Biology subjects

Siegfried, K. R.

Publications and source records attributed to Siegfried, K. R..

3 recordsLinked to original sources

bioRxiv2022

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

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 an adad1 mutant 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 in adad1 mutant testes, the spermatocytes failed to progress beyond the zygotene stage. Thus, Adad1 is 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. Author SummaryInfertility is a serious problem for millions of couples who wish to have children. Globally more than 10% of couples suffer from infertility due to genetic, epigenetic, and environmental factors. Among these about 50% of cases occur due to genetic factors such as aneuploidy and genetic mutations affecting development of the gametes (i.e. sperm and eggs). Although many genes are known to be involved in germ cell development, genetic causes of infertility are still largely unexplained. Therefore, it is imperative to investigate genes involved in reproductive processes. In this study, we report that the adad1 gene is essential for germ cell maintenance and fertility in zebrafish. Our analysis of zebrafish adad1 mutants demonstrates that it is required for maintenance of the germline stem cells and for completion of meiosis. This is in contrast to mouse Adad1, which functions later in gamete development to regulate differentiation of haploid sperm. Our work on zebrafish adad1 has uncovered previously unknown roles of adad1 function in germline stem cell maintenance.

developmental biology↗

bioRxiv2022

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

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.

developmental biology↗

bioRxiv2021

The zebrafish meiotic cohesion complex protein Smc1b is required for key events in meiotic prophase I

The eukaryotic structural maintenance of chromosomes (SMC) proteins are involved in key processes of chromosome structure and dynamics. SMC1{beta} was identified as a component of the meiotic cohesion complex in vertebrates, which aids in keeping sister chromatids together prior to segregation in meiosis II and is involved in association of homologous chromosomes in meiosis I. The role of SMC1{beta} in meiosis has primarily been studied in mice, where mutant male and female mice are infertile due to germ cell arrest at pachytene and metaphase II stages, respectively. Here, we investigate the function of zebrafish Smc1b to understand the role of this protein more broadly in vertebrates. We found that zebrafish smc1b is necessary for fertility and has important roles in meiosis, yet has no other apparent roles in development. Therefore, smc1b functions primarily in meiosis in both fish and mammals. In zebrafish, we showed that smc1b mutant spermatocytes initiated telomere clustering in leptotene, but failed to complete this process and progress into zygotene. Furthermore, mutant spermatocytes displayed a complete failure of homolog pairing and synapsis. Interestingly, meiotic DNA double strand breaks occurred in the absence of Smc1b despite failed pairing and synapsis. Overall, our findings point to an essential role of Smc1b in the leptotene to zygotene transition during zebrafish spermatogenesis. In addition, ovarian follicles failed to form in smc1b mutants, suggesting an essential role in female meiosis as well. Our results indicate that there are some key differences in Smc1b requirement in meiosis among vertebrates: while Smc1b is not required for homologue pairing and synapsis in mice, it is essential for these processes in zebrafish.

developmental biology↗