The authors would like to thank Guido Verhoeven and Ludo Deboel, Leuven, who were extremely helpful in establishing the isolation of primary Sertoli cells in the Meinhardt lab in Giessen. Monika Fijak, Gie&#223;en, is acknowledged for her help with figure 1A and advice. Studies were supported by the Deutsche Forschungsgemeinschaft through grant BH 93/1-1 (S.B.) and funding of the International Research Graduate College JLU Gie&#223;en (Germany)/Monash University (Melbourne, Australia) GRK 1871 (S.B.). Support was also obtained from a grant of the State of Hessen within the program &#34;Landes-Offensive zur Entwicklung Wissenschaftlich-&#246;konomischer Exzellenz&#34; (LOEWE) called &#34;M&#228;nnliche Infertilit&#228;t bei Infektion &#38; Entz&#252;ndung&#34; (MIBIE).

1. Animal Ethics Statement
Experiments described here were performed according to the guidelines of the Regierungspr&#228;sidium Giessen, Germany, as the local authority and confirm to the German Code of Practice for the Care and Use of Animals for Experimental Purposes (Permission no: GI 20/23 Nr. A 31/2012).
2. Preparation of Media, Enzyme Solutions and Animals
<ol>
	<li>Prepare 1% iodine alcohol by dissolving 1 g of iodine in 100 ml ethanol.</li>
	<li>Prepare 2x 500 ml Dulbecco&#180;s phosphate buffered saline (PBS) without Ca2+ and Mg2+ supplemented each with 7.5 ml D-glucose (100 g/L) and 5 ml 100x Penicillin/Streptomycin stock solution (15 mg/ml D-glucose, 50 U/ml Penicillin and 50 &#181;g/ml Streptomycin final).</li>
	<li>Supplement 1x 500 ml Roswell Park Memorial Institute (RPMI) 1640 medium with 5 ml 100x Penicillin/Streptomycin stock solution (50 U/ml Penicillin and 50 &#181;g/ml Streptomycin final).</li>
	<li>Prepare a Trypsin-DNase I solution (2.5 mg/ml Trypsin and 10 &#181;g/ml DNase I) by adding 25 mg Trypsin and 0.1 ml DNase I (1 mg/ml DNase I) to 9.9 ml PBS.</li>
	<li>Dissolve 50 mg Trypsin inhibitor in 5 ml PBS for Trypsin inhibitor A solution (10 mg/ml). Dissolve 25 mg Trypsin inhibitor in 10 ml PBS for Trypsin inhibitor B solution (2.5 mg/ml).</li>
	<li>For a Collagenase-Hyaluronidase-Deoxyribonuclease I (DNase I) solution dissolve 10 mg Collagenase A (1 mg/ml final), 10 mg Hyaluronidase (1 mg/ml final) and 0.1 ml DNase I (1 mg/ml DNase I) (10 &#181;g/ml final) in 10 ml PBS.</li>
	<li>Prepare a Hyaluronidase-DNase I solution by adding 10 mg Hyaluronidase (1 mg/ml final) and 0.1 ml DNase I solution (1 mg/ml) (10 &#181;g/ml final) to 9.9 ml PBS.</li>
	<li>Order Wistar rats on time so that they are 19 days old on the day of Sertoli cell isolation. 
	Note: The described protocol is designed for 10 rats but can be modified for a minimum of 5 and a maximum of 20 rats. The volumes of all solutions have to be adjusted accordingly.</li>
	<li>Prepare Oil Red O stock solution by dissolving 0.35 g Oil Red O in 100 ml isopropanol. Stir O/N, filter through 0.2 &#181;m and store at RT for up to one year.</li>
	<li>Prepare Oil Red O staining solution by mixing 6 ml Oil Red O stock solution with 4 ml water (3:2). After 10-20 min filter through 0.2 &#181;m. Use within the next 2 hr.</li>
	<li>Prepare a 4% (w/v) formaldehyde solution in a ventilated hood by adding 4 g paraformaldehyde powder (PFA) to 80 ml of 1x PBS while stirring gently. Heat to ~60 &#176;C, add 1 ml of 1 M NaOH and continue stirring until the paraformaldehyde is dissolved. Let the solution cool down to RT, adjust the pH to 7.4 with 1 M HCl and the volume to 100 ml with 1x PBS. Filter the solution (0.45 &#181;m) and use fresh or store in aliquots at &#8722;20 &#176;C for several months. 
	Note: Polymerized formaldehyde (PFA) depolymerizes to monomeric formaldehyde in water. This process is catalyzed by moderate heating and a slightly alkaline pH. Prepare all enzyme solutions freshly and filter sterilize (0.2 &#181;M) them on the day of use. If a different manufacturer for an enzyme (see Materials/Equipment Table) is used, carefully adjust its concentration/activity. 
	In the following protocol a &#34;pipette&#34; stands for a serological pipette.</li>
</ol>
3. Preparation of Seminiferous Tubules
<ol>
	<li>Anesthetize 10 male Wistar rats one by one in a desiccator using CO2 and a flow rate that displaces at least 20% of the chamber volume per minute. Wait until breathing stops, test anesthesia by squeezing a foot pad, and if there is no reaction euthanize each rat by cervical dislocation. Drain the blood by sectioning jugular vein and carotid artery (vagina carotica) under flowing water. Disinfect the abdomen by wiping with 70% ethanol.</li>
	<li>Using forceps lift the skin from the abdominal muscles and cut out an oval shaped skin lobe that is extending from the pubic symphysis to the sternum (Figure 2A) and flap it upwards onto the chest. Next, grab the abdominal muscles and make a medial incision from the pubic symphysis to the sternum.
	<ol>
		<li>Squeeze the abdomen with the thumbs from pelvis upwards in order to push the testes out of the lower pelvis. Pick the epididymal fat pad (Figure 2B) with forceps for pulling up the testis further. Cut the spermatic cord (Figure 2B), leaving the tunica albuginea intact, and collect all testes in 20 ml PBS in a 50 ml conical tube (Figure 2C).</li>
	</ol>
	</li>
	<li>After collecting all testes, disinfect them by adding an equal volume (20 ml) of 1% (w/v) iodine in ethanol. Invert the tube twice and decant the supernatant immediately. Quickly wash the testes twice with 25 ml PBS each (Figure 2D).</li>
	<li>Transfer the testes to a Petri dish containing 15 ml PBS (Figure 2E). Grasp each testis firmly by forceps at one end, make a small incision into the tunica albuginea at the opposite end, and squeeze out the tubules using closed scissor blades as a scraping tool. 
	Note: The tubules should still form a compact testicle-shaped bundle with only a few tubules extending into the solution in order to enable homogeneous enzymatic digestion (Figure 2F).</li>
	<li>Transfer the de-capsulated testes to a 100 ml screw-cap bottle containing 10 ml Trypsin-DNase I solution. Perform the digestion in a shaking water bath at 32 &#176;C (120 oscillations/min). After 4 min evaluate the tubules with the naked eye for dispersion of tubules. Once tubules start to disperse into the solution, stop the digestion immediately. If necessary, continue incubation for up to 2 min.</li>
	<li>Stop trypsin digestion by adding 5 ml of trypsin inhibitor solution A. Thoroughly mix the solution by pipetting up and down 3-4 times using a 10 ml pipette and transfer it to a 50 ml conical tube.</li>
	<li>After 5 min of incubation observe the tubules settle by unit gravity, carefully remove the supernatant by using the 10 ml pipette from step 3.6 and add 10 ml of trypsin inhibitor B solution in order to fully stop trypsin digestion. Resuspend the tubules by pipetting up and down 2-3 times.</li>
	<li>In order to remove contaminating interstitial cells wash the tubules 9 times with 25 ml PBS each. Resuspend tubules in each wash by using a 25 ml pipette and allow them to settle by unit gravity for 10 min. Always use the same 25 ml pipette for pipetting PBS, resuspension and removal of the supernatant.</li>
</ol>
4. Removal/Isolation of Peritubular Cells (PTCs)
<ol>
	<li>Transfer all tubules to a 100 ml screw-cap bottle and add the Collagenase-Hyaluronidase-DNase I solution (Figure 3A). Perform digestion for 10 min in a shaking water bath at 32 &#176;C (120 oscillations/min).</li>
	<li>Pipet a drop of the suspension on a glass slide and quickly check the tubules under an inverted light microscope for routine cell culture at 100x magnification. If digestion is not advanced enough continue incubation for additional 2 min (do not count the time needed for the microscopical check). 
	Note: During this step all tubules get shortened in length and tubule edges should become rough (Figure 3B and C). Rough edges are indicative for the release of peritubular cells.</li>
	<li>Transfer the suspension with digested tubules to a 50 ml conical tube using the 25 ml pipette from step 3.8. Wash the 100 ml bottle with 10 ml PBS and add it to the tubules in the conical tube. Allow the digested tubules to settle by unit gravity for 10 min, and carefully aspirate the supernatant, containing the PTCs, using the 25 ml pipette again, and transfer it to a new conical tube. For aspiration of the last few milliliters use a 5 ml pipette in order to prevent any carry-over of tubules.</li>
	<li>Add 20 ml RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) to the collected tubule supernatants, mix and centrifuge at 300 x g for 10 min at RT without using the break.</li>
	<li>Carefully decant the supernatant and resuspend the pelleted PTCs in 20 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS and seed 4 ml each on five T75 flasks containing 16 ml medium each. Incubate at 37 &#176;C in 5% CO2 atmosphere (Figure 4A).</li>
	<li>On the 3rd day of culture trypsinize the PTCs and split them 1:2 on T75 flasks containing 16 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS each (yields 10 flasks altogether) (Figure 4B). Continue incubation at 37 &#176;C in 5% CO2 atmosphere.</li>
	<li>On the 5th day of culture trypsinize the PTCs again and plate them on 6-well or 24-well plates depending on the experiment (one T75 flask per plate). Experiments can be performed on day 6.</li>
</ol>
5. Isolation of Sertoli Cells (SCs)
<ol>
	<li>Resuspend the settled tubules from step 4.3 thoroughly in 25 ml of PBS by using a 25 ml pipette and allow them to settle by unit gravity for 12 min. Use the same 25 ml pipette for pipetting PBS, resuspension of tubules and removal of supernatants. Repeat the wash 3 times (4 washes in total).</li>
	<li>After the last wash transfer the digested tubules to another 100 ml screw-cap bottle containing the Hyaluronidase-DNase I solution. Perform the digestion in a shaking water bath at 32 &#176;C (120 oscillations/min).</li>
	<li>After 5 min check the tubules again by light microscopic inspection at 100X magnification as described in step 4.2. 
	Note: Short tubules, tubular aggregates and released cells should be visible (Figure 3D).
	<ol>
		<li>Allow the tubular aggregates settle for 10 min, and aspirate the supernatant using a 25 ml pipette. Wash aggregates 4 times using 25 ml PBS and a sedimentation time of 10 min for each washing step (Figure 3E).</li>
	</ol>
	</li>
	<li>Add 20 ml of RPMI 1640 medium and pass the suspension 10 times through an 18 G needle mounted on a 20 ml syringe. Avoid air bubbles. 
	Note: Pulling the suspension in and out through the needle is considered as one pass. The disruption and homogenization of cells by hydrodynamic shearing is a standard technique in the preparation of cells that tend to aggregate. The passing of cells through a hypodermic needle of small inner diameter is unlikely to have an impact on their functional properties25. Figure 5C and D shows the normal ultrastructure of purified Sertoli cells on day 4 of culture.
	<ol>
		<li>Pipet a drop of the suspension on a glass slide and quickly check the tubules under an inverted light microscope for routine cell culture at 10X magnification if the aggregates are all dispersed (Figure 3F). Filter the cell suspension through a 70 &#181;m cell strainer in order to obtain a pure single cell suspension in the filtrate.</li>
	</ol>
	</li>
	<li>Centrifuge the filtrate of step 5.4 at 200 x g for 10 min at RT without using the break. Carefully aspirate the supernatant using a 25 ml pipette, and resuspend the Sertoli cells in 40 ml RPMI 1640 medium (serum-free).</li>
	<li>Mix 100 &#181;l cell suspension with 100 &#181;l of Trypan Blue Stain and count cells with a Neubauer Improved chamber. Adjust the cell concentration to 3 x 106 cells/ml according to the counting result. Seed 1 ml per well on a 6-well plate (=day 1). If an Oil Red O staining (step 6) is planned put a cover slip into one or a few wells before seeding Sertoli cells. 
	Note: Sertoli cells in suspension settle quickly so that the tube should be briefly shaken every time before a cell aliquot is taken with the pipette. Until day 4 they firmly attach to the substratum.</li>
	<li>In order to remove floating or adherent germ cells on day 4 (Figure 4C) wash each well of the 6-well plates used for plating isolated Sertoli cells (step 5.6) with PBS (Figure 4D). Aspirate the medium, add 1-2 ml of PBS to each well and shake the 6-well plates horizontally on a table vigorously but without spilling PBS. Repeat the wash 3 times and perform the same washing procedure on day 5 and 6 (Figure 4E). 
	Note: On day 4 Sertoli cells have firmly attached and show a cobblestone like pattern under the inverted light microscope (Figure 4C). Experiments can be performed from day 7 onwards.
	<ol>
		<li>(Alternative for step 5.7) For the removal of floating and adherent germ cells perform a hypotonic shock 3 days after seeding on 6-well plates. Remove the medium and add 1 ml of 20 mM Tris pH 7.5 taken directly out of the fridge. Aspirate the Tris buffer after 90 to 120 sec but not later. Wash cells twice with PBS, and add 2 ml of RPMI 1640 medium (serum-free). Optionally, perform experiments the next day. 
		Note: The longer the duration of the hypotonic shock is the more germ cells can be removed but the more Sertoli cells get lost as well. The first PBS wash should be performed quickly but with care in order not to displace any Sertoli cells. Directly after hypotonic treatment numerous vacuoles of different sizes can be observed in the cytoplasm by phase contrast microscopy. Vacuoles disappear within a few hours after hypotonic treatment.</li>
	</ol>
	</li>
</ol>
6. Oil Red O Staining
<ol>
	<li>Perform all steps at RT. On day 7 wash Sertoli cells grown on a cover slip (step 5.6) with PBS twice and fix them with 10% formalin in PBS. After 10 min replace formalin with fresh solution, and continue fixation for another 60 min. 
	Note: All solutions are added to the well(s) with a cover slip and aspirated before the next step.</li>
	<li>Aspirate formalin, wash cells with water twice and add 60% isopropanol. After 5 min allow cells to air dry for a few minutes.</li>
	<li>Add 1 ml Oil Red O staining solution per well and incubate for 10 min. Aspirate the solution, and wash cells immediately 4 times with water and mount cover slips in glycerol-PBS (9:1). Take bright field images with a routine light microscope (Figure 4F).</li>
</ol>
7. Immunofluorescence Staining
<ol>
	<li>Wash PTCs (from step 4.5) or Sertoli cells (from step 5.5) grown on a cover slip with PBS twice and fix them with 4% PFA for 10 min at RT.</li>
	<li>Incubate with 5% BSA in PBS for 1 hr at RT. Discard the BSA-PBS solution and add fresh BSA-PBS solution containing actin (smooth muscle) (for PTCs) or vimentin (for Sertoli cells) primary antibody (dilution 1:100 for both antibodies) and incubate at 4 &#176;C O/N. 
	Note: Incubation with BSA blocks non-specific binding of the primary antibody.</li>
	<li>Wash cover slips 3 times for&#160; 5 min in PBS-0.1% Tween 20 and incubate with green-fluorescent dye conjugated goat anti-mouse secondary antibody (dilution 1:1,000) at RT for 1 hr in the dark.</li>
	<li>Wash cover slips 3 times for 5 min in PBS-0.1% Tween 20 and mount them in DAPI mounting medium.</li>
</ol>
8. Ultrastructural Analysis
<ol>
	<li>Wash Sertoli cells (from step 5.6) grown on a 6 cm cell culture plate with PBS and fix them for 2 hr&#160;at 4 &#176;C in a mixture of 2.5% glutaraldehyde, 2.5% paraformaldehyde and 0.05% picric acid in 67 mM cacodylate buffer (pH 7.4) according to Ito and Karnovsky26.</li>
	<li>Perform post-fixation in 1% osmium tetroxide followed by an O/N incubation in 0.3% uranyl acetate dissolved in 50 mM maleate buffer (pH 5). Embed samples in embedding media according to standard procedures. Cut thin sections, contrast them with lead citrate and examine with an electron microscope.</li>
</ol>

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<li>Bhushan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uropathogenic+Escherichia+coli+block+MyD88-dependent+and+activate+MyD88-independent+signaling+pathways+in+rat+testicular+cells%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Uropathogenic Escherichia coli block MyD88-dependent and activate MyD88-independent signaling pathways in rat testicular cells.</a> J. Immunol. 180, 5537-5547 (2008).</li>
<li>Meinhardt, A., Hedger, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunological%2C+paracrine+and+endocrine+aspects+of+testicular+immune+privilege%5BTitle%5D)+AND+%22Mol.+Cell.+Endocrinol%22%5BJournal%5D)">Immunological, paracrine and endocrine aspects of testicular immune privilege.</a> Mol. Cell. Endocrinol. 335, 60-68 (2011).</li>
<li>Fijak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+Testosterone+on+Inflammatory+Response+in+Testicular+Cells+and+Expression+of+Transcription+Factor+Foxp3+in+T+Cells%5BTitle%5D)+AND+%22Am.+J.+Reprod.+Immunol%22%5BJournal%5D)">Influence of Testosterone on Inflammatory Response in Testicular Cells and Expression of Transcription Factor Foxp3 in T Cells.</a> Am. J. Reprod. Immunol. , 12-25 (2015).</li>
<li>Mamidi, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+passing+mesenchymal+stem+cells+through+smaller+bore+size+needles+for+subsequent+use+in+patients+for+clinical+or+cosmetic+indications%5BTitle%5D)+AND+%22J.+Transl.+Med%22%5BJournal%5D)">Impact of passing mesenchymal stem cells through smaller bore size needles for subsequent use in patients for clinical or cosmetic indications.</a> J. Transl. Med. 10, 229(2012).</li>
<li>Ito, S., Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formaldehyde-glutaraldehyde+fixatives+containing+trinitro+compounds%5BTitle%5D)+AND+%22J.+Cell.+Biol%22%5BJournal%5D)">Formaldehyde-glutaraldehyde fixatives containing trinitro compounds.</a> J. Cell. Biol. 39, 168-169 (1968).</li>
<li>Galdieri, M., Ziparo, E., Palombi, F., Russo, M. A., Stefanini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pure+Sertoli+cell+cultures%3A+a+new+model+for+the+study+of+somatic-germ+cell+interactions%5BTitle%5D)+AND+%22J.+Androl%22%5BJournal%5D)">Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions.</a> J. Androl. 5, 249-254 (1981).</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+%22Immunol.+Rev%22%5BJournal%5D)">The testis in immune privilege.</a> Immunol. Rev. 213, 66-81 (2006).</li>
<li>Jegou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Sertoli+cell+in+vivo+and+in+vitro%5BTitle%5D)+AND+%22Cell+Biol.+Toxicol%22%5BJournal%5D)">The Sertoli cell in vivo and in vitro.</a> Cell Biol. Toxicol. 8, 49-54 (1992).</li>
<li>Pineau, C., Le Magueresse, B., Courtens, J. L., Jegou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+in+vitro+the+phagocytic+function+of+Sertoli+cells+in+the+rat%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Study in vitro the phagocytic function of Sertoli cells in the rat.</a> Cell Tissue Res. 264, 589-598 (1991).</li>
<li>Ren, Y., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis%3A+the+importance+of+being+eaten%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">Apoptosis: the importance of being eaten.</a> Cell Death Differ. 5, 563-568 (1998).</li>
<li>Ducray, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+a+mouse+Sertoli+cell+line+producing+rat+androgen-binding+protein+%28ABP%29%5BTitle%5D)+AND+%22Steroids%22%5BJournal%5D)">Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP).</a> Steroids. 63, 285-287 (1998).</li>
<li>Konrad, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+Sertoli+cells+express+epithelial+but+also+mesenchymal+genes+after+immortalization+with+SV40%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40.</a> Biochim. Biophys. Acta. 1722, 6-14 (2005).</li>
</ol>

The authors have nothing to disclose.

The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, when Sertoli cells are isolated using the procedure described here they get deprived of their intimate contacts to neighboring PTCs and spermatogenic cells. Nevertheless they appear to retain many properties that are characteristic for them in vivo like hormone responsiveness and expression of marker genes although in a time-dependent manner and on a lower level. 
Primary Sertoli cells develop large neutral lipid droplets after six days of culture. One of the vital functions of Sertoli cells is phagocytosis of degenerating and apoptotic germ cells and of germ cell material like the so called residual bodies29. It is known that residual bodies and cytoplasts from elongated spermatids adhere to isolated Sertoli cells and get phagocytosed30. As it is impossible to remove all germ cells during the isolation procedure we therefore hypothesize that contaminating germ cells can also adhere to Sertoli cells after plating and get phagocytosed. Since residual bodies contain small lipid droplets it is easily conceivable that Sertoli cells acquire large lipid droplets after extensive phagocytosis of adhering and damaged germ cells. There is also evidence that phagocytotic clearance of apoptic cells actively downregulates inflammatory and immune responses31 which could be important for establishing and maintaining the testis as an immune priviliged organ.
When isolated Sertoli cells are plated on top of a layer of primary peritubular cells both cell types survive longer and do form a basal lamina between them. Morphologically they are able to form structures reminiscent of seminiferous tubules indicating that both cell types have retained their capacity to properly interact7. Like freshly isolated primary Sertoli cells the frequently used mouse Sertoli cell line TM4 does respond to FSH with increased production of cAMP18, but contrary to primary cells it is not able to produce androgen binding protein32. Moreover it has been shown that the mouse Sertoli cell line 1 lacks the immunoprotective properties associated with primary Sertoli cells12. 
Similarly the rat Sertoli cell line SCIT-C8 expresses the epithelial markers transferrin, clusterin and stem cell factor but has lost androgen receptor expression and androgen responsiveness whereas it has aquired expression of some mesenchymal markers like fibronectin and entactin-133. Therefore, primary Sertoli cells, although expensive and tedious to isolate, appear to retain significantly more qualitative in vivo properties during cell culture than cell lines do. 
Future applications of primary Sertoli cells will focus on immuno-protection of co-cultured or co-transplanted cells, on experiments studying cell-cell interactions, and on investigating the immune privilege of testis.

Testes produce male gametes and sexual hormones, i.e., androgens. The organ is composed of two compartments. In the interstitial compartment, that represents about 10-12% of total testicular volume1, steroidogenesis takes place within the Leydig cells. The tubular compartment represents about 60-80% of the testicular volume1 and contains germ cells and two types of somatic cells - peritubular cells and Sertoli cells. The testis is divided by connective tissue septa into 250-300 lobules, each containing 1-3 highly convoluted seminiferous tubules. These tubules are enclosed by a basal membrane, a sheet of collagen, and a circumferential layer of peritubular cells (Figure 1A).
The germinal epithelium is located on the luminal side of the basal membrane. Sertoli cells are large elongated cells that span the whole germinal epithelium from the basal membrane to the lumen. They are strongly attached to the basal membrane and form a continuous cellular sheet through basolateral organized tight junctions that occludes the germinal epithelium from the interstitium and represents the blood-testis barrier. Sertoli cells have prominent cytoplasmic projections and ramifications that enable them to get into tight morphological and functional contact with a species-specific but constant number of germ cells. Diploid germinal stem cells proliferate and differentiate into spermatogonia.
During meiosis I short-lived tetraploid spermatocytes are generated that develop further into four haploid spermatids during meiosis II. All germ cells are interconnected by cytoplasmic bridges so that they form a cellular net. The principal event during maturation of spermatids is the extrusion of large parts of the cytoplasm, forming residual bodies, in a process called spermiogenesis. Residual bodies are phagocytosed by Sertoli cells. Late spermatids are then released into the tubular lumen and transported into the epididymis for further maturation. Sertoli cells and germ cells appear to mutually coordinate spermatogenesis topographically and functionally.
Preparation of individual testicular cell types started almost a century ago when small testicular pieces were cultivated and cell types identified by microscopy2. By careful dissection of the tubules after opening the tunica albuginea using fine-tipped forceps it was later possible to separate tubular and interstitial compartments3. In 1975 Welsh and Wiebe introduced a collagenase treatment in order to free the tubules from adhering interstitial tissue and a pancreatin treatment for removal of the outer peritubular cell layer4. From early on young immature rats (around 20 days old) had been used in which the Sertoli cells comprise a large fraction of the tubular cell population because spermatogenesis has not begun yet. At this age rat Sertoli cells cease to divide, and tight junctions between neighboring cells form so that the blood-testis barrier is established5.
Independent of Welsh and Wiebe Dorrington et al. introduced a combination of trypsin and deoxyribonuclease followed by soybeen trypsin inhibitor and collagenase treatment that was published in the same year6. Both groups also used mechanical force (drawing the digested tubular fragments repeatedly through a syringe needle or a Pasteur pipette respectively) in order to produce a homogeneous cell suspension for plating that contains approximately 70% Sertoli cells. After 3 days in culture, using a medium that is serum-free, the percentage of Sertoli cells increases to approximately 90%. This could be largely attributed to the death of contaminating germ cells. Residual peritubular cells (PTCs), however, stay firmly attached via their extracellular matrix. PTCs, but not Sertoli cells, are known to produce fibronectin that can serve as a marker protein for estimating contamination with PTCs. Therefore, Tung et al. reasoned that an additional hyaluronidase treatment might improve the purity of the Sertoli cell fraction7. Indeed they could show that an additional treatment was able to reduce contamination by PTCs approximately 20-fold, which becomes particularly evident when in comparison a serum-containing medium is used for cultivating purified Sertoli cells. From then on the improved procedure of Tung et al. became the prevailing protocol and was extensively used by other major groups in the field8 (Figure 1B).
During collagenase treatment the majority of PTCs are released and can be isolated in parallel to Sertoli cells. Whereas the PTCs vigorously proliferate and do not respond to follicle-stimulating hormone (FSH), Sertoli cells do not undergo mitosis anymore and respond to FSH by characteristic morphological changes and an increase in cyclic adenosine monophosphate (cAMP) concentration9. Very similar enzymatic digestion protocols can be used for the isolation of primary Sertoli cells from other animals like man10, mouse11,12, Siberian hamster13 or yak14. For the removal of large amounts of contaminating germ cells a hypotonic shock can be employed at the end of the isolation procedure15. This allows also the efficient isolation of Sertoli cells from adult rat testes16. An enriched Sertoli cell suspension can also be separated from germ cells by plating the suspension on lectin dishes coated with Datura Stramonium agglutinin17. Only Sertoli cells and a few residual PTCs adhere to the lectin plates.
Primary Sertoli cell cultures, mainly from the rat, have been used initially for investigating responsiveness to hormones or establishing cell lines like the mouse Sertoli cell line TM418. This cell line has been investigated in more than a hundred studies until today. In a translational approach Sertoli cells have been utilized for immuno-protection of co-cultured cells and tissues like in the co-transplantation of xeno- or allogeneic pancreatic islets for long-term graft survival without systemic immunosuppression19. Isolated Sertoli cells have been also used in co-culture experiments for studying epithelial-mesenchymal (Sertoli cells - PTCc) and somatic-germ cell (Sertoli cells - germ cells) interactions20,21. Recently primary Sertoli cells have been employed for investigating the expression of Toll-like receptors and the secretion of proinflammatory cytokines as well as the signal transduction cascades leading to cytokine expression following infection with nonpathogenic and uropathogenic E. coli22. Other recent investigations were using Sertoli cells for studying testicular immune privilege23 and demonstrated that testosterone pre-treatment suppresses the LPS-induced inflammatory response24.

<table><tbody><tr><td>Actin (smooth muscle) antibody clone 1A4</td><td>Dako</td><td>M0851</td><td>Monoclonal mouse anti human antibody.&lt;br/&gt; Use 1:100 dilution for immunofluorescence.</td></tr><tr><td>Albumin bovine fraction V&lt;br/&gt; Standard grade, lyophilized</td><td>Serva</td><td>11930.04</td><td>Filter (0.45 &amp;micro;m) BSA solutions used for immunofluorescence.</td></tr><tr><td>Corning Cell Strainers 70&amp;nbsp;&amp;micro;m</td><td>Corning</td><td>431751</td><td>White color</td></tr><tr><td>Collagenase A from&lt;em&gt; Clostridium histolyticum&lt;/em&gt;</td><td>Roche</td><td>10103586001</td><td></td></tr><tr><td>DAPI mountant ProLong Gold Antifade</td><td>Life technologies</td><td>P-36931</td><td></td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G8644</td><td>100 g/L</td></tr><tr><td>Dulbecco&amp;acute;s PBS without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Electron microscope</td><td>Zeiss</td><td>EM 109S</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axioplan 2</td><td></td></tr><tr><td>Hyaluronidase from bovine testis</td><td>Sigma</td><td>H3506</td><td></td></tr><tr><td>Inverted light microscope</td><td>Olympus</td><td>CKX41</td><td>Routine cell culture microscope</td></tr><tr><td>Mouse IgG / IgM (H+L) polyclonal&lt;br/&gt; secondary Antibody&amp;nbsp;</td><td>Life technologies</td><td>A-10684</td><td>Alexa Fluor 488 conjugate</td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625</td><td>Has replaced Sudan III and Sudan IV because of brighter color.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Penicillin (5,000&amp;nbsp;U/ml)/&lt;br/&gt; Streptomycin (5,000 &amp;micro;g/ml)</td><td>Gibco</td><td>15070-063</td><td>100x solution</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>21875-034</td><td>Contains 300 mg/L L-glutamine</td></tr><tr><td>Trypsin from porcine pancreas</td><td>Sigma</td><td>T5266</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Trypsin inhibitor from soybean</td><td>Sigma</td><td>T6522</td><td>The Sigma product is considerably cheaper than the previously used&lt;br/&gt; BPTI (Aprotinin) from Roche.</td></tr><tr><td>Vimentin antibody (V9)</td><td>Sigma</td><td>V6630</td><td>Monoclonal mouse anti pig vimentin antibody.&amp;nbsp; Use 1:100 dilution for immunofluorescence.</td></tr><tr><td>Wistar WU rats</td><td>Charles River</td><td></td><td>Should be 19 days old on day of experiment.</td></tr></tbody></table>

isolation of sertoli cells and peritubular cells from rat testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

The described procedure allows isolation of approximately 12 x 107 Sertoli cells from 10 rat testes. 3 x 106 cells are plated per well on a 6-well plate so that six to seven 6-well plates are available for experiments on day 7. The Trypsin-DNase I digestion is the most critical step during Sertoli cell isolation. If digestion at this point is advancing too far, the ratio of germ cells/Sertoli cells will disproportionally increase up to the end. During day 3-6 of culture it is important to carefully wash away as many non-adhering or loosely attached germ cells as possible. As an alternative to extensive washings over 4 days germ cells can be removed by hypotonic treatment on day 327. An advantage of this method is that the time of Sertoli cell&#160;culture is kept to a minimum so that Sertoli cell-specific functions are potentially better retained than after a prolonged period of culture.
Although the Hyaluronidase treatment serves to remove most residual peritubular cells, a few cells will survive the treatment. Purity of the isolated Sertoli cells is &#62;95% as can be demonstrated by vimentin immunolabeling (Figure 5B). Because peritubular cells have a strong proliferative potential, Sertoli cell culture has to be performed in the absence of serum. In the presence of 10% serum PTCs would constitute approximately 20% of all cells after 6 days of culture whereas its fraction can be expected to be below 1% in the absence of serum7. Virtually all isolated Sertoli cells are viable as judged by Trypan blue staining (not shown). Around day 7 of culture the isolated Sertoli cells are least contaminated with germ cells and peritubular cells (Figure 4), so that experiments should be performed around this day if possible. For good reproducibility it is important to repeat experiments always on the same day of culture. If transfections are planned they should be performed on day 4 or 5, and cell extracts should be made on day 5 or 6.
Over at least two weeks in serum-free conditions Sertoli cells are responsive to FSH and produce more androgen-binding protein, plasminogen activator and transferrin upon FSH treatment. For longer culture a feeder layer of peritubular cells or serum is required. After four weeks cultures deteriorate, and cells detach from the substratum7.
On day 6 of culture most Sertoli cells have acquired lipid droplets. In the majority of cells a single large vacuole has formed (Figure 5C and 5D). The neutral lipid content can be easily visualized by Oil Red O staining (Figure 4F).
In addition to Sertoli cells the removed PTCs can be cultured as well (Figure 4A and 4B). PTCs from 10 testes yield five T75 flasks that can be expected to form a confluent layer after 2 days of culture. Like isolated Sertoli cells freshly isolated PTCs are contaminated by germ cells that can be largely removed by passaging. Flasks are split 1:2 by standard trypsinization so that 10 flasks will be confluent on day 5 after isolation. Purity of isolated PTCs is &#62;95% and can be confirmed using &#945;-smooth muscle actin immunolabeling (Figure 5A). From this day onwards PTCs can be used for experiments. One flask is sufficient for approximately one 6-well plate, and wells should be confluent the next day. Cells can be passaged many times, but experiments should be performed always at the same passage in order to make sure that cells behave in a reproducible manner.
<img alt="Figure 1" src="/files/ftp_upload/53389/53389fig1.jpg" /> 
Figure 1.&#160;Morphology of the testis and workflow of the procedure. (A) A sector of a seminiferous tubule with adjacent interstitial tissue is schematically shown. Seminiferous tubules are enclosed by a basal membrane (BM) and a circumferential layer of myoid peritubular cells (PTC). The germinal epithelium is located on the luminal side of the basal membrane. Sertoli cells (SC), spanning the whole germinal epithelium are strongly attached to the BM and interconnected via basolateral positioned occluding junctions representing the blood-testis barrier (BTB). Their irregular nucleus (N) shows one or more fissure-like indentations and contains a nucleolus. Germ cells (GC) are in intimate contact with SCs at all stages of their development. The interstitial compartment between the tubules contains Leydig cells (LC) and immune cells such as macrophages (M&#934;) as well as blood vessels (BV). Figure adapted from Fijak and Meinhardt28. (B) Workflow of the procedure; color coding is like in (A). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53389/53389fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53389/53389fig2.jpg" /> 
Figure 2. Testes from Wistar rats are excised and decapsulated. (A) The peritoneal cavity is opened through a longitudinal incision as described in the text. An asterisk (*) indicates the epidydimal fat pad. (B) Testes are removed by cutting the spermatic cord and (C) collected in a 50 ml conical tube containing 20 ml PBS. When all testes have been collected they are disinfected in 20 ml of 1% (w/v) iodine in ethanol. For removal of the iodine they are quickly washed twice with 25 ml PBS each. (D) Testes after first PBS wash. (E) Testes are transferred to a Petri dish (on the right), and seminiferous tubules are squeezed out of the opened tunica albuginea by means of closed scissor blades (on the left) as described in the text. (F) The decapsulated testes still form a compact testicle-shaped mass with single tubules protruding. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53389/53389fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53389/53389fig3.jpg" /> 
Figure 3. Isolation of Sertoli cells. (A) After removal of interstitial cells by Trypsin-DNase I digestion and washing 9x with PBS tubules become mobilized and look clean. (B) During collagenase-hyaluronidase-DNase I treatment peritubular cells from the outer layer and germ cells are released. The tubules get shortened and obtain a rough appearance. (C) Tubular fragments after washing 4x with PBS. (D) Hyaluronidase-DNase I treatment releases residual peritubular cells as well as germ cells. Tubules get further shortened, and tubular aggregates form. (E) Tubular aggregates after washing 4x with PBS. (F) Single Sertoli cells are produced by passing the aggregates 10x through an 18G needle. Scale bars in (A-E) = 200 &#181;m, in (F) = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53389/53389fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53389/53389fig4.jpg" /> 
Figure 4. Culture of peritubular cells (A-B) and Sertoli cells (C-F) and removal of contaminating germ cells. (A) Resuspended PTCs from step 4.5 were seeded immediately and photographed the next day. (B) After splitting on day 3 contamination with germ cells is reduced. (C) On day 4 Sertoli cells show a typical cobblestone-like pattern. Floating and adhering germ cells are visible before washing with PBS. (D) The same well after washing 3x with PBS. The number of contaminating germ cells is reduced. Washing thrice with PBS is repeated on day 5 and 6. (E) On day 6 of culture virtually all germ cells have been removed. Sertoli cells have formed a unique looking cell layer, and most cells have acquired a single large vacuole. (F) Oil red O staining shows that these vacuoles are lipid droplets containing largely neutral lipids. Scale bars in (A-D) = 200 &#181;m, in (E) = 50 &#181;m and in (F) = 25 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53389/53389fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53389/53389fig5.jpg" /> 
Figure 5. Immunofluorescence staining. Staining of purified primary peritubular cells with actin (smooth muscle) antibody (A) and of Sertoli cells with vimentin antibody (B). No contaminating cells are visible in both fields of view. Scale bar in (A) and (B) = 10 &#181;m. (C) and (D) Electron microscopic pictures of Sertoli cells after 4 days in culture. (C) Note the close contact of adjacent cells (arrowheads) that leads to the cobblestone-like pattern as seen in Figure 4C. A single lipid droplet (L) is observed in the cytoplasm of each cell. (D) Most abundant organelles are mitochondria (M) and the Golgi apparatus (G). The nucleus (N) contains a nucleolus (Nc) and shows the typical fissure-like indentation. Scale bar in (C) = 1 &#181;m and in (D) = 0.25 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53389/53389fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

isolation-of-sertoli-cells-and-peritubular-cells-from-rat-testes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

22.4K Views.  Justus-Liebig-Universität Gießen.  Primaire Sertoli cellen nodig zijn voor het bestuderen van testis-immune privilege, signaaltransductie tijdens ontsteking of infectie, en het gebruik van hun immunobeschermende eigenschappen. Hier beschrijven we een enzym-gebaseerd protocol voor de isolatie van zeer zuivere primaire Sertoli cellen en peritubulaire cellen van ratten testes. 

Video: Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Developmental Biology

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 ...

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.

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.

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

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

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 ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

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

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

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