Exposure Of Spermatozoa Research Articles

Potential role of the acrosome of sturgeon spermatozoa in the fertilization process

Sturgeon gametes are unique in that their spermatozoa possess acrosome containing acrosomal enzymes whereas their eggs are perforated by numerous micropyles. Thus the function of acrosome, which undergoes acrosomal reaction and filament formation, is unclear. The acrosomal reaction of sterlet Acipenser ruthenus spermatozoa was induced by sperm exposure to the solution containing Ca2+ and high pH. The rate of affected spermatozoa was evaluated using Soybean Trypsin Inhibitor-Alexa conjugate fluorescent staining. Fertilization and hatching test using sperm of this way disrupted acrosome and non-treated sperm as a control proofed necessity of acrosome integrity for fertilization. Increasing percentage of spermatozoa with disrupted acrosome highly decreased percentage of hatching, in spite of electron microscopy verification that the spermatozoa bearing such disrupted acrosomes were able to reach the micropyle. In addition the significant positive correlation between the ability of spermatozoa to induce acrosomal reaction by activation solution and sperm fertilizing ability suggested that spermatozoa, which are able to activate the acrosomal reaction, are more successful in fertilization.

Mechanism of action of mercury on sperm morphology, adenosine triphosphate content, and motility in Perca fluviatilis (Percidae; Teleostei)

The main objectives of the present study were to investigate the performance of mercury chloride (HgCl2 ) on sperm function and structure, identify sites of action of HgCl2 , and investigate the mechanism of action of HgCl2 on fish (Perca fluviatilis L.) spermatozoa. Direct exposure of nonincubated sperm decreased sperm motility and velocity in a dose-dependent manner and was totally suppressed at 250 µM HgCl2 . Adenosine-5'-triphosphate (ATP) content of sperm after activation in an activation medium (AM) containing more than 25 µM HgCl2 did not differ compared with nonactivated sperm. Motility and velocity of demembranated sperm decreased after activation in an AM containing 62 µM HgCl2 , and was totally suppressed at 250 µM HgCl2 . Incubation of sperm in an immobilizing medium (IM) containing HgCl2 enhanced HgCl2 effects after sperm activation in an AM containing HgCl2 . Sperm motility of incubated sperm in an IM without HgCl2 was totally suppressed at 125 µM HgCl2 after 3 h incubation. In case of incubated sperm in an IM containing HgCl2 , sperm motility was totally suppressed at 31 µM HgCl2 . Adenosine-5'-triphosphate content of sperm was significantly lower in an IM containing HgCl2 greater than 3 µM compared with those of the control (no HgCl2 ) and lower HgCl2 concentrations. Damage to the plasma membrane and axoneme were observed in sperm incubated in an IM containing HgCl2 compared with the control, when HgCl2 concentration and incubation time increased. In conclusion, HgCl2 acts on sperm through disruption of function of the plasma membrane, axoneme, and ATP content.

Prostasomes as Potential Modulators of Tyrosine Phosphorylation in Human Spermatozoa

Prostasomes, vesicles present in human semen, are known to play a role in male fertility. However, the mechanisms involved are still poorly understood. The present study looks at the direct influence of different concentrations of prostasomes on human sperm function in conditions supporting capacitation in vitro. Five million Percoll selected spermatozoa were incubated for 3 h at 370C under an atmosphere of 5% CO2 in air, in 100 µl Biggers Whitten Whittingham's medium (BWW) containing polyvinyl alcohol (PVA, 1 mg/ml) and bovine serum albumin (BSA; 3 mg/ml) in the absence or presence of increasing concentrations of prostasomes (expressed in terms of their cholesterol content: 15; 30; 45 nmoles per 100 µL of incubation medium). After in vitro exposure of spermatozoa to prostasomes, our data indicate that i) tyrosine phosphorylation intensity of the 107 KDa protein band was dose dependently lower and ii) the percentage of viable and progressive motile spermatozoa was unchanged and the percentage of non-progressive motility decreased. In addition, the incubation of prostasomes with spermatozoa resulted in an enrichment of their lipid content. Our experiments suggest that adhesion of prostasomes to spermatozoa could be responsible for the decrease in Tyrosine phosphorylation and the alteration of the mean curvilinear velocity (VCL) and the average path velocity (VAP).

Metabolism of motile zebrafish sperm

As metabolism of motile fish sperm is not well understood, the current study examined the metabolism of saline-activated zebrafish (Danio rerio) sperm. Activation of sperm with inhibitors of oxidative phosphorylation (potassium cyanide, 2,4 dinitrophenol or carbonyl cyanide 3-cholorophenylhydrazone) negatively impacted sperm motility by 60–90s postactivation. Incubation of quiescent sperm with 2,4 dinitrophenol prior to activation resulted in a 67% decrease in the percent motile sperm assessed 15s postactivation. Thus, production of ATP in quiescent sperm is important for motility upon activation and nascent ATP production via oxidative phosphorylation by motile sperm appears important at 60–90s postactivation. Exposure of sperm to iodoacetamide, an inhibitor of creatine kinase, at activation was without effect. However, incubation of quiescent sperm with iodoacetamide prior to activation resulted in a 77% reduction in percent motile sperm and decreased velocity and wobble at 15s postactivation. These results suggest that creatine kinase and phosphocreatine shuttle are physiologically important at, or shortly after the initiation of motility. Finally, sperm were exposed to lactate, pyruvate, or acetate as well as to several monosaccharides upon activation. The results provided no evidence supporting any metabolic role of exogenous organics (potentially from the female via ovarian fluid) in sperm once motility has begun.

Involvement of mitochondrial activity in mediating ELF‐EMF stimulatory effect on human sperm motility

It has recently been reported that the exposure of human spermatozoa to an extremely low frequency (ELF) electromagnetic field (EMF) with a square waveform of 5 mT amplitude and frequency of 50 Hz improves sperm motility. The functional relationship between the energy metabolism and the enhancement of human sperm motility induced by ELF-EMF was investigated. Sperm exposure to ELF-EMF resulted in a progressive and significant increase of mitochondrial membrane potential and levels of ATP, ADP and NAD(+) that was associated with a progressive and significant increase in the sperm kinematic parameters. No significant effects were detected on other parameters such as ATP/ADP ratio and energy charge. When carbamoyl cyanide m-chlorophenylhydrazone (CICCP) was applied to inhibit the oxidative phosphorylation in the mitochondria, the values of energy parameters and motility in the sperm incubated in the presence of glucose and exposed to ELF-EMF did not change, thus indicating that the glycolysis was not involved in mediating ELF-EMF stimulatory effect on motility. By contrast, when pyruvate and lactate were provided instead of glucose, the energy status and motility increased significantly in ELF-EMF-treated sperm. Under these culture conditions, the inhibition of glycolitic metabolism by 2-deoxy-D-glucose (DOG) again resulted in increased values of energy and kinematic parameters, indicating that gluconeogenesis was not involved in producing glucose for use in glycolysis. We concluded that the key role in mediating the stimulatory effects exerted by ELF-EMF on human sperm motility is played by mitochondrial oxidative phosphorylation rather than glycolysis.

Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen supplemented with catalase or Trolox

Semen manipulation and cryopreservation-thaw procedures may accelerate the generation of reactive oxygen species (ROS). Sperm exposure to large amounts of ROS has been shown to cause membrane lipid peroxidation and cellular injury to the sperm. The objective of this study was to overcome the ROS production in frozen-thawed ram semen by the addition of the antioxidants catalase or Trolox to semen following thawing. Frozen-thawed ram semen (100 × 10 6 sperm/straw) was supplemented with PBS (control group), 100 μg/ml catalase, or 100 μM Trolox/10 8 sperm (catalase and Trolox being dissolved in PBS) and incubated (37 °C) for 5 min. Under the experimental conditions used in this study, the catalase and Trolox antioxidants failed to protect the sperm from the spontaneous production of ROS. However, when lipid peroxidation was induced by iron (FeSO 4), the addition of Trolox promoted a reduction ( P < 0.05) in the formation of TBARS in the semen, compared to the control and catalase semen samples. The generation of TBARS and H 2O 2 occurred in the extender alone, without the presence of sperm cells. In conclusion, the addition of Trolox to frozen-thawed ram semen could be beneficial as it decreases the production of TBARS when oxidative stress is induced. It is possible that a longer incubation period could lead to different results. The concentration of catalase also needs to be further evaluated. The extender could contribute to the oxidative stress of sperm, as it is a source of ROS during the cryopreservation of semen.

High endogenous melatonin concentrations enhance sperm quality and short‐term in vitro exposure to melatonin improves aspects of sperm motility

Although human seminal fluid contains melatonin and spermatozoa reportedly possess membrane melatonin receptors, there are no experimental studies that have ascertained the relationship between melatonin and male infertility. This study evaluated whether urinary 6-sulfatoxymelatonin and urinary total antioxidant capacity correlate with different seminal parameters including sperm concentration, motility and morphology. Also, the in vitro effects of melatonin on human sperm motility were investigated. Semen samples from 52 men who were counselled for infertility were obtained. Sperm concentration was determined using the haemocytometer method, motility kinematic parameters were assessed using a computer-aided semen analysis system, while morphology and vitality were evaluated after Diff-Quick and Eosin-Nigrosin vital staining, respectively. For the quantification of urinary 6-sulfatoxymelatonin, a commercial ELISA kit was used, and urinary total antioxidant capacity was evaluated by means of a colorimetric assay kit. For the in vitro effects of melatonin, samples were incubated for 30min in the presence or absence of 1mm melatonin. Both urinary 6-sulfatoxymelatonin and total antioxidant capacity levels positively correlated with sperm concentration, motility and morphology, as well as negatively correlated with the number of round cells. Additionally, 30-min exposure of sperm to 1mm melatonin improved the percentage of motile and progressively motile cells and decreased the number of static cells, thereby promoting the proportion of rapid cells. Therefore, melatonin improves semen quality, which is important because melatonin supplementation may be potentially used to obtain a successful assisted reproductive technique outcome.

Energetic metabolism and human sperm motility: impact of CB₁ receptor activation.

It has been reported that the endocannabinoid anandamide (AEA) exerts an adverse effect on human sperm motility, which has been ascribed to inhibition of mitochondrial activity. This seems to be at variance with evidence suggesting a major role of glycolysis in supplying ATP for sperm motility; furthermore, the role of AEA-binding receptors in mediating mitochondrial inhibition has not yet been explored. In this study, human sperm exposure to Met-AEA (methanandamide, nonhydrolyzable analog of AEA) in the micromolar range significantly decreased mitochondrial transmembrane potential (ΔΨm), similarly to rotenone, mitochondrial complex I inhibitor. The effect of Met-AEA (1 μm) was prevented by SR141716, CB(1) cannabinoid receptor antagonist, but not by SR144528, CB(2) antagonist, nor by iodoresiniferatoxin, vanilloid receptor antagonist. The effect of Met-AEA did not involve activation of caspase-9 or caspase-3 and was reverted by washing. In the presence of glucose, sperm exposure either to Met-AEA up to 1 μm or to rotenone for up to 18 h did not affect sperm motility. At higher doses Met-AEA produced a CB(1)-independent poisoning of spermatozoa, reducing their viability. Under glycolysis blockage, 1 μm Met-AEA, similarly to rotenone, dramatically abolished sperm motility, an effect that was prevented by SR1 and reverted by washing. In conclusion, CB(1) activation induced a nonapoptotic decrease of ΔΨm, the detrimental reflection on sperm motility of which could be revealed only under glycolysis blockage, unless very high doses of Met-AEA, producing CB(1)-independent sperm toxicity, were used. The effects of CB(1) activation reported here contribute to elucidate the relationship between energetic metabolism and human sperm motility.

Reduced Fertility In Vitro in Mice Lacking the Cystatin CRES (Cystatin-Related Epididymal Spermatogenic): Rescue by Exposure of Spermatozoa to Dibutyryl cAMP and Isobutylmethylxanthine1

The cystatin CRES (cystatin-related epididymal spermatogenic; Cst8) is the defining member of a reproductive subgroup of family 2 cystatins of cysteine protease inhibitors and is present in the epididymis and spermatozoa, suggesting roles in sperm maturation and fertilization. To elucidate the role of CRES in reproduction, mice lacking the Cst8 gene were generated and their fertility examined. Although both male and female Cst8(-/-) mice generated offspring in vivo, spermatozoa from Cst8(-/-) mice exhibited a profound fertility defect in vitro. Compared to spermatozoa from Cst8(+/+) mice, spermatozoa from Cst8(-/-) mice were unable to undergo a progesterone-stimulated acrosome reaction and had decreased levels of protein tyrosine phosphorylation, suggesting a defect in the ability of Cst8(-/-) spermatozoa to capacitate. Incubation of Cst8(-/-) spermatozoa with dibutyryl cAMP and 3-isobutyl-1-methylxanthine rescued the fertility defect, including the capacity for sperm protein tyrosine phosphorylation. Both untreated Cst8(+/+) and Cst8(-/-) spermatozoa, however, exhibited similar increased total levels of cAMP and protein kinase A (PKA) activity throughout the capacitation time course compared to spermatozoa incubated under noncapacitating conditions. Taken together, these results suggest that mice lacking CRES may have altered local levels of cAMP/PKA activity, perhaps because of improper partitioning or tethering of these signaling molecules, or that the CRES defect does not directly involve cAMP/PKA but other signaling pathways that regulate protein tyrosine phosphorylation and capacitation.

Laptop expositions affect motility and induce DNA fragmentation in human spermatozoa in vitro by a non-thermal effect: a preliminary report

OBJECTIVE: The use of laptops has drastically increased in the last years. These devices are commonly used on the lap near the groin area and may expose the human testes to radio frequency electromagnetic waves (WiFi mode) as well as to high temperatures. There is weak scientific information about the possible impact of exposition to laptops on male reproduction. Therefore, we have assessed the sperm exposure to laptops in an in vitro study.DESIGN: An in vitro prospective study.MATERIALS AND METHODS: Semen samples from 15 men were evaluated. Semen parameters (concentration, motility, morphology and vitality) were assessed. Motile sperm were selected by swim up and separated in two fractions and incubated 4 hours at controlled temperature (25°C). The first aliquot was exposed to the laptop during the incubation times. The second fraction was incubated without exposition (control group). Motility, vitality and sperm DNA fragmentation (TUNEL) was evaluated after incubation in all samples. Comparisons between groups were performed by Student's t test. Data is expressed as mean ± SD.RESULTS: Our results showed decrease progresive sperm motility (73,5 ± 8,2 vs 63,6 ± 7,3; p < 0,05), increase sperm immotily (18,8 ± 6,9 vs 28,3 ± 7,3; p < 0,05) and increase of sperm DNA fragmentation (6,3 ± 8,1 vs 13,1 ± 9,2; p < 0,05) in the exposed group vs non exposed. Levels of non progressive sperm motility and vitality did not show significant difference between the two groups.CONCLUSION: To the best of our knowledge, this is the first study to evaluate the impact of laptops on human spermatozoa. We have demonstrated that exposure to laptops decrease progresive motility and induce DNA fragmentation in human spermatozoa in vitro by a non-thermal effect. We speculate that keeping the laptops (WiFi mode) on the lap near the testes may result in decreased male fertility. Further studies are needed to test this hypothesis and identify the causes why sperm quality is affected by laptops exposition. OBJECTIVE: The use of laptops has drastically increased in the last years. These devices are commonly used on the lap near the groin area and may expose the human testes to radio frequency electromagnetic waves (WiFi mode) as well as to high temperatures. There is weak scientific information about the possible impact of exposition to laptops on male reproduction. Therefore, we have assessed the sperm exposure to laptops in an in vitro study. DESIGN: An in vitro prospective study. MATERIALS AND METHODS: Semen samples from 15 men were evaluated. Semen parameters (concentration, motility, morphology and vitality) were assessed. Motile sperm were selected by swim up and separated in two fractions and incubated 4 hours at controlled temperature (25°C). The first aliquot was exposed to the laptop during the incubation times. The second fraction was incubated without exposition (control group). Motility, vitality and sperm DNA fragmentation (TUNEL) was evaluated after incubation in all samples. Comparisons between groups were performed by Student's t test. Data is expressed as mean ± SD. RESULTS: Our results showed decrease progresive sperm motility (73,5 ± 8,2 vs 63,6 ± 7,3; p < 0,05), increase sperm immotily (18,8 ± 6,9 vs 28,3 ± 7,3; p < 0,05) and increase of sperm DNA fragmentation (6,3 ± 8,1 vs 13,1 ± 9,2; p < 0,05) in the exposed group vs non exposed. Levels of non progressive sperm motility and vitality did not show significant difference between the two groups. CONCLUSION: To the best of our knowledge, this is the first study to evaluate the impact of laptops on human spermatozoa. We have demonstrated that exposure to laptops decrease progresive motility and induce DNA fragmentation in human spermatozoa in vitro by a non-thermal effect. We speculate that keeping the laptops (WiFi mode) on the lap near the testes may result in decreased male fertility. Further studies are needed to test this hypothesis and identify the causes why sperm quality is affected by laptops exposition.

Cytogenetic and developmental toxicity of cerium and lanthanum to sea urchin embryos

The aim of this study was to evaluate the toxicity of two rare earth elements (REE), cerium and lanthanum on sea urchin embryos and sperm. Sea urchin ( Paracentrotus lividus) embryos were reared for 72 h in Ce(IV)- or La(III)-contaminated seawater at concentrations ranging from 10 −8 to 10 −5 M. Cleaving embryos (5 h post-fertilization) were submitted to cytogenetic analysis, scoring mitotic activity and a set of mitotic aberrations. Embryological analysis was carried out to determine percent developmental anomalies and/or embryonic mortality. P. lividus sperm were suspended in Ce(IV) or La(III) (10 −8–10 −5 M) for 1 h, and percent fertilized eggs were scored in cleaving embryos that were cultured up to pluteus stage to score any developmental defects. Embryos reared in 10 −5 M Ce(IV) resulted in 100% embryonic mortality, whereas 10 −5 M La(III) induced 100% developmental defects, without causing any embryonic mortality. A significant concentration-related mitotoxic effect and induction of mitotic aberrations were observed in Ce(IV)-exposed, but not in La(III)-exposed embryos, at concentrations ranging from 10 −7 M to 3 × 10 −6 M. Following sperm exposure, both Ce(IV) and La(III) induced a decrease in sperm fertilization success at the highest tested concentration (10 −5 M). The offspring of Ce(IV)-exposed, but not of La(III)-exposed sperm displayed a significant concentration-related increase in developmental defects. The results may suggest adverse impacts in REE-exposed biota and warrant further studies of a more extended REE series.

Comparative toxicities of aluminum and zinc from sacrificial anodes or from sulfate salt in sea urchin embryos and sperm

The toxicity of aluminum or zinc from either sacrificial anodes (SA) or their sulfate salts (SS) was evaluated in sea urchin (Paracentrotus lividus) embryos or sperm exposed to Al(III) or Zn(II) (SA or SS, 0.1–10μM), scoring developmental defects (DDs), fertilization rate (FR), and mitotic abnormalities. A significant DD increase was observed in SS, but not SA Al(III)- and Zn(II)-exposed embryos vs. controls. Both Al(III) and Zn(II), up to 10μM, from SA and SS, inhibited mitotic activity and induced mitotic aberrations in exposed embryos. SA-Al(III)-exposed sperm displayed a significant FR increase, unlike Al(III) sulfate overlapping with controls. Both SA-Zn(II) and Zn(II) sulfate sperm exposure resulted in a significant FR increase. The offspring of SA-Al(III)-exposed sperm displayed a significant DD decrease, unlike Al(III) sulfate exposure. Zinc sulfate sperm exposure resulted in a significant increase in offspring DDs, whereas SA-Zn(II) sperm exposure decreased DDs. Together, exposures to SA-dissolved Al(III) or Zn(II) resulted in lesser, if any toxicity, up to hormesis, compared to SS. Studies of metal speciation should elucidate the present results.

Potassium ions Improve the Progesterone-Induced [Ca2+]I Increase and Acrosome Reaction as well as the Potassium-Induced [Ca2+]I Increase in Human Spermatozoa

Ejaculated sperm undergo a series of physiological changes in the female reproductive tract to become capable of fertilization. These changes, called capacitation can be induced in vitro in appropriate media and culminate in hyperactivated motility and the ability of the sperm to acro-some-react. These downstream effects of capacitation are mediated by increased intracellular Ca2+ concentration ([Ca2+]i). Exposure of sperm to progesterone (P4) and potassium (K+) induce an increase in [Ca2+]i. The effects of incubating sperm in media containing different potassium concentrations on the P4-induced [Ca2+]i increase and AR as well as the effect of K+ on [Ca2+]i increase were investigated. Swum-up spermatozoa were capacitated for about 6 h in culture me-dia containing either 5.4 (control), 25 or 116.4 mM K+ before being stimulated with P4. P4 evoked a biphasic [Ca2+]i response in spermatozoa incubated in each of the media. The amplitude of the transient P4-induced increase in [Ca2+]i and the percentage of P4-induced AR were significantly lower in sperm incubated in media with very low or very high K+ concentration. Absence of K+ in the incubating medium significantly inhibited the amplitude of both the P4- and K+-induced [Ca2+]i increases. Application of K+ after P4 application evoked a second biphasic [Ca2+]i re-sponse. The results suggest that i) the presence of K+ in the incubating medium is important in the K+-induced [Ca2+]i increase as well as the P4-induced [Ca2+]i increase and AR, ii) human sperm capacitates better in an incubating medium with a K+ concentration around that of ovi-ductal fluid and iii) P4- and K+ utilize different channels for inducing Ca2+ influx.

In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 μM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 μM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE–DNA adducts as measured with 32P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

In vitro Effect of Produced Water on Cod, Gadus morhua, Sperm Cells and Fertilization

The in vitro effect of produced water released by oil and gas platforms was assessed by exposing cod sperm cells to realistic concentrations of this mixture (100, 200, 500 ppm). We investigated produced water impact on enzymes of the aerobic (citrate synthase) and glycolytic metabolism (lactate dehydrogenase), lipid catabolism (lipase), as well as an anti-oxidant enzyme (catalase). Fertilization rates, viability, respiration, ATP, and total motility duration were also evaluated. To explore correlations between these parameters, we have also tested the effect of conserving sperm for 24 h at 4 degrees C. After conservation, fertilization success was decreased but other parameters were not affected. Produced water did not result in a significant change in fertilization; a significant increase in sperm protein amounts and citrate synthase activity can be observed. No correlations are found between parameters showing that sperm viability and unchanged energy levels do not translate into equivalent fertilization capacity. To conclude, exposure of sperm to produced water resulted only in subtle effects on cells. These findings bring information on the effect of produced water on sperm itself rather than on spermatogenesis or testis development of an exposed fish.

349 EFFECT OF CUMULUS CELLS AND MATURATION CULTURE PERIOD OF OOCYTES ON THE SEX RATIO OF IN VITRO-FERTILIZED BOVINE EMBRYOS

Studies have reported a high proportion of males from IVP of embryos systems. Factors such as the stage of oocyte maturation at the time of insemination and conditions of the IVC may influence the proportion of sex. Although cumulus cells (CC) improve the fertilization ability of sperm cells, there are some evidence that exposure of sperm to CC increases the proportion of male embryos produced in vitro. The objective of this study was to investigate the effects and interaction between CC and IVM culture period of bovine oocytes on the sex ratio of in vitro-produced embryos. COCs (n = 781) were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (18, 20, 22, and 24 h). Maturation medium consisted of TCM-199 with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL-1 gentamicin, 0.5 μg mL-1 FSH, 100 IU mL-1 hCG, and 10% FBS. After maturation culture, oocytes were inseminated with frozen-thawed spermatozoa. Oocytes were inseminated included in CC (COC) or denuded of such cells (denuded oocytes, DO). All zygotes were then co-cultured in vitro with CC in SOF medium with 0.5% BSA and 2.5% FBS. Cultures were carried out at 38.5°C and 5% CO2 in air. Cleavage and blastocyst development rates were observed, respectively, at 48 (Day 2) and 168 (Day 7) h post-insemination (hpi). Blastocysts were harvested on Day 7, and the sex of the embryos was examined using the pair of primers S4B (Kageyama S et al. 2004 Theriogenology 66, 509-514). The data (mean ± SEM) were analyzed by ANOVA, in which the effects of the existence of CC (COC v. DO) and IVM culture periods (18 h, 20 h, 22 h v. 24 h), and interaction between these variables were evaluated. Multiple comparisons of means were followed using Tukey’s test (P &lt; 0.05). The chi-square test was utilized to test the difference in the proportion of sex of embryos. Independently of the presence or absence of CC during IVF, there was no effect of IVM culture duration (P &gt; 0.05) on cleavage (81.5 to 94.8% for COC; and 71.7 to 75.8% for DO) and blastocyst rates (20.0 to 35.9% for COC; and 10.0 to 15.5% for DO). But independently of IVM culture duration, the comparison between COC and DO groups revealed that absence of CC adversely affected (P &lt; 0.05) the cleavage (87.0 and 73.3%, respectively; averaged results) and blastocyst rates (24.5 and 13.5%, respectively; averaged results). In COC, the proportion of male blastocysts increased with the progression in maturation culture period from 18 up to 22 h (54.2, 65.9, and 83.3%, respectively). But for DO, the proportion of females was higher, with exception of 22 h IVM group (65.4, 32.3, 53.3, and 60.0%, respectively from 18 to 24 h of IVM). These results indicate that the sex ratio of in vitro-fertilized embryos is apparently influenced by the maturation culture period of the oocytes and by the presence of CC. FAPESP.

A short period of ejaculatory abstinence before intrauterine insemination is associated with higher pregnancy rates

An ejaculatory abstinence period of <or=2 days before IUI produced the highest pregnancy rates per cycle compared with longer intervals of ejaculatory abstinence. This higher conception rate occurred despite a lower total number of motile spermatozoa inseminated.

Development of a novel, physiologically important calcium and magnesium ion containing non-spermicidal vaginal lubricant

OBJECTIVE: The primary objective of this study was to evaluate a novel calcium and magnesium containing non-spermicidal vaginal lubricant using in vitro assays, including its effectiveness in maintaining sperm viability and motility. Trying to conceive couples seek a vaginal lubricant that mimics natural fluids and is not toxic to the sperm. DESIGN: Semen samples were incubated in the optimum sperm media, in the absence or presence of the newly designed lubricant, and the sperm viability and motility was evaluated. MATERIALS AND METHODS: The motile sperm fraction was collected according to literature protocols. Spermatozoa were incubated for 24h in optimum sperm medium alone or in medium containing 10% (v/v) of the newly designed non-spermicidal lubricant and the sperm viability and motility was measured as a percentage of motile spermatozoa in a blinded fashion by a trained technician using the WHO protocol for the assessment of sperm motility. RESULTS: 83% of spermatozoa remained motile upon exposure to 10% (v/v) of the newly developed calcium and magnesium containing non-spermicidal lubricant, as compared to 85% motile sperm observed after incubation without any lubricant, suggesting that exposure of sperm to the newly developed lubricant did not result in any significant decrease in the sperm motility and viability (which is essential for motility) as compared to the treatment with optimum culture medium alone. CONCLUSIONS: The newly designed non-spermicidal lubricant from Aquatrove contains the physiologically important calcium and magnesium ions to better mimic the natural fluids and does not harm viability or motility of spermatozoa. Data from additional independent in vitro tests, which will also be presented, show that the new lubricant does not create a barrier against sperm motility and does not affect the process of fertilization between mouse ova and sperm that are exposed to this lubricant nor affects their development to the blastocyst stage.

Processing of semen can result in increased sperm DNA fragmentation

Processing of semen for assisted reproductive technologies (ART) entails a number of procedures that include semen liquefaction, removal of seminal plasma by centrifugation, incubation, and cryopreservation. The results of this study indicate that incubation of semen at room temperature and semen cryopreservation can result in increased levels of sperm DNA fragmentation.

Characterization of the Endocannabinoid System in Human Spermatozoa and Involvement of Transient Receptor Potential Vanilloid 1 Receptor in Their Fertilizing Ability

Human spermatozoa express type-1 cannabinoid receptor (CB1), whose activation by anandamide (AEA) affects motility and acrosome reaction (AR). In this study, we extended the characterization of the AEA-related endocannabinoid system in human spermatozoa, and we focused on the involvement of the AEA-binding vanilloid receptor (TRPV1) in their fertilizing ability. Protein expression was revealed for CB1 ( approximately 56 kDa), TRPV1 ( approximately 95 kDa), AEA-synthesizing phospholipase D (NAPE-PLD) ( approximately 46 kDa), and AEA-hydrolyzing enzyme [fatty acid amide hydrolase (FAAH), approximately 66 kDa]. Both AEA-binding receptors (CB1 and TRPV1) exhibited a functional binding activity; enzymatic activity was demonstrated for NAPE-PLD, FAAH, and the purported endocannabinoid membrane transporter (EMT). Immunoreactivity for CB1, NAPE-PLD, and FAAH was localized in the postacrosomal region and in the midpiece, whereas for TRPV1, it was restricted to the postacrosomal region. Capsazepine (CPZ), a selective antagonist of TRPV1, inhibited progesterone (P)-enhanced sperm/oocyte fusion, as evaluated by the hamster egg penetration test. This inhibition was due to a reduction of the P-induced AR rate above the spontaneous AR rate, which was instead increased. The sperm exposure to OMDM-1, a specific inhibitor of EMT, prevented the promoting effect of CPZ on spontaneous AR rate and restored the sperm responsiveness to P. No significant effects could be observed on sperm motility. In conclusion, this study provides unprecedented evidence that human spermatozoa exhibit a completely functional endocannabinoid system related to AEA and that the AEA-binding TRPV1 receptor could be involved in the sperm fertilizing ability.