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 (Figure 1A). Test and adjust the settings depending on the capillary puller machine that is being used.</li>
		<li>Break the pipette tips with forceps to use to obtain capillaries of a 50-&#181;m diameter at the tip. 
		Note: The tips may be too long to penetrate the testis.</li>
		<li>Sharpen the tip in a 30&#176; angle by using a micropipette beveler (Figure 1C).</li>
	</ol>
	</li>
	<li>Reagents
	<ol>
		<li>Prepare 1% pentobarbital sodium: dissolve pentobarbital sodium 100 mg in 10 mL of phosphate-buffered saline (PBS) (step 2.3.3).</li>
		<li>Prepare 10 mg/mL inulin-FITC: dissolve inulin-FITC 1 mg in 100 &#956;L of PBS (step 2.1.6).</li>
		<li>Prepare 4% paraformaldehyde: dissolve paraformaldehyde (PFA) 4 g in 100 mL of PBS (step 2.3.3).</li>
		<li>Prepare 30% sucrose: dissolve sucrose 3 g in 10 mL of PBS (step 2.3.4).</li>
		<li>Prepare 0.1% cadmium chloride: dissolve cadmium chloride 10 mg in 10 mL of PBS (step 2.1.1).</li>
	</ol>
	</li>
</ol>
2. Methods
<ol>
	<li>Anesthesia and presurgery preparation
	<ol>
		<li>Weigh 8-week-old C57BL/6 male mice and calculate the required dose of CdCl2. Treat a group of mice (n = 3) with CdCl2 (5 mg/kg b.w., i.p.) for 3 d before surgery. Treat another group of 8-week-old C57BL/6 male mice (n = 3) with PBS as control.</li>
		<li>Perform surgery under aseptic conditions by using sterile syringe, needle, scissors, and forceps.</li>
		<li>Prepare anesthesia working solution freshly. The required dose of anesthesia is 70 mg of pentobarbital sodium/kg of body weight. On average, an adult C57BL/6 mouse at 8 weeks of age weighs ~25 g, which corresponds to 175 &#956;L of 1% pentobarbital sodium (1.75 mg per mouse). 
		NOTE:&#160;The pentobarbital sodium is dissolved in sterile phosphate-buffered saline (PBS). The solution is filtered by 0.22 um filter unit before being used.</li>
		<li>Clean the area for surgery with 75% ethanol and cover the area with a clean tissue towel.</li>
		<li>Turn on the thermostatic heater and adjust the temperature to 37 &#176;C.</li>
		<li>Prepare inulin-FITC working solution (10 mg/mL) on the day of the surgery.</li>
	</ol>
	</li>
	<li>Surgical procedure
	<ol>
		<li>Weigh 8-week-old C57BL/6 male mice and calculate the required dose of anesthesia.</li>
		<li>Perform an intraperitoneal injection of pentobarbital sodium using the 1-mL sterile syringe. Keep the mouse in a clean cage.</li>
		<li>Observe the eye reflex response and breathing pattern of the mouse to confirm that it is under complete anesthesia. 
		NOTE: Usually, 10 - 15 min are needed until the mouse is deeply anesthetized, with a total lack of toe pinch response and maintenance of slow, steady breathing.</li>
		<li>Move the mouse to the operation area and cover its eye area with a moist tissue paper to avoid dryness during anesthesia (Figure 2A).</li>
		<li>Shave its abdominal hair with a&#160;shaver and disinfect the surgery area with 75% ethanol.</li>
		<li>With sharp scissors, make a 1-cm skin incision above the preputial glands to expose the abdominal wall. Lift the abdominal wall with small forceps and make a 0.5-cm incision to expose the peritoneal cavity (Figure 2B).</li>
		<li>Use forceps to search for fat pads around the epididymis and testis. Carefully pull the fat pads out to expose the attached testis clearly (Figure 2C and 2D). Usually, operate on one testis at a time. 
		NOTE: Avoid touching the fat pads and testis with hands.</li>
		<li>Place a paper (9 cm in diameter) underneath the fat pads and testis (Figure 2C).</li>
		<li>Inject inulin-FITC into a microinjection pipette and connect it to the micromanipulator unit (Figure 1D). Gently insert the microinjection pipette under the tunica albuginea and load a total of 20 &#956;L of inulin-FITC into the interstitium of the testis (Figure 2E and 2F). 
		NOTE: Carefully depress the microinjection pipette to avoid moving it.</li>
		<li>Monitor the movement of inulin-FITC in the testis (Figure 2G).</li>
		<li>Immediately put the testis back into the abdominal cavity after the completion of the injection.</li>
		<li>Repeat the procedure on the contralateral testis. This testis is injected with 20 &#956;L of PBS as a control.</li>
		<li>Close the skin with a surgical suture and move the mouse to a heat pad of a thermostatic heater (Figure 1B).&#160;Administer a dose of 1 mg of analgesic (buprenorphine) per 1 kg of body weight via subcutaneous injection.</li>
	</ol>
	</li>
	<li>Harvesting the testes and frozen sections preparation
	<ol>
		<li>40 min after the injection, euthanize the recipient mouse by cervical dislocation according to animal care and use guidelines.</li>
		<li>Use a pair of sharp scissors to collect the testes. Place the testes into 1 mL of ice-cold PBS to remove any blood contamination.</li>
		<li>Fix the testes in 4% paraformaldehyde (PFA) at 4 &#176;C for 12 - 24 h. Discard the 4% PFA and wash the tissue 3x with 1.5 mL of 1% PBS at room temperature.</li>
		<li>Dehydrate the tissue in 30% sucrose overnight. Put the testis in the embedding frame and cover the tissue with optimal cutting temperature compound (OCT). After the OCT is frozen, fill the embedding frame with OCT so that the whole testis can be covered with OCT.</li>
		<li>Cut 5-&#956;m-thick, frozen cross sections of the testes in a cryostat at -20 &#176;C and let them adhere to microscope slides. 
		NOTE: Refreezing embedded tissues in dry ice may increase the rigidity for cutting.</li>
	</ol>
	</li>
	<li>Image requisition
	<ol>
		<li>Place the slides in a humidified box. Warm the slides at room temperature for 10 min.</li>
		<li>Wash the sections 3x with Tris-buffered saline (TBS) at room temperature.</li>
		<li>Air-dry for 5 min in the dark. Wipe off any residual TBS with dust-free paper.</li>
		<li>Cover the cross sections with 4&#8217;,6-diamidino-2-phenylindole (DAPI). Place the inverted coverslip on a microscope slide.</li>
		<li>Acquire images using a confocal microscope. A total of 20X magnification is generally sufficient for detecting the brightly fluorescent signal.</li>
	</ol>
	</li>
</ol>

<ol><li>Mruk, D. D., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sertoli-Sertoli+and+Sertoli-germ+cell+interactions+and+their+significance+in+germ+cell+movement+in+the+seminiferous+epithelium+during+spermatogenesis%5BTitle%5D)+AND+%22Endocrine+Reviews%22%5BJournal%5D)">Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis.</a> Endocrine Reviews. 25 (5), 747-806 (2004).</li>
<li>Wen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transport+of+germ+cells+across+the+seminiferous+epithelium+during+spermatogenesis-the+involvement+of+both+actin-+and+microtubule-based+cytoskeletons%5BTitle%5D)+AND+%22Tissue+Barriers%22%5BJournal%5D)">Transport of germ cells across the seminiferous epithelium during spermatogenesis-the involvement of both actin- and microtubule-based cytoskeletons.</a> Tissue Barriers. 4 (4), e1265042(2016).</li>
<li>Wang, C. Q., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+seamless+trespass%3A+germ+cell+migration+across+the+seminiferous+epithelium+during+spermatogenesis%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((The+testis+in+immune+privilege%5BTitle%5D)+AND+%22Immunological+Reviews%22%5BJournal%5D)">The testis in immune privilege.</a> Immunological Reviews. 213, 66-81 (2006).</li>
<li>O'Bryan, M. K., Hedger, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+Networks+in+the+Control+of+Spermatogenesis+Chronic+Inflammation+in+an+Immunologically+Privileged+Tissue%3F%5BTitle%5D)+AND+%22Molecular+Mechanisms+In+Spermatogenesis%22%5BJournal%5D)">Inflammatory Networks in the Control of Spermatogenesis Chronic Inflammation in an Immunologically Privileged Tissue?</a> Molecular Mechanisms In Spermatogenesis. 636, 92-114 (2008).</li>
<li>Li, N., Wang, T., Han, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+cellular+and+molecular+aspects+of+immune+privilege+in+the+testis%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Structural cellular and molecular aspects of immune privilege in the testis.</a> Frontiers in Immunology. 3, 152(2012).</li>
<li>Mruk, D. D., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Mammalian+Blood-Testis+Barrier%3A+Its+Biology+and+Regulation%5BTitle%5D)+AND+%22Endocrine+Review%22%5BJournal%5D)">The Mammalian Blood-Testis Barrier: Its Biology and Regulation.</a> Endocrine Review. 36 (5), 564-591 (2015).</li>
<li>Govero, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zika+virus+infection+damages+the+testes+in+mice%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Zika virus infection damages the testes in mice.</a> Nature. 540 (7633), 438-442 (2016).</li>
<li>Jenabian, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+tolerance+properties+of+the+testicular+tissue+as+a+viral+sanctuary+site+in+ART-treated+HIV-infected+adults%5BTitle%5D)+AND+%22AIDS%22%5BJournal%5D)">Immune tolerance properties of the testicular tissue as a viral sanctuary site in ART-treated HIV-infected adults.</a> AIDS. 30 (18), 2777-2786 (2016).</li>
<li>Holembowski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TAp73+is+essential+for+germ+cell+adhesion+and+maturation+in+testis%5BTitle%5D)+AND+%22Journal+Of+Cell+Biology%22%5BJournal%5D)">TAp73 is essential for germ cell adhesion and maturation in testis.</a> Journal Of Cell Biology. 204 (7), 1173-1190 (2014).</li>
<li>Legendre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+engineered+3D+blood-testis+barrier+model+for+the+assessment+of+reproductive+toxicity+potential%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential.</a> Biomaterials. 31 (16), 4492-4505 (2010).</li>
<li>Setchell, B. P., Waites, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+the+permeability+of+the+testicular+capillaries+and+of+the+%27blood-testis+barrier%27+after+injection+of+cadmium+chloride+in+the+rat%5BTitle%5D)+AND+%22Journal+of+Endocrinology%22%5BJournal%5D)">Changes in the permeability of the testicular capillaries and of the 'blood-testis barrier' after injection of cadmium chloride in the rat.</a> Journal of Endocrinology. 47 (1), 81-86 (1970).</li>
<li>Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+central+role+of+Sertoli+cells+in+spermatogenesis%5BTitle%5D)+AND+%22Seminars+in+Cell+%26+Developmental+Biology%22%5BJournal%5D)">The central role of Sertoli cells in spermatogenesis.</a> Seminars in Cell & Developmental Biology. 9 (4), 411-416 (1998).</li>
<li>Cheng, C. Y., Mruk, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+blood-testis+barrier+and+its+implications+for+male+contraception%5BTitle%5D)+AND+%22Pharmacological+Reviews%22%5BJournal%5D)">The blood-testis barrier and its implications for male contraception.</a> Pharmacological Reviews. 64 (1), 16-64 (2012).</li>
<li>Mruk, D. D., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro+system+to+study+Sertoli+cell+blood-testis+barrier+dynamics%5BTitle%5D)+AND+%22Methods+Molecular+Biology%22%5BJournal%5D)">An in vitro system to study Sertoli cell blood-testis barrier dynamics.</a> Methods Molecular Biology. 763, 237-252 (2011).</li>
<li>Orth, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proliferation+of+Sertoli+cells+in+fetal+and+postnatal+rats%3A+a+quantitative+autoradiographic+study%5BTitle%5D)+AND+%22Anatomical+Record%22%5BJournal%5D)">Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study.</a> Anatomical Record. 203 (4), 485-492 (1982).</li>
<li>Lee, N. P. Y., Mruk, D., Lee, W. M., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+the+cadherin%2Fcatenin+complex+a+functional+unit+of+cell-cell+actin-based+adherens+junctions+in+the+rat+testis%3F%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Is the cadherin/catenin complex a functional unit of cell-cell actin-based adherens junctions in the rat testis?</a> Biology of Reproduction. 68 (2), 489-508 (2003).</li>
<li>Bai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Germline-Specific+Role+for+the+mTORC2+Component+Rictor+in+Maintaining+Spermatogonial+Differentiation+and+Intercellular+Adhesion+in+Mouse+Testis%5BTitle%5D)+AND+%22Molecular+Human+Reproduction%22%5BJournal%5D)">A Germline-Specific Role for the mTORC2 Component Rictor in Maintaining Spermatogonial Differentiation and Intercellular Adhesion in Mouse Testis.</a> Molecular Human Reproduction. 24 (5), 244-259 (2018).</li>
<li>Korhonen, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DICER+Regulates+the+Formation+and+Maintenance+of+Cell-Cell+Junctions+in+the+Mouse+Seminiferous+Epithelium%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">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/pubmed?term=((Trout+Sertoli+cells+and+germ+cells+in+primary+culture%3A+I.+Morphological+and+ultrastructural+study%5BTitle%5D)+AND+%22Gamete+Research%22%5BJournal%5D)">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/pubmed?term=((Monitoring+the+Integrity+of+the+Blood-Testis+Barrier+%28BTB%29%3A+An+In+Vivo+Assay%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Monitoring the Integrity of the Blood-Testis Barrier (BTB): An In Vivo Assay.</a> Methods in Molecular Biology. 1748, 245-252 (2018).</li>
</ol>

The authors have nothing to disclose.

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.
<p class="jove_content...

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><tbody><tr><td>Capillary puller&amp;nbsp;</td><td>SUTTER INSTRUMENT (USA)</td><td>P-97</td><td></td></tr><tr><td>10x PBS</td><td>Hyclone (USA)</td><td>SH30258.01</td><td>dilution to 1&amp;times; in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma (USA)</td><td>F6057</td><td></td></tr><tr><td>Adhesion microscope slides</td><td>CITOGLAS (China)</td><td>80312-3161</td><td></td></tr><tr><td>Cadmium chloride</td><td>Sigma (USA)</td><td>655198-5G</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss (Germany)</td><td>LSM700</td><td></td></tr><tr><td>Dust-free paper</td><td>Kimberly-Clark (USA)</td><td>34120</td><td></td></tr><tr><td>Inulin-FITC</td><td>Sigma (USA)</td><td>F3272</td><td></td></tr><tr><td>Microinjection capillaries</td><td>Zhengtianyi (China)</td><td>BJ-40</td><td>1.0 mm &amp;times; 0.8 mm&amp;nbsp; &amp;times; 100 mm</td></tr><tr><td>Micropipette beveler</td><td>NARISHIGE (JAPAN)</td><td>EG-400</td><td></td></tr><tr><td>OCT</td><td>SAKURA (JAPAN)</td><td>4583</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma (USA)</td><td>P6148</td><td></td></tr><tr><td>Pentobarbital sodium</td><td>Merck (Germany)</td><td>P11011</td><td></td></tr><tr><td>Shaver&amp;nbsp;</td><td>Yashen (China)</td><td></td><td></td></tr><tr><td>Stereo microscope</td><td>Nikon (JAPAN)</td><td>SMZ1000</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sangon Biotech (China)</td><td>A610498</td><td></td></tr><tr><td>Surgical instruments</td><td>Stronger (China)</td><td></td><td>scissors, forceps, needle holder</td></tr><tr><td>Syringe</td><td>KDL (China)</td><td>20163150518</td><td>0.45 mm &amp;times; 0.16 mm RW LB</td></tr><tr><td>thermostatic heater</td><td>KELL (Nanjing, China)</td><td>KEL-2010</td><td></td></tr><tr><td>&lt;strong&gt;10x TBS, pH 7.6&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 M Tris</td><td>Sangon Biotech (China)</td><td>A600194</td><td></td></tr><tr><td>1.37 M Nacl</td><td>Sangon Biotech (China)</td><td>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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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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10.9K Views.  Nanjing Medical University. 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.

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

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This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

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Medicine

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

ICSI-ET Mouse Model to Study Long-Term Impacts on Glucose Metabolism

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.

Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Biology

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Neuroscience

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

This article reports an in vivo inhibition of CENP-E through abdominal surgery and testicular injection of GSK923295, a valuable model for male meiotic division. Using the immunofluorescence, flow cytometry and transmission electron microscopy assays, we show that CENP-E inhibition results in chromosome misalignment and genome instability in mouse spermatocytes.

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

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

Summary

Explore More Videos

An in vivo Assay to Test Blood Vessel Permeability

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

In Vivo Microinjection and Electroporation of Mouse Testis

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland