RESUMO
Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse.
Assuntos
Capacitação Espermática , Espermatozoides , Animais , Capacitação Espermática/efeitos dos fármacos , Masculino , Cavalos/fisiologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Cálcio/metabolismo , Zona Pelúcida/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , FosforilaçãoRESUMO
This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of É-aminocaproic acid (É-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with É-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.
Assuntos
Células do Cúmulo/metabolismo , Fibrinolisina/farmacologia , Fibrinolíticos/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Plasminogênio/farmacologia , Ácido Aminocaproico/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Meios de Cultura , Células do Cúmulo/efeitos dos fármacos , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização in vitro/métodos , Fibrinolisina/antagonistas & inibidores , Oócitos/efeitos dos fármacos , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/metabolismoRESUMO
We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2-4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus-oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.
Assuntos
Anisóis/farmacologia , Antioxidantes/metabolismo , Blastocisto/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Derivados de Alilbenzenos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Bovinos , Meios de Cultura/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilização in vitro , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/metabolismoRESUMO
Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.
Assuntos
Calgranulina B/metabolismo , Epitélio/metabolismo , Oviductos/metabolismo , Capacitação Espermática , Interações Espermatozoide-Óvulo , Reação Acrossômica/efeitos dos fármacos , Adulto , Animais , Sítios de Ligação , Epitélio/efeitos dos fármacos , Feminino , Humanos , Masculino , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oviductos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Sêmen/efeitos dos fármacos , Sêmen/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/metabolismoRESUMO
The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm-zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Camelídeos Americanos/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Zona Pelúcida/efeitos dos fármacos , Animais , Bovinos , Meios de Cultura , Feminino , Sangue Fetal , Masculino , Soro , Capacitação Espermática/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , EspermatozoidesRESUMO
Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.
Assuntos
Proteínas do Ovo/genética , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular/genética , Capacitação Espermática/genética , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Reação Acrossômica/efeitos dos fármacos , Adulto , Anticorpos/farmacologia , Sítios de Ligação , Ligação Competitiva , Proteínas do Ovo/metabolismo , Proteínas do Ovo/farmacologia , Epididimo/citologia , Epididimo/efeitos dos fármacos , Epididimo/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Zona Pelúcida/efeitos dos fármacos , Glicoproteínas da Zona PelúcidaRESUMO
We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.
Assuntos
Biópsia/métodos , Embrião de Mamíferos/efeitos dos fármacos , Soluções Isotônicas , Lasers , Diagnóstico Pré-Implantação/métodos , Adulto , Embrião de Mamíferos/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/patologiaRESUMO
The acrosome reaction (AR), an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca²(+) into the spermatozoa through voltage-dependent Ca²(+) channels and store-operated channels. Maitotoxin (MTx), a Ca²(+)-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida (ZP), possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 (rhZP3), mouse ZP and two MTx channel blockers (U73122 and U73343), we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca²(+) ([Ca²(+)](i)) in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 (inactive analogue), can block phospholipase C (PLC). Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.
Assuntos
Reação Acrossômica/fisiologia , Canais de Cálcio/metabolismo , Toxinas Marinhas/farmacologia , Oxocinas/farmacologia , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Reação Acrossômica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Estrenos/farmacologia , Humanos , Masculino , Camundongos , Pirrolidinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Capacitação Espermática/fisiologia , Espermatozoides/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores , Zona Pelúcida/efeitos dos fármacosRESUMO
The anti-acrosin monoclonal antibody AcrC5F10 inhibited proacrosin activation, proacrosin-human zona pellucida glycoprotein A (ZPA) binding, and the zona pellucida (ZP)-induced acrosome reaction of the ZP-bound spermatozoa but had no significant effect on sperm-ZP binding. These results suggest that proacrosin-acrosin may play an important role in the ZP-induced acrosome reaction of spermatozoa after primary binding to the ZP.
Assuntos
Acrosina/imunologia , Reação Acrossômica/efeitos dos fármacos , Anticorpos/farmacologia , Precursores Enzimáticos/imunologia , Espermatozoides/efeitos dos fármacos , Zona Pelúcida/fisiologia , Acrosina/metabolismo , Reação Acrossômica/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas do Ovo/metabolismo , Proteínas do Ovo/farmacologia , Ativação Enzimática/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Zona Pelúcida/efeitos dos fármacos , Glicoproteínas da Zona PelúcidaRESUMO
Acrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin-like, chymotrypsin-like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim-up method (4 x 10(6) cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N-alpha-p-tosyl-l-lysine-chloromethyl-ketone (trypsin inhibitor); Trypsin inhibitor (I-S Type from soybean); N-tosyl-l-phenylalanine-chloromethyl-ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm-zona binding capacity was evaluated using cumulus cell-free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome-reacted sperm was observed only in cells incubated with trypsin (10.2 +/- 1%) and chymotrypsin inhibitors (18.5 +/- 1%) in relation to the control (52.2 +/- 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 +/- 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.