Decreasing sperm quality in mice subjected to chronic cannabidiol exposure: New insights of cannabidiol-mediated male reproductive toxicity.

Cannabidiol (CBD) is a natural cannabinoid present in the Cannabis sativa plant, widely prescribed as an anticonvulsant drug, especially for pediatric use. However, its effects on male reproduction are still little investigated. Therefore, the present study assessed the effects of CBD on the spermatogenesis and sperm quality. For this, twenty-one-day-old Swiss mice received CBD for 34 consecutive days by gavage at doses of either 15 or 30 mg/kg. Chronic exposure to CBD decreased the frequency of stages VII-VIII and XII of spermatogenesis and an increase in the frequency of stage IX were noted. Furthermore, the seminiferous epithelium height reduced at stage IX and increased at stage XII in both CBD-treated groups. There was a significant rise of sperm DNA damage, while no genotoxic effects were observed in leukocytes. The activities of superoxide dismutase and catalase decreased, while malondialdehyde levels increased in the sperm of mice treated with a higher dose of CBD. Mice exposed to 30 mg/kg of CBD showed a reduction in the mobile spermatozoa percentage and in curvilinear velocity, while straight line and average path velocity decreased in both treated groups. The number of acrosome-intact spermatozoa declined in the CBD 30 group, and the number of abnormal acrosomes raised in both CBD groups. On the other hand, the weight of reproductive organs, sperm count, and hormone levels were not affected by CBD treatment. These findings show that dysregulation of the endocannabinoid system by CBD can reduce sperm quality. The mechanisms responsible may be associated with disorders during spermatogenesis, especially during the final stages of nuclear remodelling and assembly of acrosome. However, changes in mitochondrial function, as well as the reduction on the antioxidant enzyme activities during epididymal transit, at least partly, may also be involved.

Evaluation of the novel vaginal contraceptive agent PPCM in preclinical studies using sperm hyaluronan binding and acrosome status assays.

BACKGROUND: Polyphenylene carboxymethylene (PPCM) sodium salt is a promising multipurpose technology for prevention of both sexually transmitted infections (STIs) and pregnancy. In preclinical studies, PPCM has demonstrated significant (1) antimicrobial activity against several important viral and bacterial pathogens and (2) contraceptive activity associated with premature acrosome loss. OBJECTIVE: To further evaluate a vaginal antimicrobial compound as a contraceptive agent in preclinical studies utilizing a repurposed hyaluronan binding assay (HBA). MATERIALS AND METHODS: Semen samples containing either neat semen or washed spermatozoa were treated with increasing concentrations of PPCM or calcium ionophore A23187 (positive control). Sperm inactivation was measured by two methods: (1) double acrosome staining (AS), and (2) a hyaluronan binding assay (HBA® ). Percentage of inactivated sperm was compared between untreated control sperm and those treated with PPCM or A23187. RESULTS: PPCM had a significant (p < 0.05) and dose-dependent effect on sperm inactivation in both assays, with HBA detecting a higher proportion of inactivated sperm than AS. PPCM did not affect sperm motility and exhibited equivalent responses in the neat and washed samples. DISCUSSION: Both HBA and AS confirmed that spermatozoa were rapidly inactivated at PPCM concentrations likely present in the vagina under actual use conditions and in a time-frame comparable to in vivo migration of spermatozoa out of seminal plasma into cervical mucus. CONCLUSION: PPCM vaginal gel may provide contraceptive protection as well as help with STI prevention. HBA may be a sensitive and much needed biomarker for sperm activity in future contraceptive development.

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase.

The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal-Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p < .05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p < .05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p < .05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.

Membrane lipid replacement with nano-micelles in human sperm cryopreservation improves post-thaw function and acrosome protein integrity.

RESEARCH QUESTION: Membrane lipid replacement (MLR) of oxidized membrane lipids can restore sperm cellular membrane functionality and help improve surface protein stability during cryopreservation. What are the effects of MLR with nano-micelles made from a glycerophospholipid (GPL) mixture and cholesterol-loaded cyclodextrin (CLC), on the cryosurvival and expression of acrosome-related proteins in thawed human spermatozoa? DESIGN: Twenty samples were used to determine the optimum level of nano-micelles by incubation of semen with different concentrations of GPL (0.1 and 1%) and CLC (1 and 2 mg/ml) (including GPL-0.1, GPL-1, CLC-1, CLC-2, CLC-1/GPL-0.1, CLC-2/GPL-0.1, CLC-1/GPL-1 and CLC-2/GPL-1) before cryopreservation. Then, 30 semen samples were collected, and each sample was divided into the following three aliquots: fresh, frozen control and frozen incubated with optimum level of nano-micelles (0.1% GPL and 1 mg/ml CLC). RESULTS: CLC-1/GPL-0.1 and GPL-0.1 significantly increased motility parameters. CLC-1, GPL-0.1 and CLC-1/GPL-0.1 significantly improved viability rate compared with frozen control group. Significantly higher mitochondrial activity and acrosome integrity, and a lower rate of apoptosis, were observed in the CLC-1/GPL-0.1 compared with the frozen control group. The expression ratios of arylsulfatase A (ARSA), serine protease 37 (PRSS37), serine protease inhibitor Kazal-type 2 (SPINK2) and equatorin (EQTN) significantly increased compared with the frozen control group. CONCLUSIONS: Modification of membrane cholesterol and GPL mixtures in spermatozoa enhances their acrosome protein integrity by inhibiting early apoptotic changes and spontaneous acrosome reactions.

Structure-based redesigning of pentoxifylline analogs against selective phosphodiesterases to modulate sperm functional competence for assisted reproductive technologies.

Phosphodiesterase (PDE) inhibitors, such as pentoxifylline (PTX), are used as pharmacological agents to enhance sperm motility in assisted reproductive technology (ART), mainly to aid the selection of viable sperm in asthenozoospermic ejaculates and testicular spermatozoa, prior to intracytoplasmic sperm injection (ICSI). However, PTX is reported to induce premature acrosome reaction (AR) and, exert toxic effects on oocyte function and early embryo development. Additionally, in vitro binding studies as well as computational binding free energy (ΔGbind) suggest that PTX exhibits weak binding to sperm PDEs, indicating room for improvement. Aiming to reduce the adverse effects and to enhance the sperm motility, we designed and studied PTX analogues. Using structure-guided in silico approach and by considering the physico-chemical properties of the binding pocket of the PDEs, designed analogues of PTX. In silico assessments indicated that PTX analogues bind more tightly to PDEs and form stable complexes. Particularly, ex vivo evaluation of sperm treated with one of the PTX analogues (PTXm-1), showed comparable beneficial effect at much lower concentration-slower AR, higher DNA integrity and extended longevity of  spermatozoa and  superior embryo quality. PTXm-1 is proposed to be a better pharmacological agent for ART than PTX for sperm function enhancement.

Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis discus hannai.

Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p > 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.

The use of resveratrol decreases liquid-extend boar semen fertility, even in concentrations that do not alter semen quality.

In vitro and in vivo assays were conducted to investigate the effects of trans-resveratrol (RVT) on liquid-extended boar semen during 72 h of storage at 17 °C. Thirty-six ejaculates were collected from six boars, evaluated, and extended. RVT was then added at the indicated treatment concentration (0, 0.01, 0.1 or 1 mM), and the ejaculates were cooled to 17 °C and evaluated at 0, 24, 48, and 72 h. Samples were evaluated for sperm motility, kinetics, plasma and acrosome integrity, mitochondrial membrane potential, anion superoxide levels, lipoperoxidation, and antioxidant enzyme activity. In the follow-up experiment, twenty-eight gilts were fixed-time inseminated with 0 or 0.01 mM RVT liquid-extended boar semen. After five days, they were slaughtered, and their reproductive tracts were recovered. The embryos were collected, and the pregnancy, fertility, and viable embryo rates were calculated. In the in vitro assays, total motility, plasma and acrosome membrane integrity, mitochondrial membrane potential, anion superoxide levels, and lipoperoxidation did not change at any of the evaluation times with the use of RVT up to 0.01 mM. RVT decreased SOD activity without changes in GPx. RVT used at 1 mM showed harmful effects for almost all evaluated parameters. For the in vivo assay, the same pregnancy and fertility rates were observed for both groups, while the viable embryo rate was three-fold lower in the 0.01 mM group than in the 0 mM group. The results showed a dichotomous effect of RVT; a low concentration was not harmful in vitro but was catastrophic for embryo viability.

Inhibition of Potassium Channels Affects the Ability of Pig Spermatozoa to Elicit Capacitation and Trigger the Acrosome Exocytosis Induced by Progesterone.

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa.

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.

Evaluation of cryopreserved boar semen after supplementation sericin form silkworm (Bombyx mori) in semen extender.

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post-thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%-1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing-thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%-0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing.

Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P<0.05), and RSV did not significantly increase the sperm motility (0.44 vs. 0.40, P>0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨm. RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 µmol/L vs. 0.25 µmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.

Cryopreservation of Ghagus chicken semen: Effect of cryoprotectants, diluents and thawing temperature.

The present study evaluated the effects of cryoprotectants, semen diluents and thawing temperature during Ghagus chicken semen cryopreservation. Four different experiments were conducted; Experiment 1-semen was cryopreserved using 6% dimethylacetamide (DMA) and 2% dimethylsulphoxide (DMSO) in Sasaki diluent (SD) and Lake and Ravie diluent (LR), Experiment 2 and 3-semen was cryopreserved using 8% ethylene glycol (EG) in SD, LRD and Red Fowl Extender (RFE), Experiment 4-semen was cryopreserved using 6% dimethylformamide (DMF) in SD, LR and Beltsville poultry semen extender (BPSE). Semen was cryopreserved in 0.5 ml French straws. Thawing was done at 5°C for 100 s in ice water in Experiments 1, 2 and 4, whereas in Experiment 3 thawing was done at 37°C for 30 s. The post-thaw sperm motility, viable sperm and acrosome-intact sperm were significantly (p < .05) lower in cryopreserved samples in all the experiments. No fertile eggs were obtained from cryopreserved samples in Experiments 1 and 2, except for 8% EG RFE treatment where the fertility was 0.83%. In Experiments 3 and 4, highest fertility was obtained in LR treatment 48.12 and 30.89%, respectively. In conclusion, using cryoprotectant EG (8%) and thawing at 37°C for 30 s, and DMF(6%) resulted in acceptable level of fertility in Ghagus chicken. Though the diluents influenced post-thaw in vitro semen parameters, the fertility was not affected. In addition, results indicated that thawing temperature may be a critical stage in the cryopreservation protocol.

Antibiotic-free extended boar semen preserved under low temperature maintains acceptable in-vitro sperm quality and reduces bacterial load.

This study aimed to assess the sperm quality and number of colony-forming units (CFU mL-1) in extended boar semen stored at low temperatures with or without antibiotics. Normospermic ejaculates (n = 34) were diluted in split samples with Androstar® Premium with or without antibiotics (ampicillin and apramycin sulfate). The extended semen doses were stored for 120 h under three storage temperatures (5, 10, and 17 °C). Variables were analyzed as repeated measures using the GLIMMIX procedure, in a factorial design. The extended semen doses under low-temperature storage (5 and 10 °C) had total motility above 75% throughout the storage. The interaction antibiotic × temperature was significant for total (P = 0.004) and progressive motility (P = 0.005). In extended boar semen doses with antibiotics, the total and progressive motility increased as the storage temperature increased (80.2%, 84.5%, and 89.1%; 70.5%, 76.0%, and 82.9% for total and progressive motility at 5, 10, and 17 °C, respectively; P < 0.05). In extended semen doses without antibiotics, the total and progressive motility were lower when stored at 5 °C than at 10 °C and 17 °C (81.8%, 85.4% and 86.6% and 71.9%, 76.7%, 78.9% for total and progressive motility at 5, 10, and 17 °C, respectively; P < 0.05). After the thermoresistance test, total and progressive motility of doses with antibiotics were higher at 17 °C than 5 °C (P < 0.05); however, they were not affected (P > 0.05) by storage temperature in extended semen doses without antibiotics. The number of CFU mL-1 was lower in extended semen doses without antibiotics stored at 5 and 10 °C than at 17 °C (P < 0.05); however, in extended semen doses with antibiotics, no effect of storage temperature was observed (P > 0.05). The bacterial load was greater in extended semen without antibiotics than with antibiotics, regardless of the storage temperature (P < 0.05). The acrosome and sperm membrane integrity were not influenced (P > 0.05) by using antibiotics. A higher percentage of normal acrosomes was observed as the storage temperature increased (93.6%, 94.3%, and 96.8% at 5, 10, and 17 °C, respectively; P < 0.0001). The membrane integrity was higher (P < 0.0001) in extended semen doses stored at 17 °C than at 10 or 5 °C. The pH rose throughout the storage in all the treatments, except in extended semen doses stored at 17 °C without antibiotics, in which a decrease in the pH occurred at 120 h (P < 0.05). Although the sperm quality being negatively affected by low temperatures, the storage of extended boar semen doses at 5 °C is possible since the sperm viability in vitro was maintained for up to 5 days, fulfilling the requirements of semen quality to be used in artificial insemination. Nevertheless, the use of extended semen doses without antibiotics requires the optimization of hygiene procedures during semen dose processing.

Effect of dimethylformamide on sperm quality and fertilizing ability of Indian red jungle fowl (Gallus gallus murghi).

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.

Ceramide induces a multicomponent intracellular calcium increase triggering the acrosome secretion in human sperm.

Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels. We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization.

Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking.

To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p = .027), percentage of progressively motile spermatozoa (p = .002), total sperm count (p = .039), morphology (p = .001) and higher percentage of immotile spermatozoa (p = .03), round cell (p = .045) and neutrophil count (p = .009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette.

Rutin protects boar sperm from cryodamage via enhancing the antioxidative defense.

This study was aimed to investigate whether and how Rutin protects boar sperm against cryoinjury during cryopreservation. Five concentrations of Rutin with 0.2, 0.4, 0.6, 1.0, and 2.0 mM were added to the freezing extender of boar sperm, respectively, and the effects on quality and function of boar sperm after freezing-thawing were assessed. The results showed that the sperm motility, mitochondrial activity, plasma membrane integrity, and acrosomal integrity were significantly improved in 0.4 mM and 0.6 mM Rutin groups (p < .05). Compared with ganoderma lucidum polysaccharide (GLP) or Tanshinone IIA, Rutin exhibited higher rates of mitochondrial activity and acrosome integrity (p < .05). Mechanistically, the addition of Rutin at the concentration of 0.6, 0.8, and 1.0 mM significantly attenuated ROS accumulation and MDA production by improving antioxidant enzymatic activity, including SOD, CAT, and GSH-Px (p < .05). Functionally, a higher penetration rate and the increased total efficiency of fertilization were observed in the 0.4, 0.6, and 1.0 mM Rutin groups than in the control group (p < .05). Moreover, the addition of Rutin (0.6 mM) significantly induced an increase in both the cleavage and blastocyst rates (p < .05). In summary, supplementation with Rutin in cryopreservation medium protects boar sperm against ROS attack by enhancing the antioxidative defense.

Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa.

The freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. In order to decrease the detrimental effects of cryopreservation and improve the quality of post-thawed spermatozoa, the constituents of the freezing solution attracted considerable attention. In this study, for the first time, we evaluated the efficacy of knockout serum replacement (KSR) as a substitute for human serum albumin (HSA) for cryopreservation of human spermatozoa. Twenty semen samples were collected from normozoospermic men and divided them into five equal groups. One of the aliquots was diluted with glycerol-based medium as a control group (CON). The other four aliquots were diluted with the sucrose solution containing 5% HSA (H5), 10% HSA (H10), 5% KSR (K5), and 10% KSR (K10). The diluted samples were frozen and preserved in liquid nitrogen. Post thawed sperm parameters including motion characteristics, viability, membrane integrity, mitochondrial activity, acrosome integrity and DNA intactness in all of the sucrose-based groups were comparable with glycerol-based medium. The replacement of HSA by 10% KSR in the freezing medium resulted in significantly higher post-thawed viability, acrosome integrity and DNA intactness compared with other sucrose-based groups. In conclusion, the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa and may have potential to develop chemically defined freezing medium.

A phenotypic screening platform utilising human spermatozoa identifies compounds with contraceptive activity.

There is an urgent need to develop new methods for male contraception, however a major barrier to drug discovery has been the lack of validated targets and the absence of an effective high-throughput phenotypic screening system. To address this deficit, we developed a fully-automated robotic screening platform that provided quantitative evaluation of compound activity against two key attributes of human sperm function: motility and acrosome reaction. In order to accelerate contraceptive development, we screened the comprehensive collection of 12,000 molecules that make up the ReFRAME repurposing library, comprising nearly all the small molecules that have been approved or have undergone clinical development, or have significant preclinical profiling. We identified several compounds that potently inhibit motility representing either novel drug candidates or routes to target identification. This platform will now allow for major drug discovery programmes that address the critical gap in the contraceptive portfolio as well as uncover novel human sperm biology.

Ultra-rapid cooling of ibex sperm by spheres method does not induce a vitreous extracellular state and increases the membrane damages.

Sperm cryopreservation by ultra-rapid cooling based on dropping small volumes of sperm suspension directly into liquid nitrogen, has been successful in some wild ruminant species, including the Iberian ibex (Capra pyrenaica). In ultra-rapid cooling, the contents of these droplets are expected to enter a stable, glass-like state, but to the best of our knowledge no information exists regarding the presence or absence of ice formation in the extracellular milieu when using this technique. Different modifications to the extracellular milieu likely inflict different types of damage on the plasmalemma, the acrosome and mitochondrial membranes. The aims of the present work were: 1) to examine the physical state of the extracellular milieu after cryopreservation at slow and ultra-rapid cooling rates-and thus determine whether ultra-rapid cooling vitrifies the extracellular milieu; and 2) to compare, using conventional sperm analysis techniques and scanning and transmission electron microscopy, the damage to sperm caused by these two methods. Sperm samples were obtained by the transrectal ultrasound-guided massage method (TUMASG) from anesthetized Iberian ibexes, and cryopreserved using slow and ultra-rapid cooling techniques. Sperm motility (22.95 ± 3.22% vs 4.42 ± 0.86%), viability (25.64 ± 3.71% vs 12.8 ± 2.50%), acrosome integrity (41.45± 3.73% vs 27.00 ± 1.84%) and mitochondrial membrane integrity (16.52 ± 3.75% vs 4.00 ± 0.65%) were better after slow cooling (P<0.001) than after ultra-rapid technique. Cryo-scanning electron microscopy (Cryo-SEM) suggested that the vitrified state was not achieved by ultra-rapid cooling, and that the ice crystals formed were smaller and had more stretchmarks (P<0.001) than after slow cooling. Scanning electron microscopy revealed no differences in the types of damage caused by the examined techniques, although transmission electron microscopy showed the damage to the plasmalemma and mitochondrial membrane to be worse after ultra-rapid cooling. In conclusion ultra-rapid cooling provoked more membrane damage than slow cooling, perhaps due to the extracellular ice crystals formed.