Steeman Tomás José

Neural crest cells are a transient, multipotent population that gives rise to diverse structures during vertebrate embryonic development, including craniofacial cartilage and pigment cells. Although the transcriptional regulation of neural crest development is well characterized, the role of microRNAs remains less understood. Using a double-transgenic zebrafish model expressing fluorescent reporters under the sox10 promoter, combined with fluorescence-activated cell sorting and RNA sequencing, we identified microRNAs enriched in neural crest cells. We focused on miR-133a-3p and miR-338-3p, previously linked to tumor suppression, to explore their developmental roles. The overexpression of either microRNA led to craniofacial cartilage malformations and reduced melanophore number, accompanied by decreased expression of key regulators including sox9b , sox10 , and runx3 . Reporter assays confirmed direct targeting of the sox9b 3′untranslated region. In addition, miR-338-3p overexpression increased neural crest cell numbers, suggesting a role in proliferation. These findings uncover novel functions for miR-133a-3p and miR-338-3p in vertebrate craniofacial and pigment cell development, highlighting shared regulatory features between embryogenesis and tumorigenesis.

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 NH ₄ Cl-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 mSEMG1 ^Q32-V118 and mSEMG1 ^R98-G375 , but not mSEMG1 ^Y221-G375 , inhibited sperm hyperactivation and CatSper currents to a similar extent as full-length mSEMG1. Notably, only mSEMG1 ^R98-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 statement: We 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.

Sperm membrane potential (Em) hyperpolarization during capacitation is a functional hallmark of fertilizing ability and has been proposed as a predictive biomarker for conventional in vitro fertilization (IVF) success. However, it is unclear whether Em remains stable across ejaculates over time and can reliably guide assisted reproductive technology (ART) decisions in advance. Thus, we aimed to evaluate the temporal consistency of human sperm Em within individuals and assess its utility as a prognostic marker when measured days or weeks prior to IVF procedures. Em was assessed in capacitated and non-capacitated sperm from normospermic donors at three time points over 28 days, using a fluorometric assay. Capacitated values were compared to a −48.6 mV threshold previously associated with successful fertilization. Intra-donor Em variability and coefficients of variation (CV) were analyzed statistically. Our results showed that Em values exhibited significant intra-donor variability over time (p = 0.007), with approximately half of the donors crossing the −48.6 mV functional threshold across sessions. Capacitated sperm samples showed significantly greater variability than non-capacitated ones, with several donors exceeding a 30% CV cutoff. No consistent correlation was found between CV and mean Em values. While Em remains a promising functional marker when assessed on the day of IVF, its temporal variability undermines its reliability as a predictive tool for ART decisions made in advance. These findings underscore the importance of timing in functional sperm assessments and call for further studies to identify the physiological factors influencing Em stability.

Sperm membrane potential (Em) hyperpolarization during capacitation is a functional hallmark of fertilizing ability and has been proposed as a predictive biomarker for conventional in vitro fertilization (IVF) success. However, it is unclear whether Em remains stable across ejaculates over time and can reliably guide assisted reproductive treatment (ART) decisions in advance. Thus, we aimed to evaluate the temporal consistency of human sperm Em within individuals and assess its utility as a prognostic marker when measured days or weeks prior to IVF procedures. Em was assessed in capacitated and non-capacitated sperm from normospermic donors at three time points over 28 days, using a fluorometric assay. Capacitated values were compared to a −48.6 mV threshold previously associated with successful fertilization. Intra-donor Em variability and coefficients of variation (CV) were analyzed statistically. Our results showed that Em values exhibited significant intra-donor variability over time (p=0.007), with approximately half of the donors crossing the −48.6 mV functional threshold across sessions. Capacitated sperm samples showed significantly greater variability than non-capacitated ones, with several donors exceeding a 30% CV cutoff. No consistent correlation was found between CV and mean Em values. While Em remains a promising functional marker when assessed on the day of IVF, its temporal variability undermines its reliability as a predictive tool for ART decisions made in advance. These findings underscore the importance of timing in functional sperm assessments and call for further studies to identify the physiological factors influencing Em stability.

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 (3’UTR) 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.

A key factor to the success of in vitro fertilization (IVF) is the preparation of human sperm, a critical step that directly impacts the efficacy of the procedure. This proof-of-concept study evaluated the effect of HyperSperm, a novel sperm preparation technique designed to enhance sperm function, on fertilization, embryo development, and pregnancy outcomes in both a mouse model and a first-in-human trial following IVF. In mice, HyperSperm treatment significantly increased hyperactivated motility (p < 0.05), leading to improved fertilization and blastocyst development (p < 0.05), as well as higher implantation rates (p < 0.05) and larger litter sizes (p < 0.05). Offspring displayed normal growth and fertility. In human sperm samples from normozoospermic men, HyperSperm exhibited a high safety profile, with motility, acrosome reaction, viability, and DNA fragmentation comparable to controls. A first-in-human, prospective, single-center, split-oocyte study in 10 couples undergoing IVF with donated oocytes demonstrated similar fertilization rates between HyperSperm and control groups (p = 0.425), but significantly higher usable blastocyst rates in the HyperSperm arm (43.8% vs. 67.9%, p = 0.0122). Morphokinetic parameters were comparable between groups. These results suggest that HyperSperm enhances sperm hyperactivation, a hallmark of capacitation, leading to improved embryo development in both mice and humans. This technique represents a promising approach to optimizing sperm preparation in assisted reproduction, warranting further clinical investigation.

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity rely on the utilization of [γ-³²P] ATP and the Kemptide peptide as a substrate. This strategy presents several major drawbacks, including high costs and health risks derived from the manipulation of radioactive isotopes. In this work, we introduce an enhanced non-radioactive assay to quantify PKA activity, termed kinase mobility shift assay (KiMSA), based on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that KiMSA is suitable for isolated PKA as well as for the enzyme in cell extracts. In addition, it enables quantification of PKA activity during the progression of mouse sperm capacitation. Furthermore, the assay enables monitoring the inhibition of PKA with pharmacological inhibitors in live cells. Therefore, the experimental and optimal assay conditions are set so that KiMSA can be used to assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity relies on the utilization of [γ-³²P] ATP and the Kemptide substrate. This methodology presents several major drawbacks, including high-costs and health risks derived from the manipulation of radioactive isotopes. In this work we introduce an enhanced non-radioactive assay for quantifying PKA activity, termed KiMSA which relies on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that the KiMSA assay is suitable for purified PKA, and also to address both basal and capacitation induced PKA activity in mouse sperm cells. Furthermore, the assay enables monitoring the inhibition of PKA with inhibitors such as sPKI and H-89 in live cells. Therefore, the experimental and optimal assay conditions are set so that the KiMSA assay can be used to either assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

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.

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3′UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.