Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants

Saito, Kenji; Siegfried, Kellee R.; Nüsslein‐Volhard, Christiane; Sakai, Noriyoshi

doi:10.1002/dvdy.22661

Cited by 40 publications

(86 citation statements)

References 60 publications

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“…4B, compare with SSC clumps under SSCs propagating condition shown in Fig. 4C), as observed in intact zebrafish testis (Ozaki et al, 2011;Saito et al, 2011). Sperm-like cells appeared after 17 days of transfer (Fig.…”

Section: Differentiation Of Cultured Sscs To Functional Sperm In Culturesupporting

confidence: 60%

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

2015

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Molecular dissection and chemical screening on a complex process such as spermatogenesis could be facilitated by cell culture approaches that allow easy access for experimental manipulation and live imaging of specific molecules; however, technical limitations have thus far prevented the complete reconstruction of spermatogenic events in cell culture. Here, we describe the production of functional sperm from self-renewing spermatogonial stem cells (SSCs) in cell culture conditions, using zebrafish testicular hyperplasia cells that accumulate early stage spermatogonia. By serially transplanting hyperplasias into immunodeficient rag1 mutant zebrafish, we succeeded in long-term maintenance and efficient production of starting material for SSC culture. Through improvements of culture conditions, we achieved efficient propagation of SSCs derived from the hyperplasia. When SSCs that underwent the SSC-propagating step for 1 month were transferred onto Sertoli feeder cells, they differentiated into functional sperm that gave rise to offspring. Oxygen at the concentration of air proved to be detrimental for sperm differentiation from SSCs, but not for propagation of SSCs. These results indicate that the whole spermatogenic process can be represented in cell culture in zebrafish, facilitating analyses of the molecular mechanisms of spermatogenesis in vertebrates.

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Section: Differentiation Of Cultured Sscs To Functional Sperm In Culturesupporting

confidence: 60%

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

2015

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“…Two meiotic mutants of zebrafish have been isolated, namely, its , which has a defect in the preleptotene stage, and imo , which has a defect in the zygotene stage (Saito et al, ). We have not identified the mutated genes in these mutants or the functional consequences of these mutations.…”

Section: Resultsmentioning

confidence: 99%

“…We recently isolated several zebrafish mutants that have defects in spermatogenesis using an N‐ethyl‐N‐nitrosourea mutagenesis screen and a three‐generation mating scheme (Saito et al, ). The mutant phenotypes in testicular sections indicated that meiotic defects are the most common abnormalities in these mutants.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, we examined telomere behavior in spermatocytes of two previously described mutants, its and imo . Both mutants are sterile: the its mutant arrests at the preleptotene stage of meiotic prophase I, whereas the imo mutant arrests at the zygotene stage (Saito et al, ). This analysis showed that telomere clustering occurs independently of the formation of axial elements of the SC, allowing screening for mutants that have primary defects in telomere clustering.…”

Section: Introductionmentioning

confidence: 99%

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Telomere distribution pattern and synapsis initiation during spermatogenesis in zebrafish

Saito

Sakai

Kawasaki

et al. 2014

Developmental Dynamics

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Background: Telomeres are located at ends of eukaryotic chromosomes and can affect proper chromosomal positioning. During spermatogenesis, the appropriate dynamics and behavior of chromosomes is crucial to generate haploid cells through meiosis. Here, we describe telomere distribution patterns during spermatogenesis in zebrafish, especially during meiotic prophase I, using fluorescence in situ hybridization. This was combined with synaptonemal complex protein 3 immunostaining, which allows the staging of spermatocytes. Results: During spermatogonial proliferation and the preleptotene stage, telomeres were dispersed throughout the nucleus. During the leptotene stage, telomeres temporarily moved to one pole of the nucleus at which g-tubulin was located, forming the telomere bouquet. The cluster lasted until the onset of zygotene where it coincided with terminal synapsis initiation. They then spread around the periphery of the nucleus during the zygotene to pachytene stages. During postmeiotic stages, telomeres in spermatids and sperm were again dispersed throughout the nuclei. Application of this procedure in meiotic mutants confirmed that meiotic telomere clustering is independent of axial element formation of the synaptonemal complex. Conclusions: These data clearly showed the clustering and distributions of telomeres throughout spermatogenesis in zebrafish. This procedure could be used to screen for mutants that have primary defects in telomere clustering.

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“…Microarray analysis evidenced a downregulation of cyp19a1a , as previously found by qPCR analyses, and an upregulation of a set of genes involved in the promale pathway. These male genes were amh , sycp3, a gene that is a spermatocyte marker for meiotic cells (Ozaki, Saito, Shinya, Kawasaki, & Sakai, ; Saito, Siegfried, Nüsslein‐Volhard, & Sakai, ), sox3 , a gene associated with male development (Sutton et al, ), and spata6 , necessary for the correct progression of spermatogenesis (Yuan et al, ). However, other genes were downregulated: the steroidogenic factor 1 ( nr5a1 ), required for testis development (Wilhelm & Englert, ), and the androgen receptor.…”

Section: Discussionmentioning

confidence: 99%

Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations

2019

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Sex is remarkably plastic in fish and can be easily influenced by environmental cues, in which temperature has been the most studied abiotic factor. However, it has been shown that elevated population densities can increase the number of males in several species but little is known about the underlying molecular mechanisms and whether general patterns exist. Here, we studied the long‐term effects of population density on the gene expression program in zebrafish gonads. The ovarian transcriptome of females exposed to high versus low population densities contained 4,634 differentially expressed genes. Among them, a set of promale genes (amh, sypc3, spata6, and sox3) were upregulated in the high‐population density group. Next, we compared the transcriptomes of ovaries of female zebrafish resistant to the masculinizing effects of either high density or elevated temperature. Results showed a set of 131 and 242 common upregulated and downregulated genes, respectively, including the upregulation of known male‐related genes (e.g., amh and sycp3) but also genes involved in other functions (e.g., faima, ccm21, and ankrd6b) and a downregulation of cyp19a1a together with other genes (e.g., lgals9l1 and ubxn2a). We identified the common Gene Ontology terms involved in the reproduction and sexual development that were consistently affected in both environmental factors. These results show that regardless of the environmental perturbation there are common genes and cellular functions involved in the resistance to masculinization. These altered gene‐expression profiles can be used as markers indicative of previous exposure to environmental stress independent of conspicuous alterations in sex ratios or gonadal morphology.

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Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants

Cited by 40 publications

References 60 publications

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

Telomere distribution pattern and synapsis initiation during spermatogenesis in zebrafish

Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations

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