Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors -phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) -all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca 2+ rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca 2+ oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.
Recent loss-of-function and gain-of-function studies have revealed that transcription factor Sox9 is required for testis formation by governing Sertoli cell differentiation, and thereafter regulating transcription of Sertoli marker genes. In the present study, we identified a novel isoform of Vinexin, which is expressed in somatic cells but not germ cells of sexually indifferent stages of fetal gonads. After the sex is determined, the expression continues in testicular Sertoli cells. Immunohistochemical analyses with a specific antibody to Vinexin indicated that Vinexin γ γ γ γ is localized in the cytoplasm. Functional studies with C3H10T1/2 cells showed that Vinexin γ γ γ γ acted as a scaffold protein to activate MEK and ERK through interaction with c-Raf and ERK. Ultimately, Sox9 transcription was induced by Vinexin γ γ γ γ . This up-regulation of Sox9 expression disappeared when the cells were treated with a specific MEK inhibitor, U0126. To determine the role of Vinexin γ γ γ γ during gonad formation, the gene was disrupted by targeted mutagenesis. The phenotype displayed by the mice indicated that ERK activation was decreased in the Vinexin γ γ γ γ -/-XY gonads, and Sox9 expression was down-regulated. Thus, Vinexin γ γ γ γ seems to be implicated in regulation of Sox9 gene expression by modulating MAPK cascade in mouse fetal gonads.
Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).
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