zebrafish fertilization

TMEM95 Is a Sperm Membrane Protein Essential for Mammalian Fertilization. Total protein was visualized by Ponceau staining before blocking with 5% milk powder in 0.1% Tween in 1 TBS (TBST). Representative tracks of wild-type and spaca6/ sperm. Measurements of the head area (left) and tail length (right) of wild-type and spaca6/ sperm. Wild-type sperm were frequently found bound to the oolemma. Data was plotted as the number of sperm bound per 100m of egg membrane for 2minutes. Bioinformatics 14, 755763. Please click here to activate your free 2-hour trial. In short, wild-type eggs were activated with water and manually dechorionated to expose the entire egg surface. Images were acquired until sperm were no longer motile (5min). (2021). Changes in gene expression over time and in specific organs are easily observed and provide insight to developmental processes. 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. Science 287, 321324. Mouse Spaca6 KO sperm can penetrate the zona pellucida and bind to the oolemma, but fail to fuse with the egg (Barbaux et al., 2020; Noda et al., 2020). A subscription to JoVE is required to view this content.You will only be able to see the first 20 seconds. Cel Dev. As observed in the sperm approach assay, spaca6/ sperm was able to reach the egg, but failed to stably bind to the egg surface (Figure 4B, Supplementary Movie S3). R. Soc. The InterPro Protein Families and Domains Database: 20 Years on. Deneke, V. E., and Pauli, A. Expression and genomic features of zebrafish spaca6. For western blot analysis, sperm from 3-6 males was sedimented at 3,000rpm for 3.5min. Nature 596, 583589. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Coordinates of the sperm cells were used to calculate average sperm speed and displacement. In support of this notion, IZUMO1 protein levels as well as IZUMO1 relocation during the acrosome reaction were reported to be normal in Spaca6 KO sperm (Barbaux et al., 2020; Inoue et al., 2021). 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). Unactivated, mature eggs were squeezed from a wild-type female fish and activated by addition of E3 medium. doi:10.1093/nar/gkaa977. doi:10.1126/science.287.5451.321, Nishimura, K., Han, L., Bianchi, E., Wright, G. J., de Sanctis, D., and Jovine, L. (2016). One possibility is that mouse Spaca6 KO sperm is still able to bind to the oocyte due to the presence of IZUMO1, thereby providing a redundant role in sperm adhesion to the egg. Together, this study and studies in mice point towards a co-regulation of SPACA6 and DCST1/2. doi:10.1242/dev.094854, Inoue, N., Hagihara, Y., and Wada, I. Differential interference contrast (DIC) images did not show any gross morphological differences between spaca6/ and wild-type sperm (Figure 3A). The Pfam Protein Families Database in 2019. Tubulin protein levels of the same blot are shown as loading control. If you want more info regarding data storage, please contact gdpr@jove.com. In the rescue line, a SGGSG spacer and a part of the viral T2A protein result in a C-terminal 23-aa addition. Values were then normalized to the levels of wild-type sperm. (A). Although spaca6/ sperm drifted away after a few minutes (Figure 4A, Supplementary Movie S2), we conclude that spaca6/ sperm was able to approach and enter the micropyle similar to wild-type sperm. 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). Additionally, embryos are amenable to both physical and genetic manipulations, allowing researchers to tease apart the signals controlling developmental processes. (2020). eLife 9, e53913. Sperm from zebrafish males was collected in 3.7% formaldehyde diluted in Hanks saline solution and stored on ice for 20min to 1h. Sperm was pelleted by centrifugation at 850rpm for 3min, and the fixative was replaced with Hanks saline. This technique uses a labeled RNA molecule complementary to an mRNA of interest, to visualize gene expression throughout the entire organism. n.s, not significant (Mann-Whitney test, p > 0.05); error bars, SD; n = number of sperm. Rev. Spaca6 KO fish were generated by Cas9-mediated mutagenesis. Genomic coordinates are based on GRCz11. Sperm were isolated from 2 to 4 wild-type and mutant male fish and kept in 200l Hanks saline containing 0.5M MitoTracker Deep Red FM (Molecular Probes) on ice. The generation of zebrafish spaca6 KO fish is described below. USA 117, 1149311502. doi:10.1038/s41586-021-03819-2, Kaji, K., Oda, S., Shikano, T., Ohnuki, T., Uematsu, Y., Sakagami, J., et al. Sperm Attractant in the Micropyle Region of Fish and Insect Eggs1. Cleavage of the blastomere is then shown to produce an embryo containing thousands of cells within a matter of hours. Although zebrafish have an Izumo1 ortholog (Figure 1C), its mammalian binding partner on the egg surface, JUNO, appears to be absent in fish (Grayson, 2015). Biology II: Mouse, Zebrafish, and Chick. B., Martin, D. M. A., Clamp, M., and Barton, G. J. 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). doi:10.1093/bioinformatics/14.9.755, El-Gebali, S., Mistry, J., Bateman, A., Eddy, S. R., Luciani, A., Potter, S. C., et al. Our data shows that in zebrafish, transgenic expression of Spaca6 results in a partial rescue of fertilization (Figure 2C), which is correlated with a partial restoration of Dcst2 levels (Figure 5). Open Biol. Current gene annotations for zebrafish spaca6 (NCBI Danio rerio Annotation release 106: XM_021466914.1, seven exons; and ENSEMBL release 104: BX539313.2-201, ENSDART00000155083.2, eight exons) were found in the Genome Browser (http://genome.ucsc.edu) using the zebrafish genome release (GRCz11). Consistent with the expression of Spaca6 exclusively in the male germline (Supplementary Figure S1A), spaca6/ females were fertile (Figure 2C), revealing that Spaca6 is only required for male fertility. (2021). Please enter your Institution or Company email below to check. (D). This zygote has a few important structures, including the protective membrane surrounding the embryo called the chorion and the yolk that provides nutrients for embryonic development until the fish can feed itself. FIGURE 4. Zebrafish (Danio rerio) were raised according to standard protocols (28C water temperature, 14/10h light/dark cycle). Embryos to Study Gene Expression and Function, Histological Sample Preparation for Light Microscopy, Automated High-throughput Behavioral Analyses in Zebrafish Larvae, Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle, Generating Chimeric Zebrafish Embryos by Transplantation. 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). Zool. The dcst2/ zebrafish line has been described previously (Noda et al., 2021). Zebrafish Spaca6 is essential for male fertility. 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). 37, 391414. Most notably, mammalian testis-expressed IZUMO1 and its receptor JUNO on the egg membrane (oolemma) form a complex which is needed for binding of the two gametes prior to fusion (Inoue et al., 2013; Bianchi and Wright, 2016). Moreover, IZUMO1 was shown to be sufficient for cells to bind to the oocyte (Inoue et al., 2013; Noda et al., 2020), suggesting that one major role of IZUMO1 is to enable sperm to adhere to the egg surface. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Left: Experimental setup. Zebrafish Reproduction and Development. FIGURE 1. doi:10.1002/jez.1402270212, Herberg, S., Gert, K. R., Schleiffer, A., and Pauli, A. Quantification of fertilization rates. In this context, studies of its potential interaction partner(s) on the egg membrane in zebrafish and mice might identify new fertilization factors on the egg. Interactive Tree of Life (iTOL) V5: an Online Tool for Phylogenetic Tree Display and Annotation. (A). First, lets go over the basic steps in zebrafish development. Natl. JoVE Science Education Database. The pharyngula phase encompasses the next 24 hours until the embryos hatch into larvae. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Thanks for watching! Natl. We next tested whether spaca6 KO sperm is able to approach the micropyle, a funnel-shaped structure within the egg coat that serves as the only sperm entry point in fish (Hart and Donovan, 1983; Wolenski and Hart, 1987; Yanagimachi et al., 2013). Inoue and others showed that sperm lacking IZUMO1, DCST1 and/or DCST2 had undetectable levels of SPACA6 protein, which suggested that SPACA6 levels are dependent on the presence of each of these factors. The embryos continue to develop within their chorions until they hatch into larvae at about 3 days post fertilization. Schoch, C. L., Ciufo, S., Domrachev, M., Hotton, C. L., Kannan, S., Khovanskaya, R., et al. Early development occurs at a rapid, but predictable rate when the embryos are raised at 28 C. Amplicon sequencing of adult fin-clips identified the 86-nt deletion, which results in a frameshift mutation and a premature stop codon in intron 2 (GRCz11: Chr16:24,907,548). This method allows researchers to examine how cell interactions contribute to organ function as well as to easily visualize cell movements in vivo. Next, the dramatic cellular movements known as epiboly and gastrulation are explained, revealing how they contribute to reshaping a mass of cells into a moving embryo with a beating heart in just 1 day. FIGURE 5. (C). Finally, the notion of a membrane complex needed for binding and fusion has been previously proposed (Barbaux et al., 2020; Noda et al., 2020). (B). This video outlined the zebrafish lifecycle, covered the stages of early zebrafish development, and highlighted the power of the zebrafish as a tool in developmental biology. (2021). An 86-nt deletion in the spaca6/ line leads to a retained sequence from intron two and a premature stop-codon, resulting in a dysfunctional truncated protein (translated part of the intron is shown in black). On a molecular level the observed difference could possibly be reconciled from mouse experiments involving IZUMO1. Reprod. Ready to adopt a fish? doi:10.1002/jez.1402430211, Yanagimachi, R., Cherr, G., Matsubara, T., Andoh, T., Harumi, T., Vines, C., et al. The Structure of Sperm Izumo1 Reveals Unexpected Similarities with Plasmodium Invasion Proteins. The boxed region is shown at higher magnification below. (2020). JoVE Science Education Database. (2011). SP, signal peptide; TMD, Transmembrane domain; CD, Cytoplasmic domain. Zebrafish Spaca6 is essential for sperm-egg binding. Further investigation of the co-regulation and potential interaction between Spaca6, Dcst1/2, Izumo1 and other known essential fertilization factors may help elucidate the mechanism of gamete fusion on a molecular level. doi:10.1093/nar/gky995, Fujihara, Y., Herberg, S., Blaha, A., Panser, K., Kobayashi, K., Larasati, T., et al. The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors. 88, 47. doi:10.1095/biolreprod.112.105072, Keywords: fertilization, zebrafish, sperm-egg interaction, gamete, sperm, reproduction, Citation: Binner MI, Kogan A, Panser K, Schleiffer A, Deneke VE and Pauli A (2022) The Sperm Protein Spaca6 is Essential for Fertilization in Zebrafish. The full life cycle from fertilized egg to adult is a quick 90 days. Binding of sperm to the egg was quantified by assessing the number of stably bound sperm per 100m over a period of 2min. The amino acid position is given above the scheme. Left: Experimental setup. Scanning Electron Microscope Studies of Sperm Incorporation into the Zebrafish (Brachydanio) Egg. Finally, because zebrafish embryos are easily genetically manipulated by microinjection, researchers can examine the role of specific genes during development starting from the one-cell stage. This video provides a brief overview of the major phases of zebrafish development, with particular focus on the first 24 hours post fertilization (hpf). Direct comparisons were made between siblings of different genotypes [wild type; spaca6/; spaca6+/; spaca6/ tg(actb2:spaca6-t2a-GFP)]. Izumo1 protein family members were identified using the PFAM IZUMO HMM search tool, covering the amino-terminal conserved region of human IZUMO1 (21165, sp|Q8IYV9|IZUM1_HUMAN) in the UniProt reference proteomes databases (E-value < 0.01). Youll need a nursery to raise your fry to adulthood. Youve just watched JoVEs video on zebrafish development. Despite these recent advances, the molecular mechanisms of gamete fusion and the interplay between the various sperm and egg factors remain unclear. doi:10.1371/journal.pone.0098186, Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., and Smith, H. O.

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