Histological Analysis of Gonads in Zebrafish

Siegfried, Kellee R.; Steinfeld, Jocelyn S.

doi:10.1007/978-1-0716-0970-5_20

Cited by 10 publications

(10 citation statements)

References 10 publications

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“…Whole fish were fixed overnight in Bouin’s fixative (Cat# 112016; Fisher Scientific). Fish were then washed 4 × 4 hr in 70% ethanol before processing for paraffin embedding and 7 µm sections cut and stained with hematoxylin and eosin (H&E) as previously described ( Siegfried and Steinfeld, 2021 ). Imagers were acquired using a Zeiss Axiophot microscope equipped with a Leica DFC 500 camera.…”

Section: Methodsmentioning

confidence: 99%

Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary

Liu

Kassack

McFaul

et al. 2022

eLife

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Zebrafish are an established research organism that has made many contributions to our understanding of vertebrate tissue and organ development, yet there are still significant gaps in our understanding of the genes that regulate gonad development, sex, and reproduction. Unlike the development of many organs, such as the brain and heart that form during the first few days of development, zebrafish gonads do not begin to form until the larval stage (≥5 dpf). Thus, forward genetic screens have identified very few genes required for gonad development. In addition, bulk RNA sequencing studies which identify genes expressed in the gonads do not have the resolution necessary to define minor cell populations that may play significant roles in development and function of these organs. To overcome these limitations, we have used single-cell RNA sequencing to determine the transcriptomes of cells isolated from juvenile zebrafish ovaries. This resulted in the profiles of 10,658 germ cells and 14,431 somatic cells. Our germ cell data represents all developmental stages from germline stem cells to early meiotic oocytes. Our somatic cell data represents all known somatic cell types, including follicle cells, theca cells and ovarian stromal cells. Further analysis revealed an unexpected number of cell subpopulations within these broadly defined cell types. To further define their functional significance, we determined the location of these cell subpopulations within the ovary. Finally, we used gene knockout experiments to determine the roles of foxl2l and wnt9b for oocyte development and sex determination and/or differentiation, respectively. Our results reveal novel insights into zebrafish ovarian development and function and the transcriptome profiles will provide a valuable resource for future studies.

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Section: Methodsmentioning

confidence: 99%

Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary

Liu

Kassack

McFaul

et al. 2022

eLife

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“…Histology was performed as previously described (93). Briefly, fish were euthanized by tricaine overdose and the torso was isolated and fixed in Bouin's solution overnight at room temperature.…”

Section: Histologymentioning

confidence: 99%

The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish

Islam

Ajao

Henke

et al. 2022

Preprint

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The double stranded RNA binding protein Adad1 (adenosine deaminase domain containing 1) is a member of the adenosine deaminase acting on RNAs (Adar) protein family with germ cell-specific expression. In mice, Adad1 is necessary for sperm differentiation, however its function outside of mammals has not been investigated. Here, through an N-ethyl-N-nitrosourea (ENU) based forward genetic screen, we identified anadad1mutant zebrafish line that develop as sterile males. Further histological examination revealed complete lack of germ cells in adult mutant fish, however germ cells populated the gonad, proliferated, and entered meiosis in larval and juvenile fish. Although meiosis was initiated inadad1mutant testes, the spermatocytes failed to progress beyond the zygotene stage. Thus,adad1is essential for meiosis and germline maintenance in zebrafish. We tested if spermatogonial stem cells were affected using a label retaining cell (LRC) assay and found that the mutant testes had fewer LRCs compared to wild-type siblings, suggesting that failure to maintain the spermatogonial stem cells resulted in germ cell loss by adulthood. To identify potential molecular processes regulated by Adad1, we sequenced bulk mRNA from mutants and wild-type testes and found mis-regulation of genes involved in RNA stability and modification, pointing to a potential broader role in post-transcriptional regulation. Our findings suggest that Adad1 is an RNA regulatory protein required for fertility through regulation of spermatogonial stem cell maintenance in zebrafish.

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“…Samples were fixed either for 2-4 hours in Davidson's solution (isolated gonads) or overnight in Bouin's fixative (whole torsos) at room temperature, then embedded in paraffin. Testes were cut into 5µm sections and ovaries into 7 µm sections and were stained using Modified Harris hematoxylin and Eosin Y (H&E) (Siegfried and Steinfeld 2021).…”

Section: Histology and Ovary Quantificationmentioning

confidence: 99%

The zebrafish dmrt family genes have cooperative and antagonistic roles in sex determination and oogenesis

Steinfeld

Ameyaw

Wood

et al. 2022

Preprint

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The double-sex and mab3 related transcription factor (dmrt) gene family has conserved roles in sex determination and gonad development across metazoans. In zebrafish, dmrt1 was previously shown to function in male sex-determination and testes development. To gain a broader knowledge of this gene family in sexual development, we investigated potential roles of all zebrafish dmrt family genes in sex-determination and gonad development using mutant analysis. The dmrt2a and dmrt5 mutants conferred lethality prior to sex differentiation, whereas dmrt2b and dmrt3 mutants were viable and fertile. Dmrt2b mutants had normal sex ratios while dmrt3 showed slightly skewed sex ratios in some experiments, indicating that dmrt3 has a minor role in sex-determination. We report a previously unknown role for dmrt1 in ovary development. Although dmrt1 mutant females were fertile, oogenesis did not progress normally, as evident from abnormal proportions of differently-staged oocytes within mutant ovaries. We also asked if dmrt1 mutant phenotypes could be modified by loss of another dmrt family member. Analysis of dmrt1;dmrt2a mutants was possible as these double mutants were sub-viable, showing a partial rescue of the dmrt2a lethality in the dmrt1 mutant background. The dmrt1;dmrt2a mutants had less severe female bias than dmrt1 mutants suggesting that dmrt2a acts antagonistically to dmrt1 in sex determination. Double mutants of dmrt1 with either dmrt2a or dmrt3 had more severe oogenesis defects than dmrt1 mutants and had either sub-fertility with reduced fecundity or failed to breed, respectively. This study reveals previously unknown roles of zebrafish dmrt1, dmrt2a, and dmrt3 in oogenesis.

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Histological Analysis of Gonads in Zebrafish

Cited by 10 publications

References 10 publications

Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary

Single-cell transcriptome reveals insights into the development and function of the zebrafish ovary

The RNA-binding protein Adad1 is necessary for germ cell maintenance and meiosis in zebrafish

The zebrafish dmrt family genes have cooperative and antagonistic roles in sex determination and oogenesis

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