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1.
Anim Sci J ; 91(1): e13428, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32677083

RESUMO

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.


Assuntos
Antioxidantes , Bombyx/química , Criopreservação/métodos , Crioprotetores , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Sericinas , Motilidade Espermática/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Preservação do Sêmen/efeitos adversos , Sericinas/farmacologia , Suínos
2.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118704, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32194132

RESUMO

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.


Assuntos
Reação Acrossômica/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Espermatozoides/efeitos dos fármacos , Proteína 2 Associada à Membrana da Vesícula/genética , Acrossomo/efeitos dos fármacos , Acrossomo/metabolismo , Reação Acrossômica/efeitos dos fármacos , Adulto , Cálcio/química , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Ceramidas/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Exocitose/genética , Fertilização/genética , Humanos , Masculino , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/genética , Espermatozoides/patologia
3.
Anim Sci J ; 91(1): e13328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32219925

RESUMO

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.


Assuntos
Antioxidantes/uso terapêutico , Criopreservação , Congelamento/efeitos adversos , Rutina/farmacologia , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/patologia , Acrossomo/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismo , Superóxido Dismutase/metabolismo , Suínos
4.
Cryobiology ; 92: 208-214, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32004575

RESUMO

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.


Assuntos
Criopreservação/métodos , Crioprotetores/química , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Soro/metabolismo , Acrossomo/efeitos dos fármacos , Adulto , Animais , Congelamento , Glicerol/farmacologia , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos , Sacarose/farmacologia
5.
Cryobiology ; 92: 197-202, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31962103

RESUMO

Semen extender has a vital role in preservation of sperm cells properties in terms of sperm viability, motility, acrosome integrity, and mitochondrial membrane potential. The objective of the present study was to evaluate a new extender, known as Thai native chicken (TNC) extender compared to BHSV-based and modified Sasaki extenders for freezing chicken semen. Semen from Thai native roosters was collected, pooled, and randomly divided into three groups. Semen was frozen with a simple freezing method using nitrogen vapor and dimethylformamide. In the first experiment, post-thaw motion parameters, viability, acrosome integrity, mitochondrial function, and lipid peroxidation levels were analyzed using computer-assisted sperm analysis, propidium iodide, fluorescein isothiocyanate-conjugate peanut agglutinin, JC-1, and the thiobarbituric acid reaction. Results showed that the type of extender had no effect on the percentage of total motile and curvilinear velocity. The percentage of progressive motile, straight-line velocity, and average path velocity of post-thawed semen were significantly lower in TNC compared to the modified Sasaki extender. However, the percentages of post-thawed acrosome integrity and active mitochondria were significantly higher in TNC extender (P < 0.05). For the second experiment, semen was thawed by using each of extenders thereafter, was inseminated to 48-layer breeder hens to determine the fertility rate. Among the three extenders used, the highest fertility rate was found in TNC extender. In conclusion, TNC extender can be recommended as an appropriate and useful cryopreservation media for native chicken semen since it maintains the quality of rooster semen and fertility after freezing and thawing process.


Assuntos
Acrossomo/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Animais , Galinhas , Dimetilformamida/farmacologia , Fertilidade , Congelamento , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Sêmen/metabolismo , Análise do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Tailândia
6.
PLoS One ; 15(1): e0227946, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978160

RESUMO

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.


Assuntos
Cabras/genética , Análise do Sêmen/métodos , Preservação do Sêmen/métodos , Espermatozoides/fisiologia , Acrossomo/efeitos dos fármacos , Acrossomo/ultraestrutura , Animais , Temperatura Baixa , Criopreservação , Crioprotetores/farmacologia , Humanos , Masculino , Motilidade Espermática/efeitos dos fármacos , Vitrificação/efeitos dos fármacos
7.
Cryobiology ; 92: 146-150, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31883445

RESUMO

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3-5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.


Assuntos
Gema de Ovo/química , Lecitinas/farmacologia , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Animais , Criopreservação/métodos , Congelamento , Cabras , Masculino , Malondialdeído/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sêmen/efeitos dos fármacos , Soja/química , Espermatozoides/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Trometamina/farmacologia
8.
Int J Mol Sci ; 20(24)2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31847486

RESUMO

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K+-channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.


Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/metabolismo , Acrossomo/efeitos dos fármacos , Acrossomo/metabolismo , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/fisiologia , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Fenômenos Biomecânicos/fisiologia , Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Masculino , Lipídeos de Membrana/metabolismo , Canais de Potássio/efeitos dos fármacos , Progesterona/farmacologia , Capacitação Espermática/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos , Motilidade Espermática/fisiologia , Espermatozoides/efeitos dos fármacos , Sus scrofa , Suínos
9.
Cryobiology ; 91: 40-52, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31678073

RESUMO

The aim of this study was to investigate the effects of different concentrations of catalase in a TRIS-egg yolk extender on sperm quality and embryonic development after in vitro fertilization of frozen-thawed bull sperm. For this purpose, from each of 7 bulls 2 ejaculates were collected and diluted with a TRIS-egg yolk extender containing 0, 5, 10, 15 or 20 IU catalase/mL. Sperm quality was evaluated 0, 3, 6, 12 and 24 h after thawing by using computer assisted analysis of motility and by flow cytometric assays. Embryonic development was determined after in vitro fertilization of bovine oocytes. Semen diluted with TRIS-egg yolk extender containing different concentrations of catalase showed more motile sperm, more sperm with intact plasma membranes, acrosomes and DNA, a high mitochondrial membrane potential, a high esterase activity, a low calcium level, a lower amount of synthesis of reactive oxygen species and lower degree of lipid peroxidation of sperm compared to semen frozen without catalase (P < 0.05), but not before 3 h after thawing. There was a dose-response relationship with the most prominent effect of 20 IU catalase/mL. However, the improvement of sperm quality had no effect (P ≥ 0.05) on embryonic development after in vitro fertilization with 20 IU catalase/mL. In conclusion, the addition of catalase to the sperm extender improved sperm quality with no obvious effect on in vitro fertility.


Assuntos
Catalase/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Gema de Ovo , Preservação do Sêmen/métodos , Acrossomo/efeitos dos fármacos , Animais , Bovinos , Membrana Celular/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilidade/efeitos dos fármacos , Fertilização In Vitro , Citometria de Fluxo , Humanos , Masculino , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Sêmen/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos
10.
Reprod Domest Anim ; 54 Suppl 4: 86-89, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31625235

RESUMO

Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low-density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%) were added to control extender. Vitrification was performed in 0.25-ml straws directly plunged into liquid nitrogen. Total motility (TM, %) and progressive motility (PM, %) were analysed by CASA, and plasma membrane (IMS, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post-warmed sperm parameters were compared between treatments by ANOVA. Results were expressed as mean ± SEM. In both experiments, the minimum concentration of LDL and Pronexcell obtained significantly higher values (p < 0.01) than the control extender for TM (L1 = 52.95 ± 4.4; P1 = 58.99 ± 4.6; C = 30.88 ± 3.0), PM (L1 = 36.79 ± 5.5; P1 = 47.25 ± 4.3; C = 19.20 ± 2.4), IMS (L1 = 68.88 ± 3.6; P1 = 47.25 ± 4.3; C = 52.81 ± 2.6) and AIS (L1 = 45.88 ± 3.6; P1 = 47.25 ± 4.3; C = 26.00 ± 2.1). No differences in sperm parameters were found among different concentrations of LDL or Pronexcell. In conclusion, the addition of 0.25% LDL and 1% Pronexcell to the vitrification extender is recommended to improve the quality of stallion sperm after vitrification.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Lipoproteínas LDL/farmacologia , Proteínas do Leite/farmacologia , Preservação do Sêmen/veterinária , Acrossomo/efeitos dos fármacos , Animais , Criopreservação/métodos , Cavalos , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Vitrificação
11.
Reprod Biol Endocrinol ; 17(1): 85, 2019 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-31656198

RESUMO

BACKGROUND: Voluntary control of fertility is of paramount importance to the modern society. But since the contraceptive methods available for women have their limitations such as urinary tract infections, allergies, cervical erosion and discomfort, a desperate need exists to develop safe methods. Vaginal contraceptives may be the answer to this problem, as these are the oldest ways of fertility regulation, practiced over the centuries. With minimal systemic involvement, these are also the safest. Natural substances blocking or impairing the sperm motility offer as valuable non-cytotoxic vaginal contraceptives. Antimicrobial peptides (AMPs) isolated from plants, animals and microorganisms are known to possess sperm immobilizing and spermicidal properties. Following this, in the quest for alternative means, we have cloned, over expressed and purified the recombinant sperm agglutinating factor (SAF) from Staphylococcus warneri, isolated from the cervix of a woman with unexplained infertility. METHODS: Genomic library of Staphylococcus warneri was generated in Escherichia coli using pSMART vector and screened for sperm agglutinating factor (SAF). The insert in sperm agglutinating transformant was sequenced and was found to express ribonucleotide-diphosphate reductase-α sub unit. The ORF was sub-cloned in pET28a vector, expressed and purified. The effect of rSAF on motility, viability, morphology, Mg++-dependent ATPase activity and acrosome status of human sperms was analyzed in vitro and contraceptive efficacy was evaluated in vivo in female BALB/c mice. RESULTS: The 80 kDa rSAF showed complete sperm agglutination, inhibited its Mg2+-ATPase activity, caused premature sperm acrosomal loss in vitro and mimicked the pattern in vivo showing 100% contraception in BALB/c mice resulting in prevention of pregnancy. The FITC labeled SAF was found to bind the entire surface of spermatozoa. Vaginal application and oral administration of rSAF to mice for 14 successive days did not demonstrate any significant change in vaginal cell morphology, organ weight and tissue histology of reproductive and non-reproductive organs and had no negative impact in the dermal and penile irritation tests. CONCLUSION: The Sperm Agglutinating Factor from Staphylococcus warneri, natural microflora of human cervix, showed extensive potential to be employed as a safe vaginal contraceptive.


Assuntos
Colo do Útero/microbiologia , Anticoncepcionais Femininos/farmacologia , Aglutinação Espermática/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos , Staphylococcus/metabolismo , Acrossomo/efeitos dos fármacos , Acrossomo/fisiologia , Animais , Anticoncepcionais Femininos/metabolismo , Feminino , Biblioteca Genômica , Humanos , Infertilidade Feminina , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Staphylococcus/genética
12.
ACS Chem Biol ; 14(10): 2295-2304, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31525885

RESUMO

ABHD2 is a serine hydrolase that belongs to the subgroup of the α,ß-hydrolase fold-containing proteins, which is involved in virus propagation, immune response, and fertilization. Chemical tools to selectively modulate the activity of ABHD2 in an acute setting are highly desired to investigate its biological role, but are currently lacking. Here, we report a library-versus-library screening using activity-based protein profiling (ABPP) to evaluate in parallel the selectivity and activity of a focused lipase inhibitor library against ABHD2 and a panel of closely related ABHD proteins. This screen resulted in the rapid identification of novel inhibitors for ABHD2. The selectivity of the inhibitor was further investigated in native mouse testis proteome by competitive ABPP, revealing a highly restricted off-target profile. The progesterone-induced acrosome reaction was reduced in a dose-dependent manner by the newly identified inhibitor, which provides further support for the key-role of ABHD2 in the P4-stimulated acrosome reaction. On this basis, the ABHD2 inhibitor is an excellent starting point for further optimization of ABHD2 inhibitors that can modulate sperm fertility and may lead to novel contraceptives.


Assuntos
Reação Acrossômica/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Piperidinas/química , Piperidinas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
13.
Cryobiology ; 90: 1-7, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31541621

RESUMO

Effect of sericin pretreatment of sperm cells on the osmotic tolerance, ability to undergo acrosome reaction induced by calcium ionophore (CI), heparin (H) or lysophosphatidylcholine (LPC), post-thaw sperm quality and in vivo fertility was evaluated in four successive experiments in rabbit. In experiment 1, fresh semen was pretreated with sericin (0, 0.1% or 0.5% w/v) before exposing to fructose solutions adjusted to either 50, 100, 290, 500 or 1000 mOsm/L. Sericin pretreatment increased sperm livability in addition to live-membrane intact and total membrane intact sperm rates (P < 0.05) in 50 and 290 mOsm/L groups. In experiment 2, sperm samples were pretreated by either 0.1 or 0.5% sericin after removal of the semen plasma. CI, H or LPC were used to induce acrosome reaction in pretreated sperm samples. Sericin pretreatment, reduced the ability of sperm cells to undergo acrosome reaction (P < 0.05) in vitro. In experiment 3, ejaculates were frozen with or without sericin pretreatment in DMSO-sucrose extender. In post-thaw samples sericin pretreatment improved total and progressive motility, livability, membrane and acrosome integrity in a dose dependent manner (P < 0.05). In vivo fertility trials by artificial inseminations revealed contradictory results in experiment 4. Although 0.5% sericin pretreatment totally inhibited fertility, 0.1% sericin provided high pregnancy rates. In conclusion; sericin pretreatment enhances osmotic tolerance and post-thaw sperm quality, but reduces the ability of rabbit sperm cells to undergo in vitro induced acrosome reaction, but this effect is restored in vivo by dose dependent manner.


Assuntos
Reação Acrossômica/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Sericinas/farmacologia , Motilidade Espermática/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Animais , Feminino , Fertilidade , Congelamento , Inseminação Artificial/métodos , Masculino , Gravidez , Coelhos , Sêmen/efeitos dos fármacos , Análise do Sêmen , Espermatozoides/efeitos dos fármacos
14.
Chemosphere ; 226: 874-882, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31509916

RESUMO

The exposure and health effects of fluoride are an ongoing topic that has attracted worldwide attention. Fluoride exposure disturbs the testicular development, sexual hormone levels and spermatogenesis. However, as to whether fluoride interferes with acrosome formation which is essential for production of capable spermatozoa during spermatogenesis still remains unclear. The objective was to determine the effects of fluoride on the acrosome formation and to further elucidate the potential mechanism of impaired reproductive function. For this, forty adult rats were assigned into four groups. The control group received distilled water, while the other three groups were treated with 25, 50 and 100 mg NaF/L via drinking water for 56 d, respectively. Testes were processed for total RNA extraction and western blot analysis. Three samples of each group were fixed in 2.5% glutaraldehyde solution for transmission electron microscopy analysis. From the results, we first found that fluoride decreased the expression of mRNA and protein levels of Zpbp1, Spaca1 and Dpy19l2 of seven markers during acrosome biogenesis in testes. Furthermore, fluoride damaged not only the acrosome structure, but also the structure of the nuclear lamina which was observed to be discontinuous and partially missing by transmission electron microscopy. Moreover, the results indicated that the altered structure in nuclear lamina maybe due to reduced LMNB2 expression in testis induced by fluoride. In a nutshell, fluoride exposure could restrain acrosome biogenesis during spermatogenesis and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.


Assuntos
Acrossomo/patologia , Fluoretos/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/patologia , Testículo/patologia , Acrossomo/efeitos dos fármacos , Acrossomo/metabolismo , Animais , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo
15.
Anim Reprod Sci ; 209: 106171, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31514920

RESUMO

The objective of the present study was to assess the effect of two different antioxidants, enzymatic compared with non-enzymatic, in a nano lecithin-based extender on post-thaw bull sperm quality. Semen samples (n = 36) were collected from six bulls. In the first experiment, 11 different extenders were prepared by adding five quantities of vitamin E (α-tocopherol) as a non-enzymatic antioxidant (VE: 0.1, 0.2, 0.4, 0.6 and 1.0 mM), or four quantities of glutathione peroxidase (GPx) as an enzymatic antioxidant (GPx: 0.5, 1, 2 and 3 mM) to the extender. Other extenders were a Control 1 (C1: Extender with ethanol) and Control 2 (C2: Extender without ethanol). Sperm motility (CASA), plasma membrane functionality test (HOST) and lipid peroxidation (MDA) were assessed to determine the optimal treatment in the first experiment. In the second experiment, the optimally supplemented group from the first experiment (GPx-1) was compared to C2 group. Apoptotic-like changes (Annexin staining), mitochondrial activity (Rhodamine-123 staining), acrosome integrity (PSA staining), DNA fragmentation (SCSA test) and in vitro embryo production capacity were evaluated. In the first experiment, there were the greatest percentages of plasma membrane functionality and least MDA (P ≤ 0.05) in sperm diluted GPx-1 group. In the second experiment, percentage of live sperm, blastocyst formation and hatching rate were greater (P ≤ 0.05) in the GPx-1 group compared with C2 group. In conclusion, data indicate adding 1.0 mM GPx as an enzymatic antioxidant to the nano lecithin-based extender can improve post-thaw quality and in vitro fertility of bull sperm.


Assuntos
Antioxidantes/farmacologia , Bovinos , Criopreservação/métodos , Crioprotetores/farmacologia , Lecitinas/farmacologia , Preservação do Sêmen/métodos , Acrossomo/efeitos dos fármacos , Animais , Antioxidantes/classificação , Células Cultivadas , Criopreservação/veterinária , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilidade/efeitos dos fármacos , Fertilização In Vitro/veterinária , Congelamento , Lecitinas/química , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Nanopartículas/química , Análise do Sêmen , Preservação do Sêmen/veterinária , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos
16.
Cryobiology ; 90: 15-20, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518561

RESUMO

The present study was designed to test the effect of different levels of idebenone, a potent antioxidant on the quality of ram semen at post thaw. Eighteen (18) ejaculates were collected and extended with tris extender supplemented with no antioxidant (CON), with 2 µM idebenone (Id2), 5 µM idebenone (Id5), 7.5 µM idebenone (Id7.5) and 10 µM idebenone (Id10). The sperm quality was determined in terms of percent sperm motility, live sperm percentage, percent hypoosmotic swelling test (HOST) positive spermatozoa and percent intact acrosome (PIA). Moreover, malondialdehyde (MDA) level, an end product of lipid peroxidation (LPO) was also measured at post thaw both in seminal plasma and sperm cell. At post thaw, the percent sperm motility was significantly higher (p < 0.05) for Id10 as compared to Id2, Id5, Id7.5 and control. The live sperm percentage was non-significantly (p > 0.05) higher for Id10 as compared to control, Id5 and Id7.5 but significantly higher than Id2. The percent HOST positive spermatozoa was significantly higher (p < 0.05) for Id10 than control, Id2 and Id5. The MDA level in seminal plasma was significantly lower (p < 0.05) for Id10 than control and Id2. The MDA level in spermatozoa did show similar trend as in seminal plasma. Further, all the sperm parameters at all idebenone levels declined significantly from pre freeze to post thaw. In conclusion, idebenone at 10 µM level improved post thaw sperm quality by mitigating peroxidative stress, hence could be considered as a promising antioxidant additive for cryopreservation of ram semen.


Assuntos
Antioxidantes/farmacologia , Crioprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Preservação do Sêmen/métodos , Ubiquinona/análogos & derivados , Acrossomo/efeitos dos fármacos , Animais , Criopreservação/métodos , Congelamento , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/análise , Sêmen/química , Análise do Sêmen , Ovinos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Ubiquinona/farmacologia
17.
Anim Sci J ; 90(9): 1161-1169, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31381235

RESUMO

Sulfanilamide (SA) is an effective broad-spectrum antibacterial agent in human and veterinary medicine. The purpose of this study was to evaluate the effects of SA on boar sperm quality during liquid storage at 17°C and determine the optimal concentration of SA and its effects on bacterial growth, microbial composition, and maternal fertility. Boar ejaculates were diluted with a basic extender, containing different concentrations of SA, and stored in a 17°C incubator for 6 days. The sperm motility, plasma membrane integrity, and acrosome integrity were measured daily. The results showed that when the concentration of SA was 0.02 g/L, the sperm quality parameters were significantly higher than those of all other treatment groups (p < .05). We also monitored the bacterial growth and compared the differences in the microbial species between the 0.02 g/L SA group and the control by 16S rDNA sequencing. The results revealed that some bacteria, such as Staphylococcus and Pseudomonas, were considerably lower in the 0.02 g/L SA group than in the control group (p < .05). In addition, preserved semen was used for artificial insemination, and results showed that 0.02 g/L SA group had a higher litter size, and its pregnancy rate was 92.5%.


Assuntos
Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Sulfanilamida/farmacologia , Acrossomo/efeitos dos fármacos , Animais , Feminino , Fertilidade/efeitos dos fármacos , Inseminação Artificial/veterinária , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Masculino , Sêmen/efeitos dos fármacos , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/microbiologia , Suínos
18.
Cryobiology ; 89: 76-81, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071309

RESUMO

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer-assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.


Assuntos
Hidroxitolueno Butilado/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Análise do Sêmen , Preservação do Sêmen/métodos , Acrossomo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Gatos , Congelamento , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Sêmen/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos
19.
Cryobiology ; 89: 90-95, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31054855

RESUMO

This is a unique study because is the first time we are adding melatonin into an extender in order to determine its influence on cryopreserved chicken semen. The primary focus of our present study was to evaluate the influence of different concentrations of Melatonin on cryopreserved chicken semen. Semen samples were allocated into four treatments, being one control and three different combinations of antioxidants and after the freeze-thaw operation, the sperm motility, plasma membrane integrity, acrosome integrity, endogenous enzymes (GSH-Px, CAT, SOD), MDA and ROS of chicken spermatozoa were all evaluated. The collection of the semen samples was from 40 Arbor Acre roosters and this procedure was repeated twice a week and then mixed in an extender that contained different MEL treatments as follows: a diluent without MEL (control, M 0), a diluent comprising 0.125 mg/mL (M 0.125) 0.25 mg/mL, (M 0.25) and 0.5 mg/mL (M 0.5). It was revealed that the supplementation of the base extender with an optimal 0.25 mg/mL MEL led to a higher significant difference in the motility of chicken sperm (P < 0.01), higher acrosome integrity (P < 0.05) and a higher plasma membrane integrity (P < 0.01) when compared to the control group at post-thaw. Furthermore, when compared to the control group, 0.25 mg/mL MEL addition into the extender significantly enhanced the activity of endogenous enzymes (GSH-Px, CAT, and SOD) in the chicken spermatozoa at post-thaw (P < 0.05). Moreover, 0.5 mg/mL MEL supplementation into the extender enhanced the GSH-Px activity in the chicken spermatozoa when compared with the control group (P < 0.05) at post-thaw. In contrast, the addition of 0.25 mg/mL MEL into the extender resulted in a significantly lower MDA in comparison to the 0.125 mg/mL, 0.5 mg/mL MEL treatment group and the control group (P < 0.05). Also, compared to the control group, MEL concentration of 0.125 mg/mL and 0.5 mg/mL MEL into the extender resulted in a significantly low ROS concentration (P < 0.05) but the addition of 0.25 mg/mL MEL concentration resulted in a significantly lower ROS level when compared to the control group (P < 0.01). In summary, MEL improved the quality of cryopreserved chicken sperm quality by decreasing oxidative stress level and the most optimal concentration was 0.25 mg/mL.


Assuntos
Antioxidantes/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Melatonina/farmacologia , Análise do Sêmen , Preservação do Sêmen/métodos , Acrossomo/efeitos dos fármacos , Animais , Membrana Celular/metabolismo , Galinhas , Masculino , Estresse Oxidativo/efeitos dos fármacos , Sêmen/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos
20.
Cell Tissue Bank ; 20(3): 367-378, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31139967

RESUMO

Cryopreservation exposes sperm to physical and chemical stresses causing cell damages and impairs sperm functions. The aim of this study was to evaluate the association between motility and sperm chromatin/DNA damage before and after cryopreservation and investigate the effects of folic acid and nicotinic acid on post-thaw sperm quality. Thirty semen samples were obtained from 30 normozoospermic men, aged between 25 and 45 years old. Each sample were divided into five aliquots to form the following groups: fresh, cryopreserved with sperm-freeze only (control), with nicotinic acid (10 mM), with folic acid (50 nM), and with a combination of folic acid (50 nM) + nicotinic acid (10 mM). Sperm viability and motility in each group were assessed by eosin-nigrosine staining and computer-aided sperm analysis respectively. Sperm chromatin quality was studied by aniline blue, toluidine blue, acridine orange staining methods and sperm chromatin dispersion test. Cryopreservation led to a significant reduction in sperm quality in comparison to fresh sample groups (p < 0.05). Sperm chromatin damage was negatively correlated with the percentage of progressively motile cells. Supplementation of the cryopreservation medium with folic acid or nicotinic acid induced a significant improvement in sperm parameters and chromatin quality, compared to control groups (p < 0.05). Meanwhile, the combination of folic acid + nicotinic acid showed a significant protective effect in post thaw sperm. In conclusion, cryopreservation generated oxidative stress, inducingsperm cryodamage, reducing progressive motility and sperm quality, as an indicator of significant chromatin/DNA damage. Folic acid and nicotinic acid exhibited a potential cryoprotective effect by enhancing sperm quality.


Assuntos
Acrossomo/efeitos dos fármacos , Cromatina/química , Criopreservação , Dano ao DNA , Ácido Fólico/farmacologia , Niacina/farmacologia , Motilidade Espermática , Adulto , Crioprotetores/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Análise do Sêmen/métodos , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos
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