Dataset Information

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

ABSTRACT:

Background

Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain.

Methods

Fhad1^-/- mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was used to evaluate changes in gene expression, with subsequent analyses of spermatogenesis and fertility. The testis phenotypes were also examined using immunofluorescence and histological staining, while sperm concentrations and motility were quantified via computer-aided sperm analysis. Cellular apoptosis was assessed using a TUNEL staining assay.

Results

The Fhad1^-/-mice did not exhibit any abnormal changes in fertility or testicular morphology compared to wild-type littermates. Histological analyses confirmed that the testicular morphology of both Fhad1^-/-and Fhad1^+/+ mice was normal, with both exhibiting intact seminiferous tubules. Relative to Fhad1^+/+ mice, however, Fhad1^-/-did exhibit reductions in the total and progressive motility of epididymal sperm. Analyses of meiotic division in Fhad1^-/-mice also revealed higher levels of apoptotic death during the first wave of spermatogenesis.

Discussion

The findings suggest that FHAD1 is involved in both meiosis and the modulation of sperm motility.

SUBMITTER: Zhang X

PROVIDER: S-EPMC10984166 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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Publications

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

Zhang Xi X Xue Jiangyang J Jiang Shan S Zheng Haoyu H Wang Chang C

PeerJ 20240329

<h4>Background</h4>Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain.<h4>Methods</h4><i>Fhad1</i><sup>-/-</sup> mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was us ...[more]

PMID: 38563001

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Project description:The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process.

Dataset Information

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

Background

Methods

Results

Discussion

Publications

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

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