RESUMO
Early cell specification in mammalian preimplantation embryos is an intricate cellular process that leads to coordinated spatial and temporal expression of specific genes. Proper segregation into the first two cell lineages, the inner cell mass (ICM) and the trophectoderm (TE), is imperative for developing the embryo proper and the placenta, respectively. Somatic cell nuclear transfer (SCNT) allows the formation of a blastocyst containing both ICM and TE from a differentiated cell nucleus, which means that this differentiated genome must be reprogrammed to a totipotent state. Although blastocysts can be generated efficiently through SCNT, the full-term development of SCNT embryos is impaired mostly due to placental defects. In this review, we examine the early cell fate decisions in fertilized embryos and compare them to observations in SCNT-derived embryos, in order to understand if these processes are affected by SCNT and could be responsible for the low success of reproductive cloning.
Assuntos
Diferenciação Celular , Técnicas de Transferência Nuclear , Placenta , Animais , Feminino , Gravidez , Blastocisto/metabolismo , Clonagem de Organismos , Embrião de Mamíferos/metabolismo , MamíferosRESUMO
Placental defects are common in bovine embryos produced using assisted reproductive techniques. A proper understanding of the events leading to inner cell mass (ICM) and trophectoderm (TE) specification could help identify the origins of such developmental failures. We focused on caudal-type homeobox transcription factor 2 (CDX2) since it has a specific role during TE differentiation in mouse embryos. Of all the preimplantation stages analyzed, CDX2 protein was present only at the blastocyst stage. To further understand the roles of CDX2 during bovine development, we depleted CDX2 mRNA; despite a significant loss of detectable protein, embryos were able to form blastocysts at the same rate as controls. Embryos lacking CDX2 did not show abnormalities in the number of TE, ICM, or total cells in the blastocyst. Expression of the developmentally important genes SOX2, POU5F1, and NANOG, or TE markers such as IFN-T and KRT18 were not affected by the reduction in CDX2 levels, nor was the localization of SOX2 and POU5F1 protein. Using a functional barrier assay, we observed that the TE epithelial layer of embryos lacking CDX2 had lost its integrity. Our results thus indicate that CDX2 is not required for TE formation during bovine development; nevertheless, it is necessary for maintaining TE integrity.
Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , RNA Mensageiro/biossíntese , Animais , Blastocisto/citologia , Bovinos , CamundongosRESUMO
Approximately 12.5% of all 9,920 extant bird species in the world are threatened with extinction, and yet conservation efforts through natural breeding of captive species continue to encounter difficulties. However, sperm cryopreservation and artificial insemination offer potential benefits over natural breeding, but their applicability is still limited in nondomestic species. In this study, we aimed to exploit the potential of germ cell xenotransplantation as an alternative tool for preserving germplasm of endangered birds. The study was designed to investigate whether transfer of either spermatogonia-enriched cell fraction (SEF) or crude testicular cell fraction (CTF) from adult Japanese quails (as a model for wild species) would result in recolonization of gamma-irradiated gonads of adult recipient chickens. One month after transplantation, 75% of recipients injected with SEF and 25% of recipients injected with CTF resumed spermatogenesis. However, it took more than 3 months for 33% of the negative controls to resume marginal production of sperm. Some SEF recipients produced more spermatozoa bearing head morphology compared with donor controls. DNA analysis using quail-specific primers did not detect donor's DNA in these recipients' semen. However, 6 months after xenotransplantation, presence of quail germ cells was demonstrated by polymerase chain reaction and by immunohistochemistry in 1 rooster injected with SEF. These findings indicate that spermatogonia from adult quails were capable of colonizing immunocompetent testis of adult chickens but failed to produce sufficient sperm. Despite this limitation, the present approach represents a potential conservation tool that may be used to rescue germ cells of endangered adult male birds.
Assuntos
Galinhas , Coturnix , Espermatogênese , Espermatogônias/transplante , Espermatozoides/transplante , Testículo/citologia , Transplante Heterólogo/veterinária , Animais , Cruzamento , Galinhas/fisiologia , Coturnix/fisiologia , Espécies em Perigo de Extinção , Feminino , Inseminação Artificial , Masculino , Espermatozoides/fisiologiaRESUMO
OBJECTIVE: To report on the development of human parthenogenetic blastocysts and an in vitro attachment that was generated from noninseminated cryopreserved human oocytes for the first time. DESIGN: Prospective study. SETTING: Department of reproductive medicine in a medical institute in Buenos Aires, Argentina. PATIENT(S): Five healthy fertile donors. INTERVENTION(S): Artificial activation of noninseminated cryopreserved human oocytes after thawing, parthenote culture, and their in vitro attachment. MAIN OUTCOME MEASURE(S): Survival rate, activation rate, cleavage rate, and blastocyst formation. RESULT(S): Thirty-six of 38 cryopreserved noninseminated oocytes survived after thawing (survival rate, 94.7%). Thirty-one of 36 oocytes showed one pronucleus (activation rate, 86.1%). Thirty of 31 cleaved (cleavage rate, 96.8%). Five of 30 showed cavitation (blastocyst rate, 16.7%). CONCLUSION(S): Noninseminated cryopreserved human oocytes showed a high survival rate after thawing. They responded very satisfactorily to artificial activation, which was followed by a high rate of parthenogenetic embryos, which can develop into blastocysts. In the future, these could be a new source for development of human parthenogenetic stem cells.