This method allows researchers to examine how cell interactions contribute to organ function as well as to easily visualize cell movements in vivo. Available at: https://www.biorxiv.org/content/10.1101/2021.04.18.440256 (Accessed October 28, 2021). (2021). Nucleic Acids Res. (2021). Larvae are placed into tanks with little to no water exchange to start, and increasing water flow as the fish improve their ability to hunt and swim. SP, signal peptide; TMD, Transmembrane domain; CD, Cytoplasmic domain. (2016). Changes in gene expression over time and in specific organs are easily observed and provide insight to developmental processes. Science 361, 10291033. Despite these recent advances, the molecular mechanisms of gamete fusion and the interplay between the various sperm and egg factors remain unclear. Tertiary structure predictions of mouse and zebrafish Spaca6. Elucidating the role of conserved fertilization factors in both mammalian and non-mammalian vertebrate species will help to uncover common themes as well as distinct functionalities. Both wild-type and mutant sperm were able to reach the micropyle within the same time-frame [number of times sperm successfully approached the micropyle out of the total number of recorded events: 7/9 (wild-type sperm) and 5/6 (spaca6/ sperm)] (Figure 4A, Supplementary Movie S2). For western blot analysis, sperm from 3-6 males was sedimented at 3,000rpm for 3.5min. (2021). Sperm were counted as bound when they remained in the same position for at least 2min following a 90-s period in which they activated and approached the egg. Spaca6 has two gene annotations in the most recent zebrafish genome release (GRCz11): a predicted protein-coding gene containing 7 exons (NCBI: XM_021466914.1) and a predicted non-protein coding gene containing 8 exons (ENSEMBL: ENSDART00000155083.2). Quantification of sperm binding. Youve just watched JoVEs video on zebrafish development. Values were then normalized to the levels of wild-type sperm. Zebrafish (Danio rerio) were raised according to standard protocols (28C water temperature, 14/10h light/dark cycle). For example, to study the effect of loss of function of a gene, zebrafish can be injected with morpholinos, which are antisense oligonucleotides designed to block protein expression. Biology II: Mouse, Zebrafish, and Chick. Nucleic Acids Res. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.806982/full#supplementary-material, Altschul, S. (1997). The extracellular domain (ECD) is shown in blue, while the Immunoglobulin-like domain (IG) is shown in orange. Tubulin protein levels of the same blot are shown as loading control. Sperm were isolated from 2 to 4 wild-type and mutant male fish and kept in 150l Hanks saline containing 0.5M MitoTracker Deep Red FM (Molecular Probes) for >10min on ice. (B). The small size and transparency of zebrafish embryos makes them amenable to RNA in situ hybridization. Additionally, the Ig-like domain annotation was derived from previously published data for mouse IZUMO1 and SPACA6 (Nishimura et al., 2016). The zebrafish (Danio rerio) has become a popular model for studying genetics and developmental biology. (C). After 2-3 months in the nursery, the fish reach adulthood, completing the lifecycle. You have already requested a trial and a JoVE representative will be in touch with you shortly. 3l of stained spermatozoa (approximately 150,000300,000 sperm) was added as close to the egg as possible during image acquisition. Bioinformatics 14, 755763. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors. Izumo1 knockout (KO) sperm can penetrate the zona pellucida (ZP), a glycoprotein coat surrounding the mammalian oocyte, but fail to fuse with the oolemma (Inoue et al., 2005). All JoVE videos and articles can be accessed for free. Important considerations for caring for larvae are incorporated, including a brief review of how fish are raised to adulthood in a dedicated facility known as the nursery. By this method, the first 24 hours are divided into five phases: Zygote, cleavage, blastula, gastrula, and segmentation. Furthermore, spaca6/ and wild-type sperm had a similar sperm head size and tail length (Figure 3B). Published RNA sequencing data from adult tissues (Herberg et al., 2018; Noda et al., 2021) was used to analyse spaca6 gene expression levels in various adult tissues and to examine the coverage tracks across spaca6 in testis and oocytes using the Integrative Genomics Viewer (IGV) (http://software.broadinstitute.org/software/igv/). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Eggs were collected and kept at 28C in E3 medium (5mM NaCl, 0.17 mM KCl, 0.33mM CaCl, 0.33mM MgSO, 0.00001% Methylene blue). Fast, Scalable Generation of High-Quality Protein Multiple Sequence Alignments Using Clustal Omega, Mol. If the problem continues, please, An unexpected error occurred. The percentage of sequence identity of the mature Spaca6 protein of different vertebrate species was derived using the Percent Identity Matrix from Clustal Omega (Sievers et al., 2011). The Conserved Fertility Factor SPACA4/Bouncer Has Divergent Modes of Action in Vertebrate Fertilization. In recent years, several proteins have been shown to be essential for sperm-egg interaction in mammals. 49, D344D354. Fine Structure of the Chorion and Site of Sperm Entry in the Egg ofBrachydanio. Left: Experimental setup. Eggs were kept in place using a petri dish with cone-shaped agarose molds (1.5% agarose in sorting medium) filled with sorting medium. FIGURE 5. The protein domain predictions for zebrafish Spaca6 and mouse SPACA6 (Uniprot ID: E9Q8Q8) were obtained from InterPro (Blum et al., 2021). B., Martin, D. M. A., Clamp, M., and Barton, G. J. Moreover, we have shown that DCST1 and DCST2 are also necessary for zebrafish fertilization (Noda et al., 2021). Despite the many factors shown to be necessary for sperm-egg fusion, the combination of these factors appears not to be sufficient to drive fusion in a heterologous system. Biol. Enzymatic Assembly of DNA Molecules up to Several Hundred Kilobases. eLife 9, e53913. 24, 279282. No, not quite like that. Unable to load video. doi:10.1038/s41586-021-03819-2, Kaji, K., Oda, S., Shikano, T., Ohnuki, T., Uematsu, Y., Sakagami, J., et al. Cells from fluorescently labeled early embryos can be transplanted into unlabeled host embryos, and tracked over time. Interestingly, the mammalian homolog of Bouncer is SPACA4, which is present in sperm and is involved in penetrating the zona pellucida (Fujihara et al., 2021). Genet. If you do not wish to begin your trial now, you can log back into JoVE at any time to begin. Out of the full set of 453 taxa containing either DC-STAMP, Izumo1 or Spaca6 proteins, 52 representative animal species were selected, and a taxonomic tree was retrieved using the NCBI Taxonomy CommonTree tool (Schoch et al., 2020). If you have any questions, please do not hesitate to reach out to our customer success team. (C). Secondary structure prediction of the zebrafish Spaca6 protein sequence is depicted above the sequence alignment (coil: alpha-helix; arrow: beta-sheet). After preparation of all samples, 1U/l benzonase (Merck) was added, and samples were incubated for 30min at room temperature. To analyze sperm-egg binding, stably bound sperm were counted. Values were compared to tubulin levels and normalized to wild type. Zebrafish lines expressing transgenic Spaca6 were generated by injecting the spaca6 expression construct with Tol2 mRNA into spaca6+/ zebrafish embryos (15ng/l of the plasmid in RNase-free water, 35ng/l Tol2 mRNA, 0.083% phenol red solution [Sigma-Aldrich]), following standard procedures. Manual Tracking. 227, 277296. (B). During the blastula period, the embryo begins to make its own RNA, thus lengthening the cell cycle. 15 Articles, This article is part of the Research Topic, https://doi.org/10.3389/fcell.2021.806982, http://software.broadinstitute.org/software/igv/, https://www.frontiersin.org/articles/10.3389/fcell.2021.806982/full#supplementary-material, https://imagej.nih.gov/ij/plugins/track/track.html, https://www.biorxiv.org/content/10.1101/2021.04.18.440256. n.s, not significant (Mann-Whitney test, p > 0.05); error bars, SD; N = number of independent experiments; n = number of sperm. DC-STAMP-like proteins (blue star) are conserved both in vertebrates and invertebrates; Izumo1 (orange circle) and Spaca6 (green square) are conserved only in vertebrates. Severely Reduced Female Fertility in CD9-Deficient Mice. The relative displacement (grey dashed line) is overlaid. (2019). JoVE, Cambridge, MA, (2022). demonstrated that DCST1/2 regulates the levels of SPACA6 (Inoue et al., 2021), and we show that zebrafish Dcst2 levels depend on the presence of Spaca6 (Figure 5). Reprod. Therefore, zebrafish represents an interesting model system for the study of fertilization; despite the presence of mammalian fertilization factors in fish (Dcst1/2, Bouncer/SPACA4), the currently characterized factors show functional divergence in the fertilization process (Fujihara et al., 2021; Noda et al., 2021). While rapidly depleting the energy stores of the yolk, the larvae soon develop specialized structures for swimming, like the swim bladder: A gas-filled organ that controls buoyancy. Profile Hidden Markov Models. Please check your Internet connection and reload this page. doi:10.1093/nar/gkaa1100, Bianchi, E., and Wright, G. J. 25, 33893402. doi:10.1093/nar/25.17.3389, PubMed Abstract | CrossRef Full Text | Google Scholar, Barbaux, S., Ialy-Radio, C., Chalbi, M., Dybal, E., Homps-Legrand, M., do Cruzeiro, M., et al. The amino acid position is given above the scheme. The full-length spaca6 coding sequence, including the spaca6 signal peptide, the extracellular region and transmembrane and intracellular domains, was amplified by PCR from cDNA derived from adult zebrafish testis (Spaca6_CDS_F and Spaca6_CDS_R) and subcloned by Gibson cloning (Gibson et al., 2009) into a vector for Tol2-mediated transgenesis along with a SG-linker-T2A-sfGFP sequence inserted in frame immediately before the stop-codon of the spaca6 sequence (resulting vector: pMTB Tol2 - actb2-promoterspaca6SG-linker-T2A-sfGFP SV40UTR). PLoS ONE 9, e98186. Inoue et al. Research Institute of Molecular Pathology (IMP), Vienna BioCenter (VBC), Campus-Vienna-Biocenter 1, Vienna, Austria. Indeed, coverage tracks derived from RNA sequencing (Herberg et al., 2018; Noda et al., 2021) show expression peaks that align to the mapped transcript as well as specific expression of Spaca6 in testis (Figure 1A, Supplementary Figure S1A), as has been previously reported in mammals (Barbaux et al., 2020; Noda et al., 2020). Proc. Science 287, 319321. Finally, the video concludes with some common techniques utilized for studying embryo development, demonstrating how zebrafish are used to help us better understand human development and disease. Furthermore, zebrafish are amenable to genetic manipulation, allowing researchers to determine the effect of gene expression on the development of a vertebrate with a high degree of genetic similarity to humans. Our study shows that Spaca6 has an essential role in fertilization outside of mammals. To test whether Spaca6 is required for fertilization in zebrafish, we assessed the fertilization rates of wild-type, heterozygous and homozygous KO fish (Figures 2B,C). Received: 01 November 2021; Accepted: 07 December 2021;Published: 03 January 2022. doi:10.7554/eLife.53913, le Naour, F., Rubinstein, E., Jasmin, C., Prenant, M., and Boucheix, C. (2000). We use cookies to enhance your experience on our website. Creative Commons Attribution License (CC BY). Sperm (approximately 5,000 sperm/l) were activated using E3 medium in a 1:4 dilution and placed onto a 10m thick chamber slide (Leja counting chamber, SC 100104-B). In order to taxonomically map Spaca6 homologues, we performed iterative BLASTP searches. After SDS-PAGE, samples were wet-transferred onto a nitrocellulose membrane. doi:10.7554/eLife.66313, Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. It is worth pointing out that a fraction of the total number of bound wild-type sperm may have not only bound but also fused to the oolemma. Upper panels: Overview images (scale bar = 5m). If that doesn't help, please let us know. Science 287, 321324. doi:10.1093/database/baaa062, Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., et al. (2014). 37, 391414. In contrast, our findings in zebrafish reveal that zebrafish sperm lacking Spaca6 is already unable to bind to the egg membrane, and is thus required in a step before fusion can take place. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript (AAM) version arising from this submission. Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. Deneke, V. E., and Pauli, A. Syst. Data was plotted as the number of sperm bound per 100m of egg membrane for 2minutes. Screening injected fish for developmental phenotypes, such as changes in the assembly of blood vessels, provides insight into the genetic control of complex developmental processes. No use, distribution or reproduction is permitted which does not comply with these terms. Noda and others expressed the essential sperm factors involved in fusion, including IZUMO1, in HEK293T cells and observed that HEK293T cells were able to bind but were unable to fuse to ZP-free eggs, indicating the possible need for additional molecules that are involved in this process (Inoue et al., 2013; Noda et al., 2020). Interactive Tree of Life (iTOL) V5: an Online Tool for Phylogenetic Tree Display and Annotation. Despite its critical role, little is known about the molecular mechanisms mediating fertilization, particularly sperm-egg interaction and fusion. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. Acad. The resulting time-lapse movies were analyzed using Fiji. (A). Nucleic Acids Res. It contains an N-terminal signal peptide, followed by an extracellular region containing an alpha-helical domain (extracellular domain, ECD) and an immunoglobulin (Ig) fold domain, a transmembrane domain and a short cytoplasmic domain (Figure 1B). (2009). (2011). Rep. 10, 5335. doi:10.1038/s41598-020-62091-y, Bateman, A., Martin, M.-J., Orchard, S., Magrane, M., Agivetova, R., Ahmad, S., et al. The supernatant was replaced with RIPA buffer (50mM Tris-HCl [pH 7.5], 150mM NaCl, 1mM MgCl, 1% NP-40, 0.5% sodium deoxycholate, 1 complete protease inhibitor [Roche]) including 1% SDS. Alignment of Spaca6 protein sequences from different vertebrates shows that Spaca6 is conserved in its amino acid sequence and secondary structure elements (Figure 1B, Supplementary Figure S1B). The editor and reviewer's affiliations are the latest provided on their Loop research profiles and may not reflect their situation at the time of review. On a molecular level the observed difference could possibly be reconciled from mouse experiments involving IZUMO1. Thanks for watching! Their rapid, external development and transparency make them uniquely suited to visualization. Zebrafish Dcst2 is predicted to be a glycoprotein, resulting in a wider area of detected signal. Another possibility is that the role of SPACA6 can be by-passed by transgenic expression of DCST1/2, suggesting an indirect role of SPACA6 in fertilization. Now that youre familiar with the major stages of zebrafish development, lets go over the first 24 hours in more detail. Zebrafish spaca6 knockout males are sterile. The uncropped immunoblot is shown in Supplementary Figure S2. If you need immediate assistance, please email us at subscriptions@jove.com. Protein folding predictions of mature Spaca6 (lacking the signal peptide sequence) were performed using AlphaFold2 (Jumper et al., 2021). (D). Requirement of CD9 on the Egg Plasma Membrane for Fertilization. Sperm were spun onto an adhesive slide using a CytoSpin 4 (Thermo Fisher Scientific) at 1,000rpm for 5min followed by permeabilization with ice-cold methanol for 5min and a wash with 0.1% Tween in 1x PBS (PBST). doi:10.1093/bioinformatics/14.9.755, El-Gebali, S., Mistry, J., Bateman, A., Eddy, S. R., Luciani, A., Potter, S. C., et al. Finally, the notion of a membrane complex needed for binding and fusion has been previously proposed (Barbaux et al., 2020; Noda et al., 2020).

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