Cyclin-dependent kinase 21 is a novel regulator of proliferation and meiosis in the male germline of zebrafish

in Reproduction
Authors:
Kaitlyn A Webster Biology Department, University of Massachusetts Boston, Boston, Massachusetts, USA

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Katrin Henke Orthopaedic Research Laboratories, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

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Delaney M Ingalls Biology Department, University of Massachusetts Boston, Boston, Massachusetts, USA

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Adeeba Nahrin Biology Department, University of Massachusetts Boston, Boston, Massachusetts, USA

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Matthew P Harris Orthopaedic Research Laboratories, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

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Kellee R Siegfried Biology Department, University of Massachusetts Boston, Boston, Massachusetts, USA

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Correspondence should be addressed to K R Siegfried; Email: kellee.siegfried@umb.edu

†(K A Webster is now at John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA)

DOI:
https://doi.org/10.1530/REP-18-0386
Volume/Issue:
Volume 157: Issue 4
Page Range:
383–398
Article Type:
Research Article
Online Publication Date:
Apr 2019
Copyright:
© 2019 Society for Reproduction and Fertility 2019
Restricted access

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Germ cell differentiation and maintenance relies on complex regulation of mitotic and meiotic progression. Cyclin-dependent kinases (CDKs) and their activating cyclin partners are known to have specialized roles in regulating cell cycle progression across tissues, including germ cells. Very little is known about CDK/cyclin function in zebrafish or the regulation of germ cell maintenance and differentiation. In a forward genetic screen for gonadogenesis defects in zebrafish, a mutation disrupting cdk21 (cyclin-dependent kinase 21) was identified, which caused gonad hypoplasia, reduced fertility and failure of female sex specification. The cdk21 gene is unique to fishes, though the encoded protein is related to the D-cyclin partners Cdk4 and Cdk6, which are known G1 cell cycle regulators. In the testis, cdk21 mutant germ cells exhibited cell cycle defects such as diminished proliferation, prolonged meiosis and delayed sperm differentiation. Furthermore, cdk21 mutants failed to maintain germ cells following breeding. Based on these findings, we propose that cdk21 regulates spermatogonial proliferation, progression through meiosis and germline stem cell activation in the testis. In addition, we investigated cdk4 and cdk6 in zebrafish development and found that each has distinct expression patterns in the gonads. Mutant analysis demonstrated that cdk6 was necessary for viability beyond larval stages. In contrast, cdk4 mutants were viable but were all male with low breeding success and sperm overabundance. Our analysis demonstrated that zebrafish harbor three genes of the cdk4/6 family, cdk4, cdk6 and cdk21, with cdk21 having an essential role in germ cell development in the testis.

Supplementary Materials

    • Supplementary Table 1. List of primers used in this paper. References are given for those primers from previous studies. *primers previously published in (Siegfried and Nüsslein-Volhard, 2008). **Primers previously published in (Knaut et al., 2000).
    • Supplementary Figure 1. Mutations generated in this study. A) The Cdk21 protein is largely composed of a kinase domain (gray). The cdk21t30421 allele is a missense mutation in the kinase domain. The cdk21umb7 mutation produced a frame shift at residue 105 and ultimately a premature stop codon. B) Mutations disrupting the cdk4 and cdk6 genes. The cdk4umb8 mutation produced a frame shift at residue 217. The cdk6umb9 allele produced a frame shift at residue 10 resulting in a premature stop codon. C) Alignments of a portion of the kinase domain of zebrafish cdk21, cdk4, and cdk6. The cdk21t30421 mutations resulted in replacement of glycine 204 with a tryptophan in a highly conserved region of the kinase domain (boxed). D) DNA sequence alignments showing mutations characterized in this paper.
    • Supplementary Figure 2. Western blots on protein extracts from wild-type and cdk21t30421 testes. A) Western blots of three biological replicates of each, immature (2 mpf) and mature (4 mpf) testes. In cdk21 mutants phosphor-Histone H3 (pH3) was decreased in testis from immature fish and elevated in testes of mature fish. B) Western blots of two biological replicates from adult fish showing elevated Sycp3 levels in mutant testes. A third biological replicate was shown in Fig. 4.

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1470-1626
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1741-7899

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