Name: preparation of meiotic chromosome spreads from zebrafish spermatocytes
Uploaded: 2020-03-03T12:00:00
Description: Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes at JoVE.com

We thank Trent Newman and Masuda Sharifi for comments on the manuscript and An Nguyen for helping to optimize methods for spreading and staining chromosomes from zebrafish meiocytes. This work was supported by NIH R01 GM079115 awarded to S.M.B.

All methods involving zebrafish were carried out using ethical standards approved by the Institutional Animal Care and Use Committee at UC Davis.
1. Chromosome spreading procedure
NOTE: The following protocol is designed to create 4-6 slides, with hundreds of spread meiotic nuclei per slide. The numbers of testes used will depend on the size of the fish. Expect to use 20 animals at ~60 days post fertilization (dpf) and 15 animals at ~6 months post fertilization (mpf)....

<ol>
	<li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nature Reviews Genetics. 13, 493-504 (2012).</li><li>Zickler, D., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination,+Pairing,+and+Synapsis+of+Homologs+during+Meiosis.">Recombination, Pairing, and Synapsis of Homologs during Meiosis.</a> Cold Spring Harbor Perspectives in Biology. 7, (2015).</li><li>Blokhina, Y. P., Nguyen, A. D., Draper, B. W., Burgess, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telomere+bouquet+is+a+hub+where+meiotic+double-strand+breaks,+synapsis,+and+stable+homolog+juxtaposition+are+coordinated+in+the+zebrafish,+Danio+rerio.">The telomere bouquet is a hub where meiotic double-strand breaks, synapsis, and stable homolog juxtaposition are coordinated in the zebrafish, Danio rerio.</a> PLoS Genetics. 15, e1007730 (2019).</li><li>Saito, K., Sakai, C., Kawasaki, T., Sakai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+distribution+pattern+and+synapsis+initiation+during+spermatogenesis+in+zebrafish.">Telomere distribution pattern and synapsis initiation during spermatogenesis in zebrafish.</a> Developmental Dynamics. 243, 1448-1456 (2014).</li><li>Oliver-Bonet, M., Turek, P. J., Sun, F., Ko, E., Martin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+progression+of+recombination+in+human+males.">Temporal progression of recombination in human males.</a> Molecular Human Reproduction. 11, 517-522 (2005).</li><li>Gruhn, J. R., Rubio, C., Broman, K. W., Hunt, P. A., Hassold, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+studies+of+human+meiosis:+sex-specific+differences+in+recombination+originate+at,+or+prior+to,+establishment+of+double-strand+breaks.">Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks.</a> PLoS One. 8, e85075 (2013).</li><li>Pratto, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+initiation+maps+of+individual+human+genomes.">Recombination initiation maps of individual human genomes.</a> Science. 346, 1256442 (2014).</li><li>Brown, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+synapsis+proceeds+from+a+limited+number+of+subtelomeric+sites+in+the+human+male.">Meiotic synapsis proceeds from a limited number of subtelomeric sites in the human male.</a> American Journal of Human Genetics. 77, 556-566 (2005).</li><li>Poss, K. D., Nechiporuk, A., Stringer, K. F., Lee, C., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+aneuploidy+in+zebrafish+with+mutations+in+the+mitotic+checkpoint+gene+mps1.">Germ cell aneuploidy in zebrafish with mutations in the mitotic checkpoint gene mps1.</a> Genes and Development. 18, 1527-1532 (2004).</li><li>Saito, K., Siegfried, K. R., N&#252;sslein-Volhard, C., Sakai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cytogenetic+characterization+of+zebrafish+meiotic+prophase+I+mutants.">Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants.</a> Developmental Dynamics. 240, 1779-1792 (2011).</li><li>Sansam, C. L., Pezza, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+by+breaking+and+repairing:+mechanisms+of+DNA+strand+exchange+in+meiotic+recombination.">Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination.</a> Febs Journal. 282, 2444-2457 (2015).</li><li>Feitsma, H., Leal, M. C., Moens, P. B., Cuppen, E., Schulz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mlh1+Deficiency+in+Zebrafish+Results+in+Male+Sterility+and+Aneuploid+as+Well+as+Triploid+Progeny+in+Females.">Mlh1 Deficiency in Zebrafish Results in Male Sterility and Aneuploid as Well as Triploid Progeny in Females.</a> Genetics. 175, 1561-1569 (2007).</li><li>Dia, F., Strange, T., Liang, J., Hamilton, J., Berkowitz, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Meiotic+Chromosome+Spreads+from+Mouse+Spermatocytes.">Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes.</a> Journal of Visualized Experiments. (129), e55378 (2017).</li><li>Moens, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+chiasmata+and+interference.">Zebrafish: chiasmata and interference.</a> Genome. 49, 205-208 (2006).</li><li>Lisachov, A. P., Zadesenets, K. S., Rubtsov, N. B., Borodin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+chromosome+synapsis+and+recombination+in+male+guppies.">Sex chromosome synapsis and recombination in male guppies.</a> Zebrafish. 12 (2), 174-180 (2015).</li><li>Ocalewicz, K., Mota-Velasco, J. C., Campos-Ramos, R., Penman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+and+DAPI+staining+of+the+synaptonemal+complex+of+the+Nile+tilapia+(Oreochromis+niloticus)+allow+orientation+of+the+unpaired+region+of+bivalent+1+observed+during+early+pachytene.">FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene.</a> Chromosome Research. 17 (6), 773 (2009).</li></ol>

Here we describe methods to probe the location of telomeres and chromosome-associated proteins in nuclear surface spreads from spermatocytes isolated from zebrafish testes. We expect that these methods will be applicable for analysis of spermatocytes in other teleost species with adjustment to the size of the testis.
While only a few antibodies have been raised to zebrafish meiotic proteins, we have had success using the following antibodies raised to human (h) or mouse (m) proteins. Our lab h...

Sexual reproduction proceeds through the combination of two haploid gametes, each carrying half the chromosome complement of a somatic cell. Meiosis is a specialized cell division that produces haploid gametes through one round of DNA replication and two successive rounds of chromosome segregation. In prophase I, homologous chromosomes (homologs) must undergo pairing, recombination, and synapsis, the latter of which is characterized by the formation of the synaptonemal complex that comprises two homolog axes bridged by the transverse filament, Sycp1 (Figure 1A,B). Failure to properly execute these processes can lead to the production of aneuploid gametes, which are a leading cause of miscarriages in humans1. Our knowledge of the coordination between pairing, recombination and synapsis has been facilitated by studies in a wide range of organisms, such as yeast, C. elegans, mouse, and Drosophila, among others2. While the general process of homologous chromosome pairing followed by segregation is well conserved, its dependency on recombination and synapsis and the order of these events varies.
Meiotic double-strand break (DSB) formation, which initiates homologous recombination, occurs near telomeres clustered in the bouquet during leptotene and synapsis ensues shortly after3,4. This configuration of DSB formation and synapsis initiation is also a characteristic of male meiosis in humans but not in mouse5,6,7,8, suggesting that zebrafish can serve as a model for human spermatogenesis. There are also several practical advantages of studying zebrafish meiosis. Both males and females undergo gametogenesis throughout adulthood, their gonads are easily accessible, and hundreds of offspring are generated from a single cross. Additionally, the embryos are transparent and develop externally, which facilitates the early detection of aberrations in embryonic development due to aneuploid gametes3,9. Disadvantages of using zebrafish are that they are slow to reach sexual maturity (~60 days) and the amount of material needed for nuclear surface spreads must be collected from ~10-20 adult animals, depending on their size.
Meiotic chromosome spread preparations are a vital tool for studying chromosome dynamics across all model organisms, since key signatures of meiotic chromosome dynamics can be probed. In zebrafish, key aspects of the progression of the meiotic program and nuclear organization have been dissected through probing nuclear surface spreads, referred to here as chromosome spreads, with antibodies for immunofluorescence detection of proteins and/or nucleic acids by FISH3,4,9,10,11,12. Indeed, the polarized localization of clustered telomeres in the bouquet can be preserved in the spread preparation (Figure 1C). Recently, we have used zebrafish spermatocyte chromosome spreads together with fluorescence detection methods and super-resolution microscopy to elucidate the detailed progression of zebrafish telomere dynamics, homologous chromosome pairing, double-strand break localization, and synapsis at key meiotic transitions3. Here we present methods to prepare chromosome spreads from spermatocytes of the zebrafish testes and subsequently stain them with fluorescent peptide nucleic acid (PNA) probes to repeated telomere sequences and immunofluorescence detection of chromosome associated proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube rack</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde, methanol-free</td>
			<td>ThermoFisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 50 mm glass coverslips</td>
			<td>Corning</td>
			<td>2980-245</td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 60 mm glasscoverslips</td>
			<td>VWR International</td>
			<td>16004-312</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>363696</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave bag</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td>Used to make plastic coverslips.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1605-100</td>
			<td>Prepare a 100 mg/ml stock solution in sterile distilled water.</td>
		</tr>
		<tr>
			<td>Cell Strainer, 100 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>CF405M goat anti-chicken IgY (H+L), highly cross-adsorbed</td>
			<td>Biotium</td>
			<td>203775-500uL</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Chicken anti-zfSycp1</td>
			<td>Generated by Burgess lab</td>
			<td>N/A</td>
			<td>Use at 1:100</td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C0130-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I, grade II from bovine pancreas</td>
			<td>Roche Diagnostics</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Fisher Scientific</td>
			<td>MT10014CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont No. 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td>Two are required for dissecting the testes.</td>
		</tr>
		<tr>
			<td>Eppendorf Tubes, 5 mL</td>
			<td>VWR International</td>
			<td>89429-308</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fisher Scientific</td>
			<td>BP228-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11039</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11042</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-hDMC1</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8973</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H3393-100KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity chamber</td>
			<td>Fisher Scientific</td>
			<td>50-112-3683</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>VWR International</td>
			<td>230401V (Model 5420)</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Model Classic C25</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimerbly-Clark Professional</td>
			<td>34155</td>
			<td>Used for the humidity chamber</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td>Any standard microscope capable of at least ~1.65X magnification is sufficient.</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-hamsterSCP3</td>
			<td>Abcam</td>
			<td>ab97672</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Mouse anti-hMLH1</td>
			<td>BD Biosciences</td>
			<td>550838</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Mouse anti-hRPA</td>
			<td>Sigma-Alrich</td>
			<td>MABE285</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Na2HPO4 &#183; 7 H2O</td>
			<td>Fisher Scientific</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-Flo 200 solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>74257</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipettes</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PNA TelC-Alexa647</td>
			<td>PNA Bio Inc</td>
			<td>F1013</td>
			<td>Prepare as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr>
			<td>PNA TelC-Cy3</td>
			<td>PNA Bio Inc</td>
			<td>F1002</td>
			<td>Prepare as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P36971</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-hRPA</td>
			<td>Bethyl</td>
			<td>A300-244A</td>
			<td>Use at 1:300</td>
		</tr>
		<tr>
			<td>Rabbit anti-hSCP3</td>
			<td>Abcam</td>
			<td>ab150292</td>
			<td>Use at 1:200</td>
		</tr>
		<tr>
			<td>Rabbit anti-hRad51</td>
			<td>GeneTex</td>
			<td>GTX100469</td>
			<td>Use at 1:300</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Supercut Scissors, 30&#176; angle, 10 cm</td>
			<td>Fisher Scientific</td>
			<td>50-822-353</td>
			<td>Can also use any pair of small scissors.</td>
		</tr>
		<tr>
			<td>Sylgard kit</td>
			<td>Fisher Scientific</td>
			<td>NC9897184</td>
			<td>Prepare as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td>Dilute in sterile distilled water to make a 20% working solution. Store at room temperature. Triton X-100 forms a precipitate when diluted in water; precipitate dissolves overnight.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Worthington Biochemical</td>
			<td>LS003708</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from chicken egg white</td>
			<td>Sigma-Aldrich</td>
			<td>T9253-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td>Dilute in sterile distilled water to make a 20% working solution. Store at room temperature.</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 4 mm (micro scissors)</td>
			<td>Fine Science Tools</td>
			<td>15018-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of meiotic chromosome spreads from zebrafish spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

We have outlined a method to prepare and visualize zebrafish spermatocyte spread preparations. When performed correctly, our procedure yields well spread, non-overlapping nuclei. To recover such nuclei, it is important to have the appropriate amount of starting material (i.e., testes), treat testes for a sufficient length of time in trypsin and an adequate number of DNase I treatments. These spreads can then be stained for telomeres and meiotic proteins to study meiotic progression during...

Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes at JoVE.com

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Zebrafish is emerging as an excellent vertebrate model for studying the chromosome events of meiosis including homologous chromosome pairing, synapsis and recombination. This nuclear surface spreading protocol has enabled us to visualize key features of meiotic chromosome architecture using super resolution microscopy. Researchers can expect hundreds of well-spread nuclei per slide with less debris than other zebrafish spread protocols.The most technically challenging part of this procedure is the dissection of the zebrafish testes as it is in close proximity to the skin and is also attached to other organs. Visualizing the dissection procedure will allow researchers to know what to expect when they see the gonad embedded in the tissue of the animal. After euthanizing male zebrafish, decapitate one fish at a time with small scissors and then use micro scissors to cut along the ventral midline to expose the body cavity.Place the fish in a silicone-coated Petri dish and cover by a shallow pool of 1X PBS. Under a microscope at 1.65X magnification, dissect the testes using forceps. Remove as much as fat and surrounding tissue from the testes as possible.The gonad will appear translucent under the dissection scope light while the surrounding fat may have a slightly darker appearance. Some amount of fat may be left on the gonad without impeding the procedure. Add each dissected testes directly into a five milliliter tube with two milliliters of DMEM and keep on ice.To dissociate the testes cells, add 200 microliters of collagenase solution to the five milliliter tube with the testes. Mix the solution by inverting it several times. Gently shake the testes in an incubator shaker horizontally at 100 RPM at 32 degrees Celsius.Rapidly invert the tube every 10 minutes to facilitate dissociation. After an hour, the DMEM is cloudy and the testes are in small chunks. While the testes are incubated in collagenase, prewarm 100 microliters of 0.1 molar sucrose solution to 37 degrees Celsius.To wash out the collagenase, add DMEM to a final volume of five milliliters and invert the tube a few times. Pellet the testes at 200 g for three minutes at room temperature. Remove three milliliters of the supernatant so that only two milliliters remain.Repeat the DMEM wash two additional times. After the last DMEM wash, remove four milliliters of the supernatant and add one milliliter of DMEM, 200 microliters of trypsin solution, and 20 microliters of DNase I solution. Invert the tube a few times to mix the solution.Horizontally shake the tube at 32 degrees Celsius at 100 RPM. Rapidly invert the tube every five minutes to facilitate dissociation. After five to 15 minutes, the DMEM solution contains only a few clumps.Add 500 microliters of the trypsin inhibitor solution and 50 microliters of DNase I solution. Invert the tube a few times to mix then briefly spin down at 200 g for three minutes at room temperature to remove liquid or clumps that may adhere to the tube cap or the side of the tube. Place the tube on ice and resuspend the cell suspension by repeatedly pipetting up and down with a plastic transfer pipette for two minutes to facilitate dissociation of any remaining clumps.Ensure that the cell suspension does not go into the bulb of the transfer pipette. Then pre-wet a 100 micron cell strainer with DMEM and place it on top of a 50 milliliter tube on ice. Transfer the cell suspension through the strainer one drop at a time using a plastic transfer pipette.Transfer the filtrate using a plastic transfer pipette to a new five milliliter tube. Be sure to collect the pooled cells attached to the underside of the filter. Add DMEM into the tube to a final volume of five milliliters.Pellet the cells at 200 g for five minutes at room temperature. Remove as much supernatant as possible without disturbing the pellet and add five microliters of DNase I solution directly into the pellet. Then mix the pellet with the DNase I solution by gently scraping the outside bottom of the tube four to five times along an empty microcentrifuge tube rack.Add DMEM to a final volume of five milliliters and mix the solution by inverting the tube several times. Pellet the cell suspension at 200 g for two minutes at room temperature. Repeat the DNase treatment an additional one to three times until the resuspended pellet does not clump upon addition of DMEM.After the last spin, remove as much as supernatant as possible without disturbing the pellet. Next, add one milliliter of 1X PBS and resuspend the pellet by gently scraping the outside bottom of the tube along an empty tube rack. Pellet the cell suspension at 200 g for five minutes and then remove as much supernatant as possible without disturbing the pellet.Cut three millimeters from the end of a 200 microliter pipette tip to widen the aperture. With the cut pipette tip, resuspend the pellet in 80 microliters of 0.1 molar sucrose solution by pipetting up and down. Let the cell suspension sit at room temperature for three minutes.Next, with a pipette tip, coat one slide with 100 microliters of 1%formaldehyde with 0.15%Triton X-100. Then add 18 microliters of the sucrose cell suspension onto the center of the slide in a straight line perpendicular to the long edge. Tilt the slide back and forth to facilitate spreading of the cell suspension to all corners.Place the slides flat down in a slightly cracked open humidity chamber to prevent the formaldehyde solution from drying. Place the humidity chamber in a dark drawer overnight. In the morning, remove the lid from the humidity chamber and allow the slides to fully dry.Place the slides in a coplin jar and then fill the coplin jar with distilled water. Incubate for five minutes with gentle shaking at room temperature. Pour out the water and fill the jar with one to 250 wetting agent.Wash two times for five minutes each with gentle shaking at room temperature. Allow the slides to fully dry and store at minus 20 degrees Celsius until they are stained. This protocol outlined a method to prepare and visualize zebrafish spermatocyte spread which yields well-spread non-overlapping nuclei.The panels on the left show three stages of meiotic prophase, leptotene, early zygotene, and pachytene. Telomeres were stained magenta for each stage. An example of poor quality spreads due to insufficient DNase I treatments are shown here.Meiotic chromosomes stained for SYCP3 indicate overlapping nuclei due to a viscous sucrose cell suspension that prevents cells from properly spreading on the slide. It is very important to start with the correct amount of starting material, treat zebrafish testes for the appropriate amount of time in trypsin, and carry out the necessary number of DNase I treatments as failure to do so can lead to very few nuclei or clumped nuclei. Proper PPE should always be worn when working with formaldehyde.Following the spreading procedure, chromosomes can be stained to visualize various chromosomal structures as well as double-strand breaks to analyze the timing dependency and localization of meiotic events. Our technique has paved the way for showing that zebrafish may be a better model of human spermatogenesis than mouse based on the chromosome features and the telomere bouquet.

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Research

JoVE Journal

Genetics

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome assays is traditionally a manual, labor-intensive process not well suited to handle the volume of samples which may require examination in the wake of a mass casualty event. Automated Dicentric Chromosome Identifier and Dose Estimator (ADCI) software automates this process by examining sets of metaphase images using machine learning-based image processing techniques. The software selects appropriate images for analysis by removing unsuitable images, classifies each object as either a centromere-containing chromosome or non-chromosome, further distinguishes chromosomes as monocentric chromosomes (MCs) or dicentric chromosomes (DCs), determines DC frequency within a sample, and estimates biological radiation dose by comparing sample DC frequency with calibration curves computed using calibration samples. This protocol describes the usage of ADCI software. Typically, both calibration (known dose) and test (unknown dose) sets of metaphase images are imported to perform accurate dose estimation. Optimal images for analysis can be found automatically using preset image filters or can also be filtered through manual inspection. The software processes images within each sample and DC frequencies are computed at different levels of stringency for calling DCs, using a machine learning approach. Linear-quadratic calibration curves are generated based on DC frequencies in calibration samples exposed to known physical doses. Doses of test samples exposed to uncertain radiation levels are estimated from their DC frequencies using these calibration curves. Reports can be generated upon request and provide summary of results of one or more samples, of one or more calibration curves, or of dose estimation.

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification ADCI and Dose Estimation

The cytogenetic dicentric chromosome (DC) assay quantifies exposure to ionizing radiation. The Automated Dicentric Chromosome Identifier and Dose Estimator software accurately and rapidly estimates biological dose from DCs in metaphase cells. It distinguishes monocentric chromosomes and other objects from DCs, and estimates biological radiation dose from the frequency of DCs.

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial patterns of gene expression. Embryoid bodies (EBs) derived from pluripotent stem cells have been a popular model to study the differentiation of the main three germ layers and to define regulatory circuits during cell fate specification. Although it is well-known that tissue-specific enhancers play an important role in these networks by interacting with promoters, assigning them to their relevant target genes still remains challenging. To make this possible, quantitative approaches are needed to study enhancer-promoter contacts and their dynamics during development. Here, we adapted a 4C method to define enhancers and their contacts with cognate promoters in the EB differentiation model. The method uses frequently cutting restriction enzymes, sonication, and a nested-ligation-mediated PCR protocol compatible with commercial DNA library preparation kits. Subsequently, the 4C libraries are subjected to high-throughput sequencing and analyzed bioinformatically, allowing detection and quantification of all sequences that have contacts with a chosen promoter. The resulting sequencing data can also be used to gain information about the dynamics of enhancer-promoter contacts during differentiation. The technique described for the EB differentiation model is easy to implement.

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

We report the application of quantitative chromosome conformation capture followed by high-throughput sequencing in embryoid bodies generated from embryonic stem cells. This technique allows to identify and quantitate the contacts between putative enhancers and promoter regions of a given gene during embryonic stem cell differentiation.

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data analysis.

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage ...

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.

CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...