Biology subjects

Steeman, T. J.

Publications and source records attributed to Steeman, T. J..

3 recordsLinked to original sources

bioRxiv2025

Semenogelin-1 Inhibition of Mouse Sperm Hyperactivation Reveals Two Functional Domains Modulating CatSper Channel

Seminal plasma is essential for sperm survival and function after ejaculation. Semenogelin-1 (SEMG1), the predominant seminal plasma protein, transiently suppresses sperm motility and hyperactivation after ejaculation through epididymal protease inhibitor (EPPIN) binding. However, the molecular mechanism underlying SEMG1-mediated inhibition of hyperactivation remains unclear. Here, we tested the hypothesis that SEMG1 inhibits CatSper, a sperm-specific calcium channel crucial for hyperactivation. Full-length recombinant mouse SEMG1 (mSEMG1; Q32-G375) inhibited both progressive motility and hyperactivation; the latter was not recovered with NH4Cl-induced alkalinization, indicating an effect downstream of capacitation-associated intracellular alkalinization. Electrophysiological recordings revealed that mSEMG1 reduced CatSper currents at physiologically relevant concentrations. Truncated mSEMG1 fragments mSEMG1Q32-V118 and mSEMG1R98-G375, but not mSEMG1Y221-G375, inhibited sperm hyperactivation and CatSper currents to a similar extent as full- length mSEMG1. Notably, only mSEMG1R98-G375 retained full EPPIN-binding capacity. Together, our findings identify two functional domains within mSEMG1 (Q32-V118 and R98-S220) that inhibit sperm hyperactivation by suppressing CatSper activity and differ in their EPPIN-binding capacities. These functional domains represent promising prototypes for the design of spermiostatic molecules, offering additional avenues for non-hormonal male contraception. Summary statementWe report a novel mechanism by which SEMG1, a major seminal plasma protein, modulates sperm hyperactivation by inhibiting a key calcium channel, offering new insights into sperm function and non- hormonal male contraception.

physiology↗

bioRxiv2025

miR-133a and miR-338-3p Shape Neural Crest Derivatives in Zebrafish

Neural crest (NC) cells are a transient, multipotent cell population that contributes to the formation of diverse tissues during vertebrate development. While numerous transcription factors and signalling pathways regulate NC specification and differentiation, the role of microRNAs (miRNAs) in these processes remains underexplored. In this study, we employed a double-transgenic zebrafish model (Tg(sox10:eGFP, sox10:mRFP)), in combination with fluorescence-activated cell sorting (FACS) and RNA sequencing, to identify miRNAs enriched in NC cells and assess their role in the development of NC derivatives. Given the parallels between NC development and tumorigenesis, we focused on a subset of miRNAs previously implicated in cancer progression. Functional assays revealed that overexpression of miR-133a and miR-338-3p resulted in significant craniofacial cartilage malformations and a reduction in melanophore numbers, correlating with the downregulation of key developmental genes, such as sox9b, sox10, and runx3. Reporter assays further demonstrated that miR-133a and miR-338-3p directly target the 3 untranslated region (3UTR) of sox9b, supporting their role in translational repression and mRNA degradation. Additionally, miR-338-3p overexpression increased the overall NC cell population, suggesting a regulatory function in cell proliferation. These findings offer new insights into the molecular mechanisms governing NC development and highlight functional parallels between miRNA-mediated NC regulation and tumour biology. Given that miR-133a and miR-338-3p are known tumour suppressors, their roles in NC differentiation and proliferation may reflect conserved pathways underlying both embryogenesis and oncogenesis. This study enhances our understanding of miRNA function in vertebrate embryogenesis and underscores their potential relevance in neurocristopathies and NC-derived cancers.

developmental biology↗

bioRxiv2023

G-quadruplexes regulate miRNA biogenesis in live zebrafish embryos

RNA guanine quadruplexes (G4s) regulate RNA functions, metabolism, and processing. G4s formed within precursors of microRNAs (pre-miRNAs) may impair pre-miRNAs maturation by Dicer, thus repressing mature miRNA biogenesis. As miRNAs are essential for proper embryonic development, we studied the role of G4s on miRNA biogenesis in vivo during zebrafish embryogenesis. We performed a computational analysis on zebrafish pre-miRNAs to find putative G4 forming sequences (PQSs). The precursor of the miRNA 150 (pre-miR-150) was found to contain an evolutionarily conserved PQS formed by three G-tetrads and able to fold in vitro as G4. MiR-150 controls the expression of myb, which shows a well-defined knock-down phenotype in zebrafish developing embryos. We microinjected zebrafish embryos with in vitro transcribed pre-miR-150 synthesized using either GTP (G-pre-miR-150) or 7-Deaza-GTP, a GTP analogue unable to form G4s (7DG-pre-miR-150). Compared to embryos injected with G-pre-miR-150, embryos injected with 7DG-pre-miR-150 showed higher levels of miRNA 150 (miR-150) and lower levels of myb mRNA and stronger phenotypes associated with myb knock-down. The incubation of pre-miR-150 prior to the injection with the G4 stabilizing ligand pyridostatin (PDS) reverted gene expression variations and rescued the phenotypes related to myb knock-down. Overall, results suggest that the G4 formed in pre-miR-150 functions in vivo as a conserved regulatory structure competing with the stem-loop structure necessary for miRNA biogenesis.

molecular biology↗