TY - JOUR
AU - O’Bryan, Moira, K
AB - Abstract The purification of individual male germ cell populations is integral for the molecular and biochemical characterisation of specific spermatogenic phases. Although a number of more contemporary techniques have been developed, velocity sedimentation using the STAPUT method remains as a gold standard for this purpose. The gentle nature of the technique, wherein germ cell subpopulations are separated by sedimentation at unit gravity, results in the isolation of viable and high-purity cells. We provide an updated and simplified step-by-step version of the STAPUT protocol for the purification of mouse male germ cells. As per the original method, the protocol described herein allows for the purification of mouse spermatocyte and round spermatids, however it also allows for successful purification of elongating, and elongated spermatid populations, and is optimised for the preservation of cellular ultrastructure. This method yields sufficient numbers of high-purity cells from one adult mouse for RNA or protein extraction or for immunolocalisation studies. spermatogenesis, spermatocyte, spermatid, male fertility, male infertility Introduction During mammalian spermatogenesis, germ cells progress through multiple distinct developmental phases (spermatogonia, spermatocytes and spermatids) to ultimately form functional spermatozoa. Each germ cell type is unique in its biochemistry and morphology. Purification of specific germ cell populations is thus a fundamental technique in spermatogenesis research. It allows the assessment of differential gene and/or protein expression relative to developmental phase, validation of gene knockout models specific to certain germ cell types, and the normalisation of expression or biochemical analyses wherein a particular germ cell type is depleted or partially depleted. Moreover, the isolation of high-quality germ cells, wherein the ultrastructure is pristinely preserved, is an essential technique for studying the unique 3D architecture of germ cell division and differentiation processes, and is of increasing importance with the increasing sensitivity and resolution of many analysis techniques. Indeed, in many instances, the limiting factor in understanding the precise subcellular localisation of targets in isolated germ cells is the quality of the cells, as opposed to microscope resolution. Within the testis, germ cells are found in the seminiferous tubules, where they are surrounded by and embedded within the cytoplasmic extensions of Sertoli cells. In addition, the testis harbours peritubular and multiple interstitial somatic cell populations, including Leydig, myoepithelial, and immune cell populations. Isolation of germ cells requires the mechanical and enzymatic digestion of testicular tissue to obtain a single cell suspension, followed by a method to purify the desired germ cell populations and remove contaminant cells. STAPUT velocity sedimentation was first developed by Miller and Phillips in 1969 (Miller and Phillips, 1969) for the fractionation of cells based on size and density and was subsequently adapted for the purification of various murine male germ cell types in the 1970s (Meistrich, 1972; Meistrich et al., 1973; Romrell et al., 1976; Bellve et al., 1977). In essence, the technique takes advantage of the fact that the different cell populations that comprise a tissue typically display distinct size and density characteristics, and as such, when loaded onto a fluid, gradient exhibit different sedimentation velocities at unit gravity (Miller and Phillips, 1969). Due to the dramatic physical differences between the various male germ cell types, this technique is readily applicable to the purification of spermatogenic cells. Despite there being a number of more contemporary techniques, STAPUT still remains the most appropriate method in many contexts. This is in large part due to the gentle nature of the technique that results in a high yield of viable high-quality germ cells. Fluorescence activated cell sorting (FACS) has been successfully employed to isolate a broad array of germ cell types at high purity (Mays-Hoopes et al., 1995; Bastos et al., 2005; Getun et al., 2011,), and similarly, magnetic-activated cell sorting can be used to purify spermatogonial populations (Schönfeldt et al., 1999); however, both methods involve greater mechanical manipulation and processing of the cells, which can affect downstream cell viability and ultrastructure integrity. As such, both methods typically have a low yield, often necessitating the use of multiple mice, and moreover, FACs requires expensive, specialised equipment. The other most commonly used technique, centrifugal elutriation, has similar drawbacks, with cells being subjected to centrifugal forces of up to 13 000 × g (Meistrich and Trostle, 1975) raising concerns about the ultrastructural quality of the isolated cells. The centrifugal elutriation method also requires the use of multiple animals per preparation, which can be particularly challenging when using age-matched genetically modified mouse models. Since STAPUT was first used for mouse germ cell purification, a number of variations have been developed. In one of the earliest successful adaptations, Romrell and colleagues (Romrell et al., 1976) used the STAPUT apparatus to isolate mouse pachytene spermatocytes, residual bodies and round spermatids with high purity (>85%) and viability (98%); however, they struggled to purify elongating spermatids (30%) and recovered negligible quantities of spermatogonia. The protocol was subsequently adapted for the purification of prepubertal Sertoli cells and germ cells (Bellve et al., 1977). While various cell types were isolated with excellent purity [Sertoli cells (>99%), spermatogonia (primitive A (91%), A (91%) and B (97%)) preleptotene spermatocytes (93%), leptotene/zygotene spermatocytes (52%) and pachytene spermatocytes (89%)], age-restricted mice had to be used for the optimal purification of each cell type. This method is thus of limited utility in situations where differences in gene expression are suspected between the first and subsequent waves of spermatogenesis, or expression is dependent on cell–cell interactions. In 1992, the method was further adapted with the advent of the ‘mini-STAPUT’ apparatus, which unlike previous iterations allowed the purification of germ cells from as few as one adult mouse, or 5–20 neonatal and prepubertal mice (McCarrey et al., 1992). Similar to previous methods, the protocol was successful in isolating spermatocytes (94% pure), round spermatids (99% pure) and residual bodies (96%) from adult testes, in addition to spermatogonia from prepubertal and spermatocytes from pubertal testes with high purity (>85%). However, again successful isolation of elongating and elongated spermatids from the testis was not described. More recently, Bryant and colleagues (Bryant et al., 2013) published an updated STAPUT protocol for the isolation of fractions containing: somatic and meiotic cells; round spermatids; and importantly elongating spermatids for cell culture. Unlike previous methods, this method is particularly useful due to its step-by-step format, allowing it to be more easily reproduced. However, it is of restricted utility for other applications outside cell culture involving experimental or genetic interventions, as it requires 11 mice per experiment. Similarly, the precise characteristics and purity of each fraction were not reported, and the presence of somatic and meiotic cells in the same fraction would confound analyses of meiotic gene and protein expression. Herein, we provide a simplified highly reproducible, step-by-step STAPUT velocity sedimentation method for the purification of mouse spermatocyte and spermatid populations, with a focus on the preservation of the complex cytoskeletal elements characteristic of these cells. The method described builds upon previous STAPUT adaptations, requiring as little as one adult male mouse for the isolation of sufficient amounts of cells for RNA or protein extraction or for immunolocalisation studies, in addition to allowing successful purification of elongating and elongated spermatids. Importantly, we show that this method is successful in isolating germ cell populations of high purity [spermatocytes (81%), round spermatids (76%) and elongating and elongated spermatids (>85%)] and thus allows for reliable protein and RNA expression comparisons between the various germ cell types. Materials and Methods Reagents Bovine serum albumin (BSA) (Sigma, A7906) Collagenase Type IV (Sigma, C5138) DNase I (Sigma, DN25) Dulbecco’s modified Eagle’s medium (DMEM), high glucose (GIBCO, 11965084) 2.5% Trypsin (GIBCO, 15090046) Optional Egtazic acid (EGTA) 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) MgCI2 Paraformaldehyde (PFA) 1X Phosphate buffered saline (PBS) 1,4-Piperazinediethanesulfonic acid (PIPES) Equipment Benchtop centrifuge capable of 100 × g and compatible with 15- and 150-ml centrifuge tubes Centrifuge tubes, 15 and 50 ml Circular spirit level Conical borosilicate 3.3250-ml separating funnel, ST/NS 29/32 joint, 4.0-mm bore (Lenz Bistabil, 4.0041.49) Fine forceps (e.g. Dumont #5, 11 252-20) Hoefer SG100 Gradient Maker Magnetic stirrer Magnetic stirring flea, small Masking tape Microcentrifuge Microcentrifuge tubes, 1.5 ml Nylon cell strainer, 70 μM Parafilm Petri dishes, 35 × 10 mm Plastic transfer pipettes, 3 ml Retort ring with bosshead, 100 mm Retort stand Rolling mixer Shaking incubator (or shaker in a 37°C room) Surgical scissors (small and large) Syringes, 50 ml Syringe filters, 0.2 μm Optional: Electrostatically charged adhesion slides (e.g. SuperFrost Plus slides (Menzel Gläser, 4951PLUS4)) Optional: Hydrophobic PAP pen (e.g. Abcam, ab2601) Reagent setup Collagenase Stock. Prepare 5-mg/ml collagenase Type IV in DMEM. Dissolve well, aliquot and store at −20°C. Use a fresh aliquot per germ cell purification. DNase Stock. Prepare 5-mg/ml DNase I in PBS. Dissolve well, aliquot and store at −20°C. Use a fresh aliquot per germ cell purification. Collagenase working solution. For each animal, add 500 μl of the 5-mg/ml collagenase stock solution and 500 μl of the 5-mg/ml DNase I stock to 4 ml of DMEM for a final concentration of 0.5-mg/ml collagenase/0.5-mg/ml DNase I. Mix solution thoroughly, and filter sterilize through a 0.2-μm syringe filter. Pre-warm solution to 37°C prior to use. Trypsin working solution. For each animal, add 500 μl of the 2.5% trypsin and 500 μl of the 5-mg/ml DNase I stock to 4 ml of DMEM for a final concentration of 0.25% trypsin/0.5-mg/ml DNase I. Mix solution thoroughly, and filter sterilize through a 0.2-μM syringe filter. Pre-warm solution to 37°C prior to use. 0.5% BSA solution. For each animal, prepare 5 ml of 0.5% BSA in DMEM. Dissolve by placing the parafilm sealed tube on a rolling mixer. Filter sterilize solution through a 0.2-μM syringe filter. Gradient solutions. For one gradient, prepare a 50 ml solution of 2% BSA in DMEM and a 50-ml solution of 4% BSA in DMEM. Dissolve both by placing parafilm-sealed tubes on a rolling mixer and filter sterilize both solutions through a 0.2-μM syringe filter. Optional 2X PHEM Buffer Stock. For the purification of cells that will ultimately undergo a cytoskeletal analysis, prepare 120-mM PIPES/50-mM HEPES/4-mM MgCI2/20-mM EGTA in ultrapure water. Bring pH to 6.9 with the addition of NaOH and dissolve by stirring. Store at 4°C or aliquot and store at −20°C. 1X PHEM Buffer Working Solution. Dilute 1:1 2X PHEM buffer with ultrapure water and filter sterilize through a 0.2-μM syringe filter. 4% PFA. Prepare 4% PFA in buffer of choice (e.g. 1X PBS or 1X PHEM buffer) by heating to 55–60°C in a fume hood. Add 1-M NaOH dropwise until solution clears. Once dissolved, cool, adjust to pH 7.4 for PFA prepared in 1X PBS or to pH 6.9 for PFA prepared in 1X PHEM. Filter sterilize through a 0.2-μM syringe filter. For best results, prepare a new preparation of PFA for each germ cell purification. Procedure Preparation of the STAPUT gradient. Assemble the separating funnel as per Fig. 1, ensuring the stopcock (tap) at the bottom is in the closed position. Clamp the retort ring onto the retort stand using the bosshead and place the separating funnel into the ring. Use the circular spirit level to ensure the separating funnel is level and to secure the funnel in this position with tape (Fig. 1A). Key tip: To prevent the gradient being disrupted, have the apparatus setup on a vibration-free bench. Assemble the gradient maker, ensuring the valves are in the closed position (Fig. 1B). Attach the gradient maker tubing, and at the distal end of the tube, insert a sterile, 10-μl pipette tip. Place a small magnetic stirring flea in the right-hand side (RHS) chamber. Load 50 ml of 4% BSA in DMEM into the RHS chamber of the gradient maker, and 50 ml of 2% BSA in DMEM into the left-hand chamber. Place the gradient maker onto a magnetic stirring plate, positioned above the separation funnel (Fig. 1B) and switch the magnetic stirrer on. Insert the 10-μl pipette tip, attached to the gradient tubing, into the separating funnel, and secure the pipette tip in place so that it opens onto the inner surface of the separating funnel (Fig. 1C). Key tip: So as not to mix the gradient as it forms, it is critical that the pipette is secured in this way so that fluid flows gently down the inner surface of the funnel. Partially open the RHS valve, approximately a quarter or half turn, allowing the 4% BSA/DMEM solution to slowly flow into the tubing. Once the fluid is half way down the tubing, fully open (approximately a full turn) the RHS valve allowing the 2% BSA/DMEM solution to flow into the RHS chamber. Check to ensure no bubbles impede the flow of the 2% BSA/DMEM solution into the RHS chamber. Once the gradient is loaded into the separation funnel, carefully remove the gradient maker tubing from the separation funnel, taking care not to disturb the gradient. Note: If samples are being collected for RNA extraction, take care to sterilize equipment appropriately prior to use. Figure 1 Open in new tabDownload slide Setup of the STAPUT velocity sedimentation device. (A) The Lenz Bistabil separating funnel is held by a retort stand and secured in place by masking tape. (B) The Hoefer SG100 Gradient Maker is placed on a magnetic stirrer and a small magnetic stirring flea is placed in the RHS chamber. (C) To make the gradient, a 10-μl pipette tip is placed in the end of the tubing connected to the gradient maker. The 10-μl pipette tip is then secured such that it opens against the inner surface of the separation funnel. (D) To load the testicular single cell suspension onto the gradient, a 3-ml plastic transfer pipette is used. The end of the pipette is held against the inside surface of the flask, slightly above the gradient, ensuring that the suspension moves down the inner surface of the flask. Figure 1 Open in new tabDownload slide Setup of the STAPUT velocity sedimentation device. (A) The Lenz Bistabil separating funnel is held by a retort stand and secured in place by masking tape. (B) The Hoefer SG100 Gradient Maker is placed on a magnetic stirrer and a small magnetic stirring flea is placed in the RHS chamber. (C) To make the gradient, a 10-μl pipette tip is placed in the end of the tubing connected to the gradient maker. The 10-μl pipette tip is then secured such that it opens against the inner surface of the separation funnel. (D) To load the testicular single cell suspension onto the gradient, a 3-ml plastic transfer pipette is used. The end of the pipette is held against the inside surface of the flask, slightly above the gradient, ensuring that the suspension moves down the inner surface of the flask. Ethics statement All animals and procedures used in the development of this protocol were approved by the Monash Animal Experimentation Ethics Committee and conducted in accordance with Australian NHMRC Guidelines on Ethics in Animal Experimentation. Preparation of testicular single cell suspension. To collect testis, humanely kill the mouse (an adult mouse, 7 weeks or older will give the best yield). Wet the abdomen with 70% ethanol and dissect out the testes. Carefully remove any connective tissue associated with the tunica albuginea (testis capsule). Immediately transfer the testes to DMEM. Key tip: For optimum preservation of certain subcellular structures, such as microtubules which are prone to cold depolymerisation at temperatures of 4°C and lower (Dustin, 2012), the DMEM must not be cold. It can be room temperature or pre-warmed (37°C). Add 1 mL of pre-warmed (37°C) collagenase solution to a 35 × 10 mm Petri dish and transfer both testes into the dish. Using two sets of fine forceps, gently tear open the tunica albuginea to release the seminiferous tubules. Discard the tunica albuginea, and use the forceps to gently tease apart the tubules. Using a wide bore 1-ml pipette tip, transfer the tubules and collagenase solution to a sterile, 15-ml centrifuge tube. Rinse the Petri disk with an additional 4 ml of collagenase solution to collect residual tubules and add it to the 15-ml tube. Secure the centrifuge tube lid and seal with parafilm. Note: If wide bore pipette tips are not available, sterile scissors can be used to remove the end of a 1-ml pipette. Incubate the tube at 37°C for 20 minutes with gentle shaking at 100 rpm. Key tip: For best results, place the tube lying horizontal during shaking, using masking tape to fasten in place. Pellet the cells by centrifugation at 100 × g for 10 minutes at room temperature. Remove the supernatant and resuspend the cells in 1 ml of pre-warmed (37°C) trypsin solution using gentle pipette action. Once resuspended, add an additional 4 ml of trypsin solution. Incubate the tube at 37°C for a maximum of 30 minutes with shaking at 100 rpm. Again, for best results, place the tube lying horizontal during shaking, using masking tape to fasten in place. Note: For juvenile testis, reduce this incubation time to 15–20 minutes depending on the size of the testis. Inactivate trypsin by adding 5 ml of 0.5% BSA solution, prior to pelleting the cells by centrifugation at 100 × g for 10 minutes at room temperature. Note: After centrifugation, the pellet can be quite loose; therefore, the supernatant should be very slowly removed and up to 1 ml can be left. If the pellet is too loose, centrifuge the samples once more before removing the supernatant. Resuspend the pellet in 1 ml of room temperature or pre-warmed (37°C) DMEM using a gentle pipette action. Once resuspended, add additional DMEM to make the cell suspension up to a total volume of 5 ml. Key tip: At this step, it is important to fully resuspend the pellet, dispersing clumps by gentle pipette action. Ensuring the solution is predominantly a single cell suspension will minimise loss during filtering and maximise the purity of the fractions. Pass the resuspended pellet through a 70-μM nylon cell strainer into a sterile, 50-ml centrifuge tube. Add additional DMEM to make the cell suspension up to a final volume of 10 ml. Key tip: For one STAPUT gradient, testis cell suspensions from two or more adult mice can be combined if a higher yield is desired. In this instance, instead of supplementing the sample with additional DMEM to make the total volume up to 10 ml, pass the 5-ml testes cell suspension from the second male through a fresh 70-μM nylon cell strainer, but into the same 50-ml centrifuge tube to ensure the final volume is still 10 ml. For more than two males, adjust resuspension volumes accordingly to again ensure the final volume is 10 ml. Velocity sedimentation of testicular single cell suspension. To load the testicular single cell suspension onto the STAPUT gradient, use a 3-ml plastic transfer pipette. Hold the pipette to the inside surface of the flask, slightly above the gradient (Fig. 1D), ensuring that the suspension is released slowly and moves down the inner surface of the flask, so that it does not billow into the gradient but instead forms a separate phase on the top of the gradient. Key tip: It should take 5–10 minutes to load the cell suspension onto the gradient. Ensuring the suspension loads on the top of the gradient as described above is essential to ensure the correct fractionation of the cells. Once the cell suspension has been loaded onto the gradient, allow it to undergo velocity sedimentation for 3.5 hours at room temperature. Once sedimentation has completed, collect fractions 1–7 in 15-mL sterile centrifuge tubes. In total seven fractions are collected, each of which is 10 mL, with the exception of fraction 4 which is 15 mL. Fraction 2 contains spermatocytes; fraction 5, round spermatids; fraction 6, elongating/elongated spermatids; and fraction 7, elongated spermatids. Fractions should be collected using a low elution speed so that the fluid drips into the tubes. Collection should also be continuous, i.e. once the specified amount is reached for the fraction, immediately switch to the next tube without turning the tap off. Key tip: The fluid speed should be just slow enough that the fluid is not forming a ‘stream’, approximately 3 drops per second. Pellet the desired fractions by centrifugation at 100 × g for 10 minutes at room temperature. Remove most of the supernatant leaving approximately 500-μl remaining. Resuspend the pellet in the remaining supernatant and transfer to a sterile, 1.5-ml microcentrifuge tube. Make up to 1.5 ml with PBS or assay-specific wash buffer. Key tip: for preservation of certain subcellular structures, specialised buffers can be used in place of PBS for the wash steps. For example, PHEM buffer is often used to provide a better preservation of microtubule networks. Microcentrifuge the suspension at 100 × g for 10 minutes at room temperature; remove 1 ml of supernatant and resuspend the pellet. Make up to 1.5 ml with PBS or assay-specific wash buffer. Repeat this step and either proceed with preparing the isolated cells for immunofluorescence labelling or for protein or RNA extraction. Figure 2 Open in new tabDownload slide Purity of the germ cells fractions obtained via STAPUT velocity sedimentation. Representative images of the cellular composition of fractions 2, 5 and 6 obtained via STAPUT velocity sedimentation are shown in (A). Cells are stained with DAPI (blue) to visualise DNA and with lectin peanut agglutinin (PNA, green) to visualise the acrosome. The upper panel shows DAPI alone, and the lower panel shows DAPI and PNA together. Scale bars = 20 μM. (B) Cellular composition of fractions 2, 5, 6 and 7 obtained as determined by the assessment of nuclear morphology. Data represent mean ± SD. N = 3–6 germ cell isolations assessed for each fraction type. S’cytes = spermatocytes, R s’tids = round spermatids, E s’tids = elongating spermatids. (C) Western blot analysis of Sertoli cell (WT1, (Pelletier et al., 1991)), spermatocyte (SYCP3, (Dobson et al., 1994; Lammers et al., 1994)) and spermatid (CRISP2, (O'Bryan et al., 2001)) enriched proteins in STAPUT fractions 2, 5, 6 and 7 and whole testis lysate. Each germ cell isolation blot was achieved using protein obtained from a single adult mouse. (D) qPCR analysis of the relative expression of spermatocyte (Sycp3, (Dobson et al., 1994; Lammers et al., 1994)), spermatid (Crisp2, (O'Bryan et al., 2001)) and elongating/elongated spermatid (Gapdhs, (Welch et al., 1992)) enriched markers in STAPUT fractions 2, 5, 6 and 7. Data represent mean ± SD and expression is normalised to Ppia. N = 3 germ cell isolations, each obtained from a single adult mouse, assessed for each fraction type. F2 = fraction 2, F5 = fraction 5, F6 = fraction 6, F7 = fraction 6 and T = whole testis. WT1 = Wilms tumour protein 1, SYCP3/Sycp3 = synaptonemal complex 3, CRISP2/Crisp2 = cysteine rich secretory protein 2, Gapdhs = Gapdh spermatogenic, Ppia = peptidyl-prolyl cis-trans isomerase A. Figure 2 Open in new tabDownload slide Purity of the germ cells fractions obtained via STAPUT velocity sedimentation. Representative images of the cellular composition of fractions 2, 5 and 6 obtained via STAPUT velocity sedimentation are shown in (A). Cells are stained with DAPI (blue) to visualise DNA and with lectin peanut agglutinin (PNA, green) to visualise the acrosome. The upper panel shows DAPI alone, and the lower panel shows DAPI and PNA together. Scale bars = 20 μM. (B) Cellular composition of fractions 2, 5, 6 and 7 obtained as determined by the assessment of nuclear morphology. Data represent mean ± SD. N = 3–6 germ cell isolations assessed for each fraction type. S’cytes = spermatocytes, R s’tids = round spermatids, E s’tids = elongating spermatids. (C) Western blot analysis of Sertoli cell (WT1, (Pelletier et al., 1991)), spermatocyte (SYCP3, (Dobson et al., 1994; Lammers et al., 1994)) and spermatid (CRISP2, (O'Bryan et al., 2001)) enriched proteins in STAPUT fractions 2, 5, 6 and 7 and whole testis lysate. Each germ cell isolation blot was achieved using protein obtained from a single adult mouse. (D) qPCR analysis of the relative expression of spermatocyte (Sycp3, (Dobson et al., 1994; Lammers et al., 1994)), spermatid (Crisp2, (O'Bryan et al., 2001)) and elongating/elongated spermatid (Gapdhs, (Welch et al., 1992)) enriched markers in STAPUT fractions 2, 5, 6 and 7. Data represent mean ± SD and expression is normalised to Ppia. N = 3 germ cell isolations, each obtained from a single adult mouse, assessed for each fraction type. F2 = fraction 2, F5 = fraction 5, F6 = fraction 6, F7 = fraction 6 and T = whole testis. WT1 = Wilms tumour protein 1, SYCP3/Sycp3 = synaptonemal complex 3, CRISP2/Crisp2 = cysteine rich secretory protein 2, Gapdhs = Gapdh spermatogenic, Ppia = peptidyl-prolyl cis-trans isomerase A. Figure 3 Open in new tabDownload slide Cytoskeletal preservation in germ cells obtained via STAPUT velocity sedimentation. Representative images of the microtubule architecture in cells obtained via STAPUT velocity sedimentation as shown by (A) confocal microscopy and (B) super resolution (stimulated emission depletion) microscopy (B). In (A) and (B), α-Tubulin is immunolabelled as a marker of microtubules (green), allowing the visualisation of the cytoplasmic microtubule architecture of pachytene spermatocytes and round spermatids, and of the microtubule manchette of elongating spermatids. In (A), nuclei also are counterstained with DAPI (blue). Scale bars = 2 μM. Figure 3 Open in new tabDownload slide Cytoskeletal preservation in germ cells obtained via STAPUT velocity sedimentation. Representative images of the microtubule architecture in cells obtained via STAPUT velocity sedimentation as shown by (A) confocal microscopy and (B) super resolution (stimulated emission depletion) microscopy (B). In (A) and (B), α-Tubulin is immunolabelled as a marker of microtubules (green), allowing the visualisation of the cytoplasmic microtubule architecture of pachytene spermatocytes and round spermatids, and of the microtubule manchette of elongating spermatids. In (A), nuclei also are counterstained with DAPI (blue). Scale bars = 2 μM. Preparation of isolated cells for immunofluorescent labelling. After the final wash and microcentrifugation of the germ cells, remove as much of the supernatant as possible and resuspend the pellets in 1 ml of 4% PFA. Incubate for 10–15 minutes at room temperature. To remove the fixative, microcentrifuge at 100 × g for 10 minutes at room temperature. Remove the supernatant and resuspend the pellet in PBS or assay-specific wash buffer. Note: At this point, the pellet is usually harder to see, therefore be sure to carefully remove supernatant by pipetting from the opposing side of the tube to the pellet. Repeat the above microcentrifugation and wash step twice more. After the final wash and microcentrifugation of the germ cells, resuspend the pellet in 200–500 μl of PBS or desired buffer. Using a hydrophobic PAP pen, draw 0.5–1 cm diameter circles onto electrostatic-adhesion slides. Aliquot 30–50 μl droplets of the purified cells into each circle and allow cells to settle onto the slides overnight at 4°C in a humidified chamber. Wash off the residual fluid using PBS or assay-specific buffer and proceed with immunostaining, making sure not to allow the cells to dry out. Note: The cells can also be used for live cell staining. In this situation, we recommend that you proceed immediately to live cell staining after pelleting. Preparation of isolated cells for RNA or protein extraction. After the final wash and microcentrifugation of the germ cells, remove all supernatant. Cells pellets can then be frozen and stored at −80°C for later extraction. Anticipated results The successful use of the protocol described here yields viable, high-quality and highly pure fractions of spermatocytes (fraction 2: 81 ± 8% pure (mean ± SD), 3.2 ± 1 × 105 cells), round spermatids (fraction 5: 76 ± 3% pure, 1.5 ± 0.4 × 106 cells), elongating and elongated spermatids (fraction 6: 85 ± 9% pure, 1.2 ± 0.2 × 106 cells) and elongated spermatids (fraction 7: 93 ± 4% pure, 1.3 ± 0.4 × 106 cells) (Fig. 2), which retain an outstanding cellular architecture (Fig. 3) (purity determined as described in Supplementary data). As shown in Figure 2, from a single adult mouse, the protocol yields a sufficient number of cells to perform either RNA or protein expression analysis; indeed, we typically obtain ~50 μg of protein or 2 ± 0.5 × 103 ng of RNA per fraction. The purity of the fractions can be determined via the assessment of nuclear morphology (using a stain such as DAPI), either with or without the aid of specific markers, such as SYCP3 (synaptonemal complex protein 3; stains meiotic cells during prophase 1) and lectin peanut agglutinin (PNA: stains the acrosome, which first begins to form in step 2 spermatids, to allow staging of the spermatids) (Fig. 2A). The appearance of spermatocyte nuclei varies depending on meiotic phase (Russell et al., 1993). The vast majority of spermatocytes obtained in fraction 2 are in pachytene and are thus characterised by a large nucleus containing a patchy array of condensed chromatin (Fig. 2A). Conversely, in the earlier phases of preleptotene, leptotene and zygotene, spermatocytes exhibit nuclei wherein chromatin is largely concentrated at the nuclear periphery. Spermatocytes undergoing metaphase and anaphase are also present and easily identified due to their highly condensed chromatin, which is characteristically aligned along the metaphase plate before being segregated into two equal halves. Round spermatids are characterised by nuclei that contain a dense circular chromocenter (Russell et al., 1993) and, from step 2–3 onwards, possess a developing PNA-positive acrosome (Fig. 2A). Early elongating spermatids exhibit a teardrop-shaped nucleus, which as the spermatids progressively elongate, is remodelled into the characteristic falciform-shape of mouse sperm, and as spermatids elongate, the chromatin also becomes increasingly condensed (Fig. 2A) (Russell et al., 1993). As shown in Figure 2, the predominant contaminants in the various fractions are other germ cell types. For example, in the spermatocyte fraction, aggregates containing two or three round spermatids are sometimes observed due to the inadequate digestion of intercellular bridges or insufficient resuspension of the pellet after the final centrifugation step. If such contamination is excessive, see Table I for troubleshooting advice. Table I Trouble shooting guide for the optimised STAPUT method for the purification of mouse spermatocyte and spermatid populations. Problem Possible causes/solutions Low purity The presence of a high amount of contaminant cell types in the fractions is most likely due to the insufficient digestion of the testicular tissue or due to issues with the preparation or loading of the gradient. Insufficient digestion will be characterised by the presence of cellular aggregates, in particular round spermatid aggregates in fraction 2. To remedy this, ensure concentrations of enzyme solutions are correct, that they are pre-warmed to 37°C prior to use, and that sample tubes are laying on their side during shaking incubation to maintain consistent exposure to the solution and prevent clumping at the tube base. Issues with digestion could also be due to insufficient mechanical separation of the seminiferous tubules prior to incubation with enzymatic solutions. If contamination is present, despite enzymatic digestion being sufficient (i.e. contaminants are single cells, as opposed to aggregates), this is likely due to incorrect preparation or loading of the gradient, or imprecise collection of the fractions. Ensure that the gradient solutions are the correct concentrations, and that the setup of the gradient maker is correct. When loading the cell suspension onto the gradient, take extra care not to disrupt the gradient, instead slowly loading the suspension such that it forms a layer on top. Also ensure the gradient cannot be disturbed once it has been prepared and during the sedimentation process (e.g. by vibrations such as those from a vortex). Low cell yield Low cell yield may be due to improper sedimentation resulting in cells not being in the correct fractions or due to the insufficient digestion of the testicular tissue, and if this is suspected, refer to the advice for low purity above. If cells are in the correct fractions, but in low concentrations, this likely due to cell loss/degradation during either preparation of the suspension or during fixation and washing of the collected cells. Ensure buffers and fixatives are properly prepared and at the correct pH, centrifugation steps are performed with a maximum force of 100 × g and all handling of cells, such as resuspension by pipette action, is performed gently. Be careful not to leave cells in the trypsin solution for longer than necessary and add the 0.5% BSA solution immediately after incubation with trypsin solution to prevent the digestion of the cells. The length of trypsin incubation can also be decreased if necessary. If cells are being lost due to lack of adherence to slides, the use of specialist electrostatic-adhesion slides, such as the recommended SuperFrost Plus slides, should prevent this. Low viability or quality of the cells Low viability or poor quality of cellular ultrastructure may be due to issues in the preparation of buffers and fixatives and of the cell suspension, similar to those described for low cell yield (see above). Problem Possible causes/solutions Low purity The presence of a high amount of contaminant cell types in the fractions is most likely due to the insufficient digestion of the testicular tissue or due to issues with the preparation or loading of the gradient. Insufficient digestion will be characterised by the presence of cellular aggregates, in particular round spermatid aggregates in fraction 2. To remedy this, ensure concentrations of enzyme solutions are correct, that they are pre-warmed to 37°C prior to use, and that sample tubes are laying on their side during shaking incubation to maintain consistent exposure to the solution and prevent clumping at the tube base. Issues with digestion could also be due to insufficient mechanical separation of the seminiferous tubules prior to incubation with enzymatic solutions. If contamination is present, despite enzymatic digestion being sufficient (i.e. contaminants are single cells, as opposed to aggregates), this is likely due to incorrect preparation or loading of the gradient, or imprecise collection of the fractions. Ensure that the gradient solutions are the correct concentrations, and that the setup of the gradient maker is correct. When loading the cell suspension onto the gradient, take extra care not to disrupt the gradient, instead slowly loading the suspension such that it forms a layer on top. Also ensure the gradient cannot be disturbed once it has been prepared and during the sedimentation process (e.g. by vibrations such as those from a vortex). Low cell yield Low cell yield may be due to improper sedimentation resulting in cells not being in the correct fractions or due to the insufficient digestion of the testicular tissue, and if this is suspected, refer to the advice for low purity above. If cells are in the correct fractions, but in low concentrations, this likely due to cell loss/degradation during either preparation of the suspension or during fixation and washing of the collected cells. Ensure buffers and fixatives are properly prepared and at the correct pH, centrifugation steps are performed with a maximum force of 100 × g and all handling of cells, such as resuspension by pipette action, is performed gently. Be careful not to leave cells in the trypsin solution for longer than necessary and add the 0.5% BSA solution immediately after incubation with trypsin solution to prevent the digestion of the cells. The length of trypsin incubation can also be decreased if necessary. If cells are being lost due to lack of adherence to slides, the use of specialist electrostatic-adhesion slides, such as the recommended SuperFrost Plus slides, should prevent this. Low viability or quality of the cells Low viability or poor quality of cellular ultrastructure may be due to issues in the preparation of buffers and fixatives and of the cell suspension, similar to those described for low cell yield (see above). Open in new tab Table I Trouble shooting guide for the optimised STAPUT method for the purification of mouse spermatocyte and spermatid populations. Problem Possible causes/solutions Low purity The presence of a high amount of contaminant cell types in the fractions is most likely due to the insufficient digestion of the testicular tissue or due to issues with the preparation or loading of the gradient. Insufficient digestion will be characterised by the presence of cellular aggregates, in particular round spermatid aggregates in fraction 2. To remedy this, ensure concentrations of enzyme solutions are correct, that they are pre-warmed to 37°C prior to use, and that sample tubes are laying on their side during shaking incubation to maintain consistent exposure to the solution and prevent clumping at the tube base. Issues with digestion could also be due to insufficient mechanical separation of the seminiferous tubules prior to incubation with enzymatic solutions. If contamination is present, despite enzymatic digestion being sufficient (i.e. contaminants are single cells, as opposed to aggregates), this is likely due to incorrect preparation or loading of the gradient, or imprecise collection of the fractions. Ensure that the gradient solutions are the correct concentrations, and that the setup of the gradient maker is correct. When loading the cell suspension onto the gradient, take extra care not to disrupt the gradient, instead slowly loading the suspension such that it forms a layer on top. Also ensure the gradient cannot be disturbed once it has been prepared and during the sedimentation process (e.g. by vibrations such as those from a vortex). Low cell yield Low cell yield may be due to improper sedimentation resulting in cells not being in the correct fractions or due to the insufficient digestion of the testicular tissue, and if this is suspected, refer to the advice for low purity above. If cells are in the correct fractions, but in low concentrations, this likely due to cell loss/degradation during either preparation of the suspension or during fixation and washing of the collected cells. Ensure buffers and fixatives are properly prepared and at the correct pH, centrifugation steps are performed with a maximum force of 100 × g and all handling of cells, such as resuspension by pipette action, is performed gently. Be careful not to leave cells in the trypsin solution for longer than necessary and add the 0.5% BSA solution immediately after incubation with trypsin solution to prevent the digestion of the cells. The length of trypsin incubation can also be decreased if necessary. If cells are being lost due to lack of adherence to slides, the use of specialist electrostatic-adhesion slides, such as the recommended SuperFrost Plus slides, should prevent this. Low viability or quality of the cells Low viability or poor quality of cellular ultrastructure may be due to issues in the preparation of buffers and fixatives and of the cell suspension, similar to those described for low cell yield (see above). Problem Possible causes/solutions Low purity The presence of a high amount of contaminant cell types in the fractions is most likely due to the insufficient digestion of the testicular tissue or due to issues with the preparation or loading of the gradient. Insufficient digestion will be characterised by the presence of cellular aggregates, in particular round spermatid aggregates in fraction 2. To remedy this, ensure concentrations of enzyme solutions are correct, that they are pre-warmed to 37°C prior to use, and that sample tubes are laying on their side during shaking incubation to maintain consistent exposure to the solution and prevent clumping at the tube base. Issues with digestion could also be due to insufficient mechanical separation of the seminiferous tubules prior to incubation with enzymatic solutions. If contamination is present, despite enzymatic digestion being sufficient (i.e. contaminants are single cells, as opposed to aggregates), this is likely due to incorrect preparation or loading of the gradient, or imprecise collection of the fractions. Ensure that the gradient solutions are the correct concentrations, and that the setup of the gradient maker is correct. When loading the cell suspension onto the gradient, take extra care not to disrupt the gradient, instead slowly loading the suspension such that it forms a layer on top. Also ensure the gradient cannot be disturbed once it has been prepared and during the sedimentation process (e.g. by vibrations such as those from a vortex). Low cell yield Low cell yield may be due to improper sedimentation resulting in cells not being in the correct fractions or due to the insufficient digestion of the testicular tissue, and if this is suspected, refer to the advice for low purity above. If cells are in the correct fractions, but in low concentrations, this likely due to cell loss/degradation during either preparation of the suspension or during fixation and washing of the collected cells. Ensure buffers and fixatives are properly prepared and at the correct pH, centrifugation steps are performed with a maximum force of 100 × g and all handling of cells, such as resuspension by pipette action, is performed gently. Be careful not to leave cells in the trypsin solution for longer than necessary and add the 0.5% BSA solution immediately after incubation with trypsin solution to prevent the digestion of the cells. The length of trypsin incubation can also be decreased if necessary. If cells are being lost due to lack of adherence to slides, the use of specialist electrostatic-adhesion slides, such as the recommended SuperFrost Plus slides, should prevent this. Low viability or quality of the cells Low viability or poor quality of cellular ultrastructure may be due to issues in the preparation of buffers and fixatives and of the cell suspension, similar to those described for low cell yield (see above). Open in new tab Authors’ roles All authors were involved in experimental conception and design, in addition to data analysis and interpretation. JEMD and AEO’C performed the experiments. JEMD and MOKB wrote the manuscript. All authors read, revised and approved the final manuscript. Funding This work was supported in part by funding from the National Health and Medical Research Council of Australia (APP1058356) and the Australian Research Council (DP160100647) to MKO’B. Conflict of interest The authors declare that they have no conflict of interests that could be perceived as prejudicing the impartiality of this manuscript. References Bastos H , Lassalle B , Chicheportiche A , Riou L , Testart J , Allemand I , Fouchet P . Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis . Cytometry A 2005 ; 65 : 40 – 49 . Google Scholar Crossref Search ADS PubMed WorldCat Bellve AR , Cavicchia J , Millette CF , O'brien DA , Bhatnagar Y , Dym M . Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization . J Cell Biol 1977 ; 74 : 68 – 85 . Google Scholar Crossref Search ADS PubMed WorldCat Bryant JM , Meyer-Ficca ML , Dang VM , Berger SL , Meyer RG . Separation of spermatogenic cell types using STA-PUT velocity sedimentation . J Visualized Exp. JoVE 2013 ; 50648 . WorldCat Dobson MJ , Pearlman RE , Karaiskakis A , Spyropoulos B , Moens PB . Synaptonemal complex proteins: occurrence epitope mapping and chromosome disjunction . J Cell Sci 1994 ; 107 : 2749 . Google Scholar PubMed WorldCat Dustin P . Microtubules . Berlin, Germany : Springer Science & Business Media , 2012 Google Preview WorldCat COPAC Getun IV , Torres B , Bois PR . Flow cytometry purification of mouse meiotic cells . J Visualized Exp. JoVE 2011 : e2602 . WorldCat Lammers JH , Offenberg HH , van Aalderen M , Vink AC , Dietrich AJ , Heyting C . The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes . Mol Cell Biol 1994 ; 14 : 1137 – 1146 . Google Scholar Crossref Search ADS PubMed WorldCat Mays-Hoopes LL , Bolen J , Riggs AD , Singer-Sam J . Preparation of spermatogonia, spermatocytes, and round spermatids for analysis of gene expression using fluorescence-activated cell sorting . Biol Reprod 1995 ; 53 : 1003 – 1011 . Google Scholar Crossref Search ADS PubMed WorldCat McCarrey JR , Berg WM , Paragioudakis SJ , Zhang PL , Dilworth DD , Arnold BL , Rossi JJ . Differential transcription of Pgk genes during spermatogenesis in the mouse . Dev Biol 1992 ; 154 : 160 – 168 . Google Scholar Crossref Search ADS PubMed WorldCat Meistrich M , Bruce W , Clermont Y . Cellular composition of fractions of mouse testis cells following velocity sedimentation separation . Exp Cell Res 1973 ; 79 : 213 – 227 . Google Scholar Crossref Search ADS PubMed WorldCat Meistrich M , Trostle PK . Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients . Exp Cell Res 1975 ; 92 : 231 – 244 . Google Scholar Crossref Search ADS PubMed WorldCat Meistrich ML . Separation of mouse spermatogenic cells by velocity sedimentation . J Cell Physiol 1972 ; 80 : 299 – 312 . Google Scholar Crossref Search ADS PubMed WorldCat Miller RG , Phillips R . Separation of cells by velocity sedimentation . J Cell Physiol 1969 ; 73 : 191 – 201 . Google Scholar Crossref Search ADS PubMed WorldCat O'Bryan MK , Sebire K , Meinhardt A , Edgar K , Keah HH , Hearn MT , de Kretser DM . Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa . Mol Reprod Dev 2001 ; 58 : 116 – 125 . Google Scholar Crossref Search ADS PubMed WorldCat Pelletier J , Schalling M , Buckler A , Rogers A , Haber D , Housman D . Expression of the Wilms' tumor gene WT1 in the murine urogenital system . Genes Dev 1991 ; 5 : 1345 – 1356 . Google Scholar Crossref Search ADS PubMed WorldCat Romrell LJ , Bellvé AR , Fawcett DW . Separation of mouse spermatogenic cells by sedimentation velocity: a morphological characterization . Dev Biol 1976 ; 49 : 119 – 131 . Google Scholar Crossref Search ADS PubMed WorldCat Russell LD , Ettlin RA , Hikim APS , Clegg ED . Histological and histopathological evaluation of the testis . Int J Androl 1993 ; 16 : 83 – 83 . Google Scholar Crossref Search ADS WorldCat Schönfeldt V , Foppiani L , Schlatt S . Magnetic cell sorting is a fast and effective method of enriching viable spermatogonia from Djungarian hamster, mouse and marmoset monkey testes . Biol Reprod 1999 ; 61 : 582 – 589 . Google Scholar Crossref Search ADS PubMed WorldCat Welch JE , Schatte EC , O’Brien DA , Eddy EM . Expression of a glyceraldehyde 3-phosphate dehydrogenase gene specific to mouse spermatogenic cells . Biol Reprod 1992 ; 46 : 869 – 878 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - An optimised STAPUT method for the purification of mouse spermatocyte and spermatid populations
JO - Molecular Human Reproduction
DO - 10.1093/molehr/gaz056
DA - 2019-11-30
UR - https://www.deepdyve.com/lp/oxford-university-press/an-optimised-staput-method-for-the-purification-of-mouse-spermatocyte-0vBQDMtogq
SP - 675
VL - 25
IS - 11
DP - DeepDyve
ER -