Centrosomes represent main microtubule organizing centers (MTOCs) in animal cells. Their duplication in S-phase enables the establishment of two MTOCs in M-phase that define the poles of the spindle and ensure equal distribution of chromosomes and centrosomes to the two daughter cells. While key functions of many centrosomal proteins have been addressed in RNAi experiments and chronic knockdown, knockout experiments with complete loss of function in all cells enable quantitative analysis of cellular phenotypes at all cell-cycle stages. Here, we show that the centriolar satellite proteins SSX2IP and WDR8 and the centriolar protein CEP135 form a complex before centrosome assembly in vertebrate oocytes and further functionally interact in somatic cells with established centrosomes. We present stable knockouts of SSX2IP, WDR8, and CEP135 in human cells. While loss of SSX2IP and WDR8 are compensated for, cep135 knockout cells display compromised PCM recruitment, reduced MTOC function, and premature centrosome splitting with imbalanced PCMs. Defective cep135 knockout centrosomes, however, manage to establish balanced spindle poles, allowing unperturbed mitosis and regular cell proliferation. Our data show essential functions of CEP135 in interphase MTOCs and demonstrate that loss of individual functions of SSX2IP, WDR8, and CEP135 are fully compensated for in mitosis.
American shad Alosa sapidissima, an anadromous clupeid, exhibits variation in reproductive strategies, including semelparity and iteroparity. It provides an excellent model for studying the behaviour of germ cells in anadromous fish during their migration from sea to river. The vasa gene was characterized in A. sapidissima as a germ-cell marker to elaborate the process of germ-cell development and differentiation in anadromous species. A complementary (c)DNA fragment of 819 bp, partial open reading frame (ORF), was cloned by degenerate PCR and named as ASvas. In adult A. sapidissima, vasa transcript was exclusively detected in gonads by reverse-transcription (RT)-PCR. Through chromogenic in situ hybridization, the vasa messenger (m)RNA was specifically detected in primordial germ cells (PGC) in embryos and germ cells at early stages in ovary and testis. Besides, the cellular distribution profile of Vasa protein also proved that vasa gene could be used as a germ-line marker to trace the PGCs migration during embryogenesis and the germ-cell differentiation during gametogenesis in A. sapidissima. During embryogenesis, the migrating PGCs were clearly detected at tail-bud stage and the PGCs reached the genital ridge at the stage of pre-hatching stage in A. sapidissima embryos. During gametogenesis, the Vasa protein was dynamically expressed in differentiating germ cells at different stages in adult gonads. As far as we know, this is the first report to demonstrate the PGCs migration and germ-cell differentiation through vasa gene expression in the anadromous species. The findings will pave a way for investigating germ-cell development and maturation in the A. sapidissima and other anadromous fish.
Hybrid sterility has been widely observed in fish species; however, the molecular mechanisms of hybrid sterility are not well understood. In this work, we compared the reproductive development of yellow catfish (Pelteobagrus fulvidraco, PF) and hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) from embryo to adult at cellular and molecular levels. Firstly, we developed a pair of genetic markers to efficiently distinguish PF and PV (Pelteobagrus vachelli) and their interspecific hybrid. Hybrid sterility was observed in hybrid yellow catfish, as indicated by the germ cell‐less phenotype and defective gonadal development. The expression levels of germ cell differentiation‐related and gonadal somatic cell development‐related genes were obviously down‐regulated in PF × PV hybrid than in PF. In addition, meiotic and mitotic defects were detected in the testis of PF × PV hybrid, since the numbers of Sycp3‐ and PH3‐positive cells were obviously reduced. As early as 4‐cell stage, defects of germplasm assembly and accelerated decay of maternal germline‐specific mRNAs were observed in hybrid yellow catfish. Our studies provide evidence that hybrid sterility in animals might be caused by the accelerated decay of germplasm mRNAs, impaired germplasm assembly and defective germ cell development.
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