Name: an in vivo method to study mouse blood-testis barrier integrity
Uploaded: 2018-12-02T13:00:00
Description: Watch this Scientific Journal Video about An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity at JoVE.com

This work was supported by the National Key R&#38;D Program of China (2016YFA0500902), the National Natural Science Foundation of China (31471228, 31771653), the Jiangsu Science Foundation for Distinguished Young Scholars (BK20150047),&#160;the Natural Science Foundation of Jiangsu Province (BK20140897, 14KJA180005) and the Innovative and Entrepreneurial Program of Jiangsu Province to K.Z.

All performed animal experiments have been approved by the Nanjing Medical University committee. Male C57BL/6 mice were kept under controlled photoperiod conditions and were supplied with food and water.
1. Preparations
<ol>
	<li>Microinjection capillaries
	<ol>
		<li>Use microinjection capillaries with an outer diameter, inner dimeter, and length of 1.0 mm, 0.8 mm, and 10.0 cm, respectively.</li>
		<li>Pull glass capillaries with a capillary puller (Fig...

<ol>
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Q., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+seamless+trespass:+germ+cell+migration+across+the+seminiferous+epithelium+during+spermatogenesis.">A seamless trespass: germ cell migration across the seminiferous epithelium during spermatogenesis.</a> Journal of Cell Biology. 178 (4), 549-556 (2007).</li><li>Fijak, M., Meinhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+testis+in+immune+privilege.">The testis in immune privilege.</a> Immunological Reviews. 213, 66-81 (2006).</li><li>O'Bryan, M. K., Hedger, M. 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M., 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=DICER+Regulates+the+Formation+and+Maintenance+of+Cell-Cell+Junctions+in+the+Mouse+Seminiferous+Epithelium.">DICER Regulates the Formation and Maintenance of Cell-Cell Junctions in the Mouse Seminiferous Epithelium.</a> Biology of Reproduction. 93 (6), 139 (2015).</li><li>Loir, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trout+Sertoli+cells+and+germ+cells+in+primary+culture:+I.+Morphological+and+ultrastructural+study.">Trout Sertoli cells and germ cells in primary culture: I. Morphological and ultrastructural study.</a> Gamete Research. 24 (2), 151-169 (1989).</li><li>Chen, H., 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=Monitoring+the+Integrity+of+the+Blood-Testis+Barrier+(BTB):+An+In+Vivo+Assay.">Monitoring the Integrity of the Blood-Testis Barrier (BTB): An In Vivo Assay.</a> Methods in Molecular Biology. 1748, 245-252 (2018).</li></ol>

Spermatogenesis takes place in the seminiferous epithelium and is a highly ordered and dynamic process that is governed by germ cells and somatic cells (e.g., Sertoli cells)13. The BTB structure, which is constructed by Sertoli cells, divides the seminiferous epithelium into a basal and an apical compartment. The development of meiotic and haploid germ cells occurs in the apical compartment which forms an immunological barrier14.
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Mammalian spermatogenesis is considered a highly structured process that encompasses spermatogonial self-renewal and differentiation through spermatocytes into haploid spermatozoa via mitosis, meiosis, and spermiogenesis, during which dramatic biochemical and morphological changes occur. Developing germ cells are progressively transported from the base of the seminiferous tubule toward the lumen. This process is regulated by cell-cell contacts between germ cells and Sertoli cells1,2. Adjacent Sertoli cells form the BTB that is located near the base of the seminiferous tubule. The BTB physically divides the epithelium into a basal and an adluminal compartment. During stages VIII - IX of the epithelial cycle, preleptotene/leptotene spermatocytes from the basal compartments migrate across the BTB, entering the adluminal compartments3. Therefore, the function of the BTB is to provide an immunoprivileged microenvironment for the completion of meiosis and spermiogenesis4,5,6. Unlike other blood-tissue barriers (e.g., blood-brain barrier) that are only composed of tight junctions (TJs), the BTB is formed by four different junctions (TJs, ectoplasmic specializations, gap junctions, and intermediate filament-based desmosomes) between Sertoli cells1,7.
Many studies have used genetically-modified mice, virus infections, and environmental toxicants to investigate mechanisms of BTB integrity7,8,9. The BTB disruption induces impaired spermatogenesis and subfertility or infertility. Since the BTB formation and integrity have been confirmed to be affected by contacts between Sertoli cells8, an in vitro model based on primary culture of isolated Sertoli cells has been used for BTB study. However, this model cannot accurately mimic BTB dynamics in vivo. Moreover, no such co-culture of germ cells with Sertoli cells has been established as capable of reflecting all relevant structural and functional components of the BTB10,11.
In general, in vivo BTB integrity assays are typically based on small molecules, such as EZ-Link Sulfo-NHS-LC-Biotin and FITC-conjugated inulin (inulin-FITC). Normally, the diffusion of biotin or inulin-FITC from the basal compartment is blocked by BTB structure. Therefore, we are able to use this method to assess the extent of BTB damage compared with control groups. While BTB can be compromised with certain types of stimuli, such as treatment with cadmium chloride (CdCl2)12, BTB becomes accessible to small molecules, which eventually enter the adluminal compartment as indicators.
An early in vivo BTB integrity assay involves injecting biotin or inulin-FITC into the jugular vein, which involves surgery, and is invasive, complicated, and time-consuming. Besides, as the reporter substances diffuse through the whole body via the circulation, the local concentration of biotin or inulin-FITC in the seminiferous tubules is limited. Moreover, systemic exposure may induce immune reactions. Here, we present a simple and effective in vivo BTB integrity assay enabling direct injection of a small aliquot of inulin-FITC into the interstitium of a testis. Using the fluorescent labeling method, the staining process is convenient, as secondary antibodies are not required. Here, the process of fluorescent dye entering the testis is visualized.

<table border="0" cellpadding="0" cellspacing="0" style="width:803px;" width="802">
	
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Capillary puller&#160;</td>
			<td style="width:231px;">SUTTER INSTRUMENT (USA)</td>
			<td style="width:139px;">P-97</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10x PBS</td>
			<td style="width:231px;">Hyclone (USA)</td>
			<td style="width:139px;">SH30258.01</td>
			<td>dilution to 1&#215; in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">F6057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesion microscope slides</td>
			<td style="width:231px;">CITOGLAS (China)</td>
			<td style="width:139px;">80312-3161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cadmium chloride</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">655198-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td style="width:231px;">Zeiss (Germany)</td>
			<td style="width:139px;">LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dust-free paper</td>
			<td style="width:231px;">Kimberly-Clark (USA)</td>
			<td style="width:139px;">34120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inulin-FITC</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">F3272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Microinjection capillaries</td>
			<td style="width:231px;">Zhengtianyi (China)</td>
			<td style="width:139px;">BJ-40</td>
			<td>1.0 mm &#215; 0.8 mm&#160; &#215; 100 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette beveler</td>
			<td style="width:231px;">NARISHIGE (JAPAN)</td>
			<td style="width:139px;">EG-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT</td>
			<td style="width:231px;">SAKURA (JAPAN)</td>
			<td style="width:139px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital sodium</td>
			<td style="width:231px;">Merck (Germany)</td>
			<td style="width:139px;">P11011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaver&#160;</td>
			<td style="width:231px;">Yashen (China)</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Stereo microscope</td>
			<td style="width:231px;">Nikon (JAPAN)</td>
			<td style="width:139px;">SMZ1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose&#160;</td>
			<td style="width:231px;">Sangon Biotech (China)</td>
			<td style="width:139px;">A610498</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Surgical instruments</td>
			<td style="width:231px;">Stronger (China)</td>
			<td style="width:139px;"></td>
			<td>scissors, forceps, needle holder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td style="width:231px;">KDL (China)</td>
			<td style="width:139px;">20163150518</td>
			<td>0.45 mm &#215; 0.16 mm RW LB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thermostatic heater</td>
			<td style="width:231px;">KELL (Nanjing, China)</td>
			<td style="width:139px;">KEL-2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td >10x TBS, pH 7.6</td>
<td></td>
<td></td>
<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M Tris</td>
			<td style="width:231px;">Sangon Biotech (China)</td>
			<td style="width:139px;">A600194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">1.37 M Nacl</td>
			<td style="width:231px;">Sangon Biotech (China)</td>
			<td style="width:139px;">A610476</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo method to study mouse blood-testis barrier integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

The experimental set-up for performing the BTB integrity assay is shown in Figure 1. Pull and sharpen microinjection capillaries with a capillary puller and micropipette beveler, respectively (Figure 1A and 1C). The thermostatic heater and equipment for microinjection are illustrated in Figure 1B and 1D.
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Watch this Scientific Journal Video about An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity at JoVE.com

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

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