RESUMO
Murine and bovine embryos at the late morula stage were cultured in medium containing high-titer rat H-Y antisera. After 12h of incubation, embryos blocked at the late morulae stage were classified as males and those at the blastocyst stage were classified as females. Sexing of murine embryos by PCR and cytogenetics revealed that 83% of the embryos classified as males and 82% of those classified as females had their sex correctly predicted (P < 0.05). Bovine embryos were transferred to recipient females. Pregnancy rates were 71.4% (10/14) for embryos classified as males and 68.8% (11/16) for embryos classified as females. The sex was correctly predicted for 80% (8/10) of the embryos classified as males and for 81.8% (9/11) of those classified as females (overall accuracy, 80.9%, P < 0.05). Therefore, the induction of developmental arrest by high-titer male-specific antisera was an efficient strategy for non-invasive embryo sexing. The procedure was straightforward and has considerable commercial potential for sexing bovine embryos.
Assuntos
Blastocisto , Bovinos/embriologia , Isoanticorpos/farmacologia , Camundongos/embriologia , Mórula , Análise para Determinação do Sexo/veterinária , Animais , Blastocisto/química , Blastocisto/efeitos dos fármacos , Blastocisto/ultraestrutura , Transferência Embrionária/veterinária , Feminino , Masculino , Mórula/química , Mórula/efeitos dos fármacos , Mórula/ultraestrutura , Reação em Cadeia da Polimerase , Gravidez , Sensibilidade e Especificidade , Análise para Determinação do Sexo/métodosRESUMO
The male-specific H-Y antigen is present on mammalian cell membranes and has been identified by various methods, including antiserum cytotoxicity. The objective of the present study was to determine the sex of in vitro produced (IVP) bovine embryos, at varying stages of development, by culturing in the presence of rat monoclonal H-Y antibodies. Embryos derived from IVM/IVF were classified according to the interval after IVF (48, 96 or 120 h) as Category 1, 2 or 3 if they had 4 to 8, <32, and >32 cells, respectively. Embryos of each category were cultured for 24h in TCM-199 supplemented with bovine oviductal epithelial cells, fetal calf serum (FCS), and antibiotics (Control group), to which the following had been added: guinea pig serum (GPS; C' group); H-Y antiserum (HY group); or GPS and H-Y antiserum (C' + HY group). After culture, embryos were designated as "affected" when development was arrested or one or more blastomeres was degenerate; embryos lacking these changes were designated "unaffected." The sex of each embryo was subsequently determined by chromosome analysis. After 48h of IVF (Category 1), within each of the four treatments, the proportion of unaffected embryos was higher than the proportion of unaffected embryos (81% versus 19%, P < 0.05). Similarly, the Control, C' and HY groups of Categories 2 and 3 embryos had different proportions of unaffected versus affected embryos (75% versus 25%, P < 0.05). In all these groups, the male:female ratio did not significantly differ from 1:1. In contrast, in the C' + HY group of Categories 2 and 3 embryos, the ratio of unaffected versus affected embryos was 41% versus 59% (P < 0.05) and the male:female ratio differed (P < 0.05) from the expected 1:1 ratio (approximately 0.3:1 and 4.5:1 for unaffected versus affected, respectively). In conclusion, when bovine embryos were cultured in the presence of rat monoclonal H-Y antibodies and compliment, alterations occurred in embryos that were beyond the 8-cell stage; we inferred that the antibodies cross-reacted with H-Y antigens.
Assuntos
Bovinos/embriologia , Fertilização in vitro/veterinária , Isoanticorpos/farmacologia , Análise para Determinação do Sexo/veterinária , Animais , Anticorpos Monoclonais/farmacologia , Técnicas de Cultura , Feminino , Masculino , Ratos , Análise para Determinação do Sexo/métodosRESUMO
Previous studies in our laboratory indicated that immunization of male and female Wistar and Lewis rats with epididymal protein DE, resulted in the development of anti-DE antibodies in over 90% of the animals, with a significant and reversible reduction of fertility. In the present study, ELISA assays performed to analyze the evolution of the immune response indicated that antibody levels in the sera of immunized animals reached a maximum at 8 weeks after the initial injection and then gradually decreased, returning to control values by the end of the sixth month. Western blot experiments demonstrated that the immune sera specifically recognized DE in epididymal sperm extracts and epididymal cytosol, while no reaction was observed with different reproductive and essential organs. The immune sera were also capable of recognizing DE on the surface of both fresh and capacitated sperm as indicated by indirect immunofluorescence experiments. Finally, the exposure of sperm to immune sera prior to uterine insemination resulted in a significant (P < 0.05) reduction in the percentage of fertilized eggs compared to controls, with no effect on sperm motility and viability, nor on their ability to undergo capacitation. Together, these results support the participation of the raised antibodies as mediators of the antifertility effect and suggest a specific interference at the sperm-egg interaction level.
Assuntos
Anticoncepção Imunológica , Epididimo/imunologia , Isoanticorpos/farmacologia , Metaloproteínas/fisiologia , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Hormônios Testiculares/fisiologia , Animais , Western Blotting , Proteínas Secretadas pelo Epidídimo , Feminino , Soros Imunes , Imunização , Isoanticorpos/biossíntese , Isoanticorpos/imunologia , Masculino , Metaloproteínas/imunologia , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Aglutinação Espermática , Capacitação Espermática , Motilidade dos Espermatozoides , Hormônios Testiculares/imunologiaRESUMO
1. Mice peritoneal mast cells previously treated with alloantibodies directed against private and public major histocompatibility antigens became partially resistant to subsequent passive sensitization with homologous IgG1-rich serum. 2. Sensitization of the mast cells was evaluated in terms of the percentage of cells that degranulated upon challenge with the specific antigen. 3. The phenomenon was immunologically specific and the responsible alloantigens were shown to be coded by the H-2K or H-2D regions.
Assuntos
Antígenos H-2/imunologia , Liberação de Histamina , Isoanticorpos/farmacologia , Mastócitos/imunologia , Anafilaxia/imunologia , Animais , Feminino , Soros Imunes , Imunização Passiva , Imunoglobulina G/imunologia , Capeamento Imunológico , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Receptores ImunológicosRESUMO
Mice peritoneal mast cells previously treated with alloantibodies directed against private and public major histocompatibility antigens became partially resistant to subsequent passive sensitization with homologous IgG1-rich serum. 2. Sensitization of the mast cells was evaluated in terms of the percentage of cells that de granulated upon challenge with the specifc antigen. 3. The phenomenon was immunologically specific and the responsible alloantigens were shown to be coded by the H-2K or H-2D regions