RESUMO
This review is intended to draw attention to the importance of the culture media composition on the health of the embryos, fetuses, newborns, and adults derived from assisted reproductive technologies (ART). Although current research and industry trends are to use chemically defined media because of their suitability for manufacturing, commercialization, and regulatory purposes, compelling evidence indicates that those media fail to adequately account for the biological demands of early embryogenesis. Here, we list the main undesirable consequences of the ART described in the literature and results we and others have obtained over the past decade exploring an alternative and more natural way to support embryo growth in vitro: inclusion of endogenous reproductive fluids as additives in the ART culture media for pigs, cows, and humans. This review systematically assesses the pros and cons of using reproductive fluid additives, as well as the requirements to implement this approach in the future.
RESUMO
This review is intended to draw attention to the importance of the culture media composition on the health of the embryos, fetuses, newborns, and adults derived from assisted reproductive technologies (ART). Although current research and industry trends are to use chemically defined media because of their suitability for manufacturing, commercialization, and regulatory purposes, compelling evidence indicates that those media fail to adequately account for the biological demands of early embryogenesis. Here, we list the main undesirable consequences of the ART described in the literature and results we and others have obtained over the past decade exploring an alternative and more natural way to support embryo growth in vitro: inclusion of endogenous reproductive fluids as additives in the ART culture media for pigs, cows, and humans. This review systematically assesses the pros and cons of using reproductive fluid additives, as well as the requirements to implement this approach in the future.(AU)
Assuntos
Animais , Técnicas In Vitro , Técnicas de Reprodução Assistida , Desenvolvimento Embrionário , Estruturas Embrionárias , Síndrome de Beckwith-Wiedemann/diagnóstico , Produtos Biológicos , EpigenômicaRESUMO
This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of â¼60 âkDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.
Assuntos
Calreticulina/fisiologia , Membrana Celular/fisiologia , Grânulos Citoplasmáticos/metabolismo , Interações Espermatozoide-Óvulo , Suínos , Animais , Calreticulina/metabolismo , Células Cultivadas , Técnicas de Cultura Embrionária , Exocitose , Fertilização , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Oócitos/citologia , Oócitos/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Suínos/metabolismo , Distribuição Tecidual , Zona Pelúcida/metabolismoRESUMO
Fertilization is a complex and fascinating biological process. The interactions between gametes transform two differentiated cells on a totipotent zygote. A few cell surface proteins in both gametes have been identified as essential for binding and fusion of gametes. At the zona pellucida level the binding is initiated by species-restricted binding of the sperm to the zona pellucida and is facilitated by the protein SED1 and or by the binding of sperm surface beta1/4-galactosyltransferase I (GaIT-I) to glycoside chains of the ZP3. This binding triggers the acrosome reaction. Among the molecules that participate on binding and fusion of gametes are included disintegrins on the sperm (ADAM1 and ADAM2) which interact with integrins (alpha6/beta-1, CD9, GPI-protein) in the egg plasma membrane, while cysteine-rich secretory proteins (CRISP) and the proteins named as Izumo participate in the fusion. The knowledge of the molecules and mechanisms involved in these processes will allow us not only a better understanding of the events underlying mammalian sperm-egg interaction, but also the development of new methods for both fertility regulation and diagnosis and clinic treatment of human and animal infertility.