apolipoprotein E genotypes of fertile and infertile men

Lipid components of spermatozoa have an important role in the functional activity of this cell. The protein, apolipoprotein E (apoE), has a central role in lipid transport. The aim of this study was to investigate the distribution of APOE genotypes, ?3?3, ?3?4, and ?2?3, and the corresponding alleles in fertile and infertile males, and to assess the semen parameters from the patients carrying the different alleles. In addition, the levels of cholesterol, phospholipid, and triacylglycerol in spermatozoa, isolated by PureSperm gradient and from seminal plasma in samples from infertile males was compared with respect to the APOE genotype. APOE genotypes were determined by PCR-RFLP on DNA extracted from peripheral blood leucocytes in 108 fertile and 107 infertile men. There was a significant difference between the distribution of APOE genotypes in fertile as compared to infertile males (χ²?=?9.1, df?=?2, p?=?0.011). The presence of genotype ?3?4 conferred a 3.82 risk factor for male infertility {Odds ratio?=?3.82 (1.46-10, p?=?0.006)}. Our findings showed that the distribution of APOE genotypes and alleles differed between fertile and infertile individuals and may be a risk factor for male infertility. We suggest that the effects of APOE genotypes may be linked to differences in the efficacy of the expressed apoE isoforms in promoting sperm maturation during epididymal transit.     

LESS NO PRODUCTION AND BETTER MOTION PARAMETER IN HUMAN SPERM BY SWIM-UP PROCESSING

Fifteen semen specimens were obtained from men for semen analysis; each was divided into two aliquots for prepararation. The motile sperm recovery rate, percentage motility, and motion parameters were measured for each semen specimen (n?=?15) before and after preparation with the use of the two methods, and cultured with different time courses (1?hr, 3?hr, and 6?hr). Nitric oxide (NO) was measured using the chemiluminscence method after centrifugation. Recovery rate of motile cell was significantly higher in direct swim-up method (69.5?±?42.4% versus 49.3?±?29.3%, p?<?0.05). In motility, direct swim-up method in the different time courses was significantly better than IxaPrep method. (1?hr: 91.1?±?5.2% vs 65.6?±?16.4%, 3?hr: 87.2?±?7.9% vs 65.2?±?16.5%, 6?hr: 86.1?±?7.5% vs 60.8?±?17.6% and prewash: 61.6?±?16.2%, p?<?0.05). In VAP and VSL, the sperm prepared by the above two methods all improved compared to pre-wash sperm (p?<?0.05), but there was no statistical significance between the two methods. NO production in the direct swim-up group was significantly lower than IxaPrep group in the first hour of culture (0.09?±?0.09?uM vs 0.15?±?0.09?uM, p?<?0.05). NO production increased as the culture time increased in swim-up group, but conversed in IxaPrep group. The lower level of NO produced in the swim-up group may suggest that better sperm quality achieved is due to the decreased NO production.     

Copper: a biphasic regulator of caprine sperm forward progression

Abstract

Copper is essential for spermatogenesis and its presence has been demonstrated in male and female reproductive fluids in several mammalian species. However, little is known about the physiological significance of this trace element in the regulation of forward progression of mammalian sperm cells which is essential for sperm fertility potential in vivo. The purpose of this investigation was to determine the physiological role of the bivalent copper ion (Cu²⁺) on mammalian sperm forward motility using a chemically-defined medium and caprine cauda epididymal sperm model. Sperm forward motility was significantly enhanced by Cu²⁺ in a dose-dependent manner; maximal activation (approx 20%) was noted at the 5?µM level of the metal. Above 10?µM Cu²⁺ sperm motility decreased, showing that Cu²⁺ exerts a biphasic regulation on sperm motility. These findings have been confirmed using a spectrophotometric motility assay, an objective method of motility analysis. At lower concentrations (up to 5?µM), copper enhanced sperm membrane lipid peroxidation as well as the level of intra-sperm cyclic adenosine mono phosphate (c-AMP), but at a higher level it caused marked inhibition of both of the biochemical parameters. The observed correlation of Cu²⁺-dependent biphasic modulation of sperm membrane lipid peroxidation and intrasperm c-AMP with sperm forward motility is consistent with the view that Cu²⁺ regulation of sperm motility is mediated by membrane lipid peroxidation, which in turn modulates the level of intra-sperm c-AMP, a well-known activator of sperm motility.     

男性不育患者精浆锌含量与精液主要参数相关性研究

目的:探讨不育男性精浆的锌含量与精液质量的关系。方法:回顾性分析2011年8月～2012年1月在广西壮族自治区妇幼保健院生殖中心就诊的343例男性不育患者的相关资料,依据精浆锌含量分为正常A组(n=274例)和异常B组(n=69例),比较两组间精液参数的差异;同时根据精液黏稠度分为黏稠C组(n=54例)与非黏稠D组(n=289例),比较两组间精浆锌含量及其他精液参数的差异。结果:A组与B组患者年龄比较差异无统计学意义(P>0.05)。A组的精液量、每次射精精子总数、前向运动精子总数显著高于B组,而黏稠精液的比例明显低于B组,差异均有统计学意义(P<0.05)。其他精液各指标比较差异无统计学意义(P>0.05);C组与D组精液圆细胞浓度、精液量、精子浓度、精子总数比较差异无统计学意义(P>0.05);D组的前向运动精子百分率、前向运动精子总数、活动率、精浆锌明显高于C组,差异有统计学意义(P<0.05)。精浆锌含量与精液量、每次射精精子总数、前向运动精子总数显著正相关,与其他参数无显著相关性。结论:精浆锌含量直接影响精液量、精子总数、前向运动精子总数和精液黏稠度,精浆锌含量是男性生殖力的重要评估指标。     

Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus)

Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 +/- 4% purity) of X- and Y-bearing spermatozoa was 3400 +/- 850 spermatozoa sex(-1) s(-1). In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen-thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.     

Flexible Eating Behavior Predicts Greater Weight Loss Following a Diet and Exercise Intervention in Older Women

Eating behaviors (cognitive restraint, flexible and rigid restraint, disinhibition, hunger) have been associated with obesity and weight loss success in middle-aged individuals, but little is known about these relationships in older adults. This study examined relationships between eating behaviors and weight loss in overweight/obese older women (n?=?61; 69?±?3.6 years; body mass index?=?31.1?±?5.0?kg/m²) completed a 6-month behavioral weight loss intervention. Baseline, postintervention, and change measures of eating behaviors (51-items Three-Factor Eating Questionnaire) were assessed for relationships with weight loss. In the final regression model, an increase in flexible restraint accompanied by a decrease in rigid restraint predicted greater weight loss (adjusted R²?=?0.21, Model F (4, 56)?=?4.97, P?P?>?0.05). Results suggest encouraging a flexible approach to eating behavior and discouraging rigid adherence to a diet may lead to better intentional weight loss for overweight and obese older women.     

Thiol oxidation by nitrosative stress: Cellular localization in human spermatozoa

Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS^® system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility.

Abbreviations: DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO^?: peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation ANOVA: analysis of variance