Flow Cytometric Characterization of Viable Meiotic and Postmeiotic Cells by Hoechst in Mouse Spermatogenesis

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1 2005 Wiley-Liss, Inc. Cytometry Part A 65A:40 49 (2005) Flow Cytometric Characterization of Viable Meiotic and Postmeiotic Cells by Hoechst in Mouse Spermatogenesis Henri Bastos, 1 Bruno Lassalle, 1 Alexandra Chicheportiche, 2 Lydia Riou, 1 Jacques Testart, 1 Isabelle Allemand, 1 and Pierre Fouchet 1 * 1 Laboratoire de Gametogenèse, Apoptose et Génotoxicité, DRR/DSV/CEA, U566 INSERM, Université Paris 7, Fontenay aux Roses, France 2 Laboratoire de Différenciation et Radiosensibilité des Gonades, DRR/DSV/CEA, U566 INSERM, Université Paris 7, Fontenay aux Roses, France Received 16 July 2004; Revision Received 12 November 2004; Accepted 16 December 2004 Background: Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. The cellular heterogeneity of testis renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to characterize each germinal differentiation step. Methods: Hoechst staining of mouse testicular cells allows characterization of an enriched population in germinal stem cell and spermatogonia, called side population. In this study, we examined the definition of the various germinal populations stained by Hoechst 33342, notably meiotic and postmeiotic cells. Results: Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids were discriminated by Hoechst staining. In addition, we associated differentiation of spermatocyte I through leptotene to diplotene with changes in Hoechst red fluorescence pattern. Conclusions: Hoechst staining of viable germinal cells constitutes a valuable tool to study normal and impaired mouse adult spermatogenesis or to isolate viable cells from various differentiation stages for studies of molecular mechanisms regulating spermatogenesis Wiley-Liss, Inc. Key terms: Hoechst 33342; spermatogenesis; meiosis; adult mouse Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. Like other self-renewing processes in adult tissues, spermatogenesis originates from a small pool of stem cells (1). The molecular mechanisms governing germinal stem cell renewal and initiation of differentiation remain barely understood. First, a phase of proliferation and differentiation of spermatogonia occurs. Various spermatogonial differentiation stages can be distinguished by morphologic criteria (2). The transition from diploid B spermatogonia to preleptotene spermatocyte and primary spermatocytes characterizes meiosis entry. A long prophase I begins at the leptotene stage, and cells sequentially differentiate in zygotene, pachytene, and diplotene steps. At diakinesis, spermatocytes I are reduced to diploid spermatocytes II, which divide to produce haploid round spermatids. Then they undergo spermiogenesis and differentiate into elongating spermatids and, finally, into spermatozoa. In adult rodents, germ cell differentiation is continuous and each step is concomitantly present inside the seminiferous tubules of testis (3). As a consequence, the germinal population is highly heterogeneous and includes diploid premeiotic and meiotic cells and haploid postmeiotic cells. This cellular heterogeneity renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to purify each germinal differentiation step. Several techniques have been developed such as sedimentation at unit gravity (4), centrifugal elution (5), and magnetic cell sorting (6), which allow the isolation of specific differentiation stages in adult spermatogenesis. Flow cytometry was also used to sort fixed germinal populations based on DNA content, light scatter parameters (7), and mitochondrial mass or activity (8,9). Recently, considerable efforts Contract grant sponsor: Electricité De France; Contract grant sponsor: Nuclear Toxicology Program of the Commissariat à l Energie Atomique. *Correspondence to: Pierre Fouchet, LGAG, DRR/DSV/CEA, U566 IN- SERM, BP Fontenay aux Roses, France. pfouchet@armoise.saclay.cea.fr Published online 18 March 2005 in Wiley InterScience (www. interscience.wiley.com). DOI: /cyto.a.20129

2 to identify and purify a germinal stem cell enriched fraction for transplantation has led to the use of flow cytometry to sort unfixed viable cells (10 12). Sorting of viable round spermatids was also achieved according to light scatter for oocyte microinjection purposes (13). Complex self-renewing cell lineages can be analyzed by flow cytometry and viable cells of interest can be purified using bis-benzamide Hoechst (Ho) dye. This dye diffuses through plasma membrane and binds with high affinity to poly(d[at]) sequences in the minor groove of DNA. Ho is generally associated with propidium iodide (PI) fluorochrome to discriminate viable from dead cells. PI is a polar nucleic acid stain that penetrates dead cells with compromised plasma membranes and is excluded from live cells with intact membranes. Ho allows the detection of variations in DNA content and chromatin structure (14). Further, stem cells of different lineages (hematopoietic, skeletal muscle, brain) share a side population (SP) phenotype based on the efflux of Ho (15 18). Some members (MDR1, BCRP1/ABCG2) of the ATP-binding cassette transporter family contribute to the dye efflux activity of the SP phenotype (19). BCRP1 was recently reported to be responsible for Ho efflux in hematopoietic stem cells, as described in Bcrp1 / mice (20). We and others have demonstrated that normal mouse testicular cells also display an SP phenotype in adult steady-state spermatogenesis (12) or during the first wave of spermatogenesis (11). SP phenotype was BCRP1 dependent at least in adult and contained spermatogonia (12). Male germinal stem cells were present in testicular SP of normal adult mice as shown by transplantation in sterile testis and spermatogenesis recovery (11,12). However, this germinal stem cell phenotype appears to be lost in mouse cryptorchid model (10). In this report, we examine further the characterization of the various germinal populations stained by Ho. We show that, apart from spermatogonia and germinal stem cell (SP phenotype), other differentiation stages can be discriminated by the same Ho staining methodology, notably meiotic stages. Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids can be discriminated. Hence, this methodology can be used as an analytical tool or as preparative methodology to isolate various differentiation stages by flow sorting. MATERIAL AND METHODS Animals and Irradiation Adult C57BL/6 males (Iffa Credo), W/W v males (all parent strains issued from the Jackson Laboratories, West Grove, PA, USA) and EGFP (Enhanced Green Fluorescent Protein) transgenic mice (C57BL/6-TgN[beta-act-EGFP]- 01Osb) were raised in our animal facilities. In the testis of EGFP transgenic mice, germ cells are labeled with EGFP except elongated spermatids and spermatozoa (21). All animal procedures described in this report were carried out in accordance with French governmental regulations (Services Vétérinaires de la Santé et de la Production HOECHST CHARACTERIZATION OF MOUSE MEIOSIS Animale, Ministère de l Agriculture, Paris, France). Mice were exposed to -irradiation using a 137 Cs source (IBL637, CIS Biointernational, Gif sur Yvette, France), with a total dose of 4 Gy and a dose rate of 0.6 Gy/min. Testicular Single-Cell Suspensions Cells were isolated from 2- to 3-month-old male mice by using a two-step enzymatic digestion to remove interstitial cells. The albuginea was removed and seminiferous tubules were dissociated by using enzymatic digestion with collagenase type I (Invitrogen, La Jolla, CA, USA) at 100 U/ ml for 25 min at 32 C in Hanks balanced salt solution (HBSS) supplemented with 20 mm HEPES (ph 7.2), 1.2 mm MgSO 4 7H 2 0, 1.3 mm CaCl 2 2H 2 0, 6.6 mm sodium pyruvate, and 0.05% lactate. A filtration step with a 40- m nylon mesh was achieved to separate, on the one hand, a fraction enriched with interstitial cells and, on the other hand, tubules retained in the filter. Tubules were collected and incubated at 32 C for 25 min in the same collagenase buffer as that used for the first step. The resulting whole cell suspension was filtered through a 40- m nylon mesh to remove cell clumps. After a wash in HBSS, the cell pellet was resuspended in incubation buffer (HBSS supplemented with 20 mm HEPES [ph 7.2], 1.2 mm MgSO 4 7H 2 0, 1.3 mm CaCl 2 2H 2 0, 6.6 mm sodium pyruvate, 0.05% lactate, glutamine, and 1% fetal calf serum). Cell concentrations were estimated with trypan blue staining ( 95% viable cells). Flow Cytometric Analysis and Cell Sorting Two million cells were diluted in 2 ml of incubation buffer and stained with Ho (5 g/ml; Sigma, Saint Quentin, France) for 1hat32 C. Before analysis, PI (2 g/ml; Sigma) was added to exclude dead cells. Analysis and cell sorting were performed on a dual-laser FACStar Plus flow cytometer (Becton Dickinson, Le Pont de Claix, France) equipped with a 360-nm ultraviolet argon laser and a 488-nm argon laser (Coherent, Orsay, France). Ho and PI were excited by a 360-nm ultraviolet laser (100 mw). A dichroic mirror (610-nm short pass filter) was used to separate blue from red fluorescence and to direct emitted light to different detectors. Ho blue was collected by using a combination of 400-nm long pass and 505-nm short pass filters in front of the first detector. PI and Ho red fluorescences were detected with a 630-nm/30-nm band pass filter in front of the second detector. EGFP was excited with the second 488-nm laser (75 mw) and fluorescence was collected with a 530-/30-nm band pass filter. For DNA content analysis using PI, flow-sorted cells were fixed in 70% ethanol for 24 h at 4 C. Fixed cells were then washed and stained with PI (50 g/ml) and RNAase (100 g/ml) in phosphate buffered saline (PBS). Flow cytometric analysis was performed with FACSCalibur (Becton Dickinson). Synaptonemal Complex Analysis Spermatocytes were spread by using 1% paraformaldehyde containing 0.2% Triton X-100 as a fixative (22). For silver staining of chromosomal proteins, chromosome 41

3 42 BASTOS ET AL. spreads were incubated in 50% AgNO 3 at 70 C for 30 min. For fluorescence immunostaining, slides were incubated in PBT (1% bovine serum albumin [BSA], 3% fetal calf serum, 0.05% Triton X-100) for 30 min before incubation with primary antibodies diluted in PBT 1 hour at 37 C. Polyclonal rabbit anti-scp3 (kind gift from C. Heyting) was used at a dilution of 1:1,000. After washes in PBS, primary antibodies were detected with donkey anti-rabbit CY3 (Jackson Laboratories) diluted 1:400 and incubated at 37 C for 1 h. After washes in PBS, slides were mounted with Vectashield (Vector, Abcys, Paris, France). Analysis and image capture were done with an Olympus AX70 epifluorescent microscope equipped with a Princeton CCD camera and IPlab software (Scanalytics, Fairfax, VA, USA). Ser10 Phosphorylated Histone H3 Immunostaining Sorted cells were deposited on L-polysine slides using Cytospin (Shandon, Waltham, MA, USA). Ser10 phosphorylated histone H3 (Upstate Biotechnology, Lake Placid, NY, USA) immunostaining was performed according to the manufacturer s protocol. Briefly, cells were fixed with 3.7% formaldehyde in PBS for 1 min at room temperature, washed with PBS, and permeablized with 0.1% Triton-100 for 3 min at room temperature. After washing, cells were incubated with 1% BSA in PBS for 1hatroom temperature. Cells were then immunostained overnight at 4 C with5to10 g/ml of anti-phospho-histone H3 (Ser10) in PBS plus 1% BSA. Cells were washed and incubated with Cy3-coupled anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA, USA). Analysis and image capture were done with an Olympus AX70 epifluorescent microscope equipped with Princeton CCD camera and IPlab software (Scanalytics). RESULTS Hoechst Staining of Adult Testicular Single-Cell Suspension Single-cell suspensions of adult mice testis were analyzed as functions of PI/Ho red fluorescence and Ho blue fluorescence (Fig. 1A). PI uptake and high red fluorescence intensity (ordinate axis) allowed discrimination of dead cells. Five major viable populations (negative for PI fluorescence) were resolved according to complex Ho red and blue fluorescences. We previously demonstrated that premeiotic spermatogonial and germinal stem cells are assigned to the SP due to an active Ho efflux (12). Postmeiotic spermatid population was assigned to subpopulation 4 according to its haploid DNA content. To assign diploid meiotic populations, we compared infertile W/W v adult males displaying an early premeiotic block in spermatogenesis due to mutations in the c-kit gene with EGFP transgenic mice whose spermatogenesis is normal (1,21). The mix of EGFP and W/W v germ cells showed the same Ho profile as the wild-type mouse (Fig. 1A,B). According to EGFP fluorescence, W/W v germ cells (EGFP negative) and EGFP cells could be discriminated (Fig. 1C) in the mix and their respective Ho profiles could be displayed (Fig. 1D,E). Figure 1 shows clearly that subpopulations 1, 2 (DNA content 4n), 3 (2n), and 4 (n) are totally absent in the infertile W/W v model containing only premeiotic cells (Fig. 1D,E). Diploid meiotic populations can therefore be assigned to subpopulations 1, 2, and 3. Discrimination of Spermatocytes I by Hoechst Staining Spermatocyte I was assigned to subpopulation 2 in adult mice due to their 4N DNA content. To analyze further this subpopulation, Ho patterns of 15- and 19-day postnatal mice were compared. Analysis of the first wave of spermatogenesis in immature males allows one to follow the meiotic progression and differentiation of spermatocyte I from leptotene to diplotene stages. A red Ho fluorescent shift was observed for spermatocyte I cells between 15 and 19 days (red box in Fig. 2A,B), as they progressed through the meiotic process. This observation emphasizes that spermatocyte I differentiation is associated with changes in Ho fluorescence pattern and that different stages of spermatocyte I can be distinguished according to red and blue Ho fluorescences in adult mice. Testicular single-cell suspensions of adult EGFP mice were then analyzed according to red and blue Ho fluorescences and EGFP fluorescence. In this transgenic model, EGFP is under the control of -actin promoter (21). During meiosis, this transgenic model exhibits a gradual increase in translation of -actin-egfp transgene as confirmed by EGFP fluorescence (23). According to the Ho red and blue fluorescences of spermatocytes I, three subpopulations could be defined: SI-Ho-red low, SI-Ho-red med, and SI-Hored hi (Fig. 2C). The Ho red fluorescent shift of spermatocyte I through meiotic progression was correlated with an increase of EGFP fluorescence (Fig 2D), confirming that Ho staining allowed the separation of different stages of spermatocytes I. The synaptonemal complex is a protein structure that tethers the homologous chromosomes together through prophase I. Analysis of the synaptonemal complex status as a marker allows discrimination of distinct substages of meiotic prophase I (24). Spermatocytes I defined in the three regions previously described were sorted. Then they were analyzed according to the synaptonemal complex (SC) formation by immunofluorescence localization of SC protein-3 (SCP3), which displays the axial elements, and the silver staining patterns of chromosome proteins (Fig. 3). Two distinct SCP3 patterns were observed in SI-Ho-red low cells (Fig. 3A,B). On the one hand, observation of uncompacted chromosomes with the beginning of axial element assembling (SCP3 fluorescence) showed that those cells are leptotene spermatocytes (Fig. 3A). On the other hand, compacting chromosomes with axial element associations observed in Figure 3B are typical of zygotene stages. Compacted, fullly synapsed chromosomes except XY chromosomes (sexual body) observed in SI-Ho-red med are characteristic of pachytene stages (Fig. 3C,C ). Compacted chromosomes with desynaptic regions showing synaptonemal complex disassembly indicated that diplotene cells were found in SI-Ho-red hi population (Fig. 3D,D ).

4 FIG. 1. Flow cytometric analysis of Hoechst and propidium iodide (PI) fluorescence. A: C57BL/6 adult murine testicular cells. B: Mix of W/W v infertile mutant and EGFP adult murine testicular cells. C: EGFP fluorescence of cells in the mix displayed in B. D: Hoechst analysis of EGFP positive cells of the mix (gate R2). E: Hoechst analysis of W/W v cells of the mix (gate R1). [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]

5 44 BASTOS ET AL. FIG. 2. Hoechst and propidium iodide (PI) fluorescence flow analysis of testicular cells from mice aged (A) 15 and (B) 19 days postpartum. Red boxes indicate the shift of red Hoechst fluorescence of spermatocytes I during meiosis. C: Hoechst and PI fluorescence flow analysis of testicular cells from adult transgenic EGFP mice. Three subpopulations of spermatocyte I are defined according to red Hoechst fluorescence: SI-Ho-red low, SI-Ho-red med, and SI-Ho-red hi are noted as low, med, and hi, respectively, on the cytogram. D: EGFP and blue Hoechst fluorescence of testicular cells from adult transgenic EGFP mice. Gated cells from SI-Ho-red low, SI-Ho-red med, and SI-Ho-red hi defined in C are labeled green, pink, and blue, respectively, on the cytogram. Preleptotene Spermatocytes Are Located in Subpopulation 1 In adult or immature mice, Ho profiles of subpopulation 1 exhibited a continuum between 2N and 4N DNA content (Figs. 1A, 2B). Those cells seemed to be actively replicating S-phase cells that would differentiate into spermatocytes I. Those results strongly suggest that subpopulation 1 corresponds to preleptotene spermatocyte stage (late S phase before meiotic prophase). To eliminate the possibility that this profile could result from active Ho efflux as for SP cells, subpopulation 1 was sorted and its cell cycle status was reanalyzed according to PI fluorescence (Fig. 4). Analysis confirmed that 83% of subpopulation 2 cells were in S phase. In addition, we observed an accumulation of cells at the end of S phase, suggesting a slowdown or a blockade in the cell cycle progress. Spermatocytes II Are Located in Subpopulation 3 Subpopulation 3 was defined as meiotic or postmeiotic cells with 2N DNA content, suggesting that they were

6 FIG. 3. Synaptonemal complex structure analysis of flow sorted as SI-Ho-red low (A, B), SI-Ho-red med (C, C ), and SI-Ho-red hi (D, D ) spermatocyte I populations according to SCP3 immunostaining (A D) and silver staining (C,D ). Arrows indicate desynapsis in diplotene spermatocyte. Sexual bodies are indicated by stars in pachytene and diplotene spermatocytes. Scale bar 10 m.

7 46 BASTOS ET AL. agreement with the high radiosensitivity of differentiating spermatogonia previously described (25). Fourteen days later, spermatocyte I population diminished dramatically (3.5% vs. 24.5%), whereas the SP recovered (3.1%). According to the timing of spermatogenesis, spermatocytes I originate from differentiating spermatogonia after 14 days. This depletion of spermatocytes I is thus linked to death of differentiating spermatogonia observed 1 day after irradiation. Twenty-nine days later, spermatocyte I was the main population (56%) because SP cells recovered at 14 days differentiated in spermatocyte I. Round spermatids were strongly depleted (17%) due to depletion of spermatocyte I at 14 days. Thus, Ho staining allows the analysis of the equilibrium between the different differentiating stages of the spermatogenesis in adult mice and the timing of spermatogenesis recovery after genotoxic stress. FIG. 4. Propidium iodide fluorescence histogram of sorted cells from subpopulation 1 (solid) and total testicular cells (open) as a control for DNA content analysis. [Color figure can be viewed in the online issue, which is available at spermatocytes II. Subpopulation 3 cells were thus sorted and analyzed by fluorescence microscopy (Fig. 5). Two kinds of cells were observed. Forty percent of the subpopulation 3 were cells in diakinesis corresponding to spermatocyte II (Fig. 5A). Observation of mitotic chromosome was confirmed by immunostaining with the mitosis marker ser10 phosphorylated histone H3 (Fig. 5B). The main population (60% of subpopulation 3) contained cells with two distinct nuclei and corresponded to spermatocytes II just before cytokinesis (Fig. 5C). Round and Elongated Spermatids Can Be Discriminated Subpopulation 4 corresponds to postmeiotic haploid spermatids. Red and blue Ho stainings were insufficient to discriminate round from elongated spermatids despite differences in chromatin compaction. Forward scatter parameter (FSC) allowed differentiation of subpopulation 4 in two subpopulations. The FSC high population consisted of round spermatids as confirmed by sorting and microscopic analysis (Fig. 6A). The FSC low population consisted of elongated spermatids and spermatozoa (Fig. 6B). Evolution of Germinal Cell Population After Irradiation as Determined by Hoechst Staining To evaluate Ho staining as a sensitive analytic tool for spermatogenetic differentiation process study, mice were exposed to 4 Gy of total body irradiation and the proportion of germinal population was determined by Ho staining at various times after irradiation (Table 1). Decrease of SP (0.6% vs. 1.6%), corresponding to spermatogonia and germinal stem cells, was observed after 24 h. This is in DISCUSSION Ho, a vital DNA dye, is largely used to study the cell cycle and apoptosis of various cell lines and to isolate stem cells (SP phenotype) in complex self-renewing lineages (15,17,18). Red and blue fluorescence emissions of Ho result from three cellular properties: (a) ploidy, (b) structure and accessibility of chromatin, and (c) Ho efflux due to ATP-binding cassette transporter activity (20). As we demonstrated previously, Ho staining of adult testicular cells allows isolation by flow cytometry of a BCRP1 effluxdependent germinal SP that contains germinal stem cells and spermatogonia (12). We show in the present work that other differentiation stages can be discriminated by the same Ho staining methodology such as preleptotene spermatocyte, spermatocyte I, spermatocyte II, and round and elongated spermatids. Switch off of BCRP1 activity and Ho efflux after spermatogonial stages turn out to be useful to characterize later spermatogenesis steps. Ploidy can only be measured on meiotic and postmeiotic cells by Ho staining, excluding spermatogonia from DNA content analysis. As a consequence, four populations are defined by their DNA content: S phase (population 1), 4N DNA content (population 2), 2N DNA content (population 3), and haploid (population 4). Our results suggest strongly that subpopulation 1 corresponds to preleptotene spermatocytes. Ho profiles show that this population constitutes a continuum between SP cells (spermatogonia) and the 4N DNA content population, corresponding to the last S phase before meiosis and differentiation in spermatocyte I. Those cells appear to be blocked at the end of the S phase. In the same way, previous work showed that DNA content of most preleptotene spermatocytes was between 3N and 4N in a vitamin A deficient rat testis model (26). Our results suggest the occurrence of a cell cycle checkpoint for S-phase preleptotene spermatocytes before the mitotic/meiotic switch and the transition to the meiotic prophase. In addition, those cells were positive for -6 integrin labeling (data not shown) like germinal SP cells (12) and can be therefore enriched by MACS sorting.

8 HOECHST CHARACTERIZATION OF MOUSE MEIOSIS 47 FIG. 5. Fluorescence microscopic analysis of sorted subpopulation 3. A: Analysis of Hoechst fluorescence shows a binucleated cell (arrow) and diakinesis (arrowhead). B: Ser10 phosphorylated histone H3 immunostaining of subpopulation 3 confirms the presence of diakinesis (arrowheads). Cells are counterstained with Hoechst Scale bar 20 m. Using immunofluorescence localization of SCP3 on flow-sorted cells, we have shown that subpopulation 3 (4N DNA content) corresponds to spermatocyte I. Those results confirm previous data showing that subpopulation 3 highly expresses meiotic differentiation markers (Crem t and Hsp70-2) but does not express spermatogonial marker (Stra8) (12). We have also observed that the differentiation of spermatocyte I through leptotene to diplotene is associated with changes in Ho red fluorescence pattern. It was recently reported that an intercalating dye, such as 7-amino-actinomycin D, allows discrimination of several spermatocyte I stages. The progressive chromatin decondensation during meiotic prophase was suggested to be responsible for the increase of fluorescence observed with 7-amino-actinomycin D (27). In the same way, the red Ho fluorescence shift might result from differences in the chromatin structure of spermatocytes I, although Ho is not an intercalating dye. A red fluorescent shift of Ho has been described in cells undergoing apoptosis compared with viable cells, suggesting that differences in spectral properties could be due to changes in chromatin structure in those cells (28). Subpopulation 3 could be subdivided in three regions, Ho-red low, Hored med, and Ho-red hi, which contained, respectively, leptotene and zygotene, pachytene, and diplotene spermatocyte I according to SCP3 immunofluorescence. EGFP transgenic mice whose EGFP expression is controlled by -actin promoter (21) was also used to characterize spermatocyte I stages. We have shown that analysis of germinal cells of this transgenic mouse constitutes a useful

9 48 BASTOS ET AL. FIG. 6. Hoechst fluorescence microscopic analysis of sorted subpopulation 4 according to FSC parameter: (A) FSC high and (B) FSC low. Scale bar 20 m. methodology to discriminate and sort spermatocyte I populations according to EGFP combined to Ho fluorescence. Subpopulation 3 was assigned to spermatocyte II populations. It confirmed previous results showing that Hsp70-2 and Crem t genes, which are meiotic and postmeiotic markers, are transcribed in the diploid subpopulation 3 (12). It was composed of two cellular types: typical spermatocyte II in diakinesis (40%) and binuclear cells (60%) corresponding to spermatocytes II undergoing cytokinesis. It was previously described that some mouse spermatocytes II complete normal nuclear divisions but fail to undergo cytokinetic cleavage (29). The high fre- Table 1 Proportion of Germinal Cell Populations After Irradiation as Determined by Hoechst Staining* -Ray dose Side population Spermatocytes I Spermatocytes II Round spermatids Control 1.6 (0.2) 24.5 (1.5) 8.3 (0.4) 58.1 (0.9) 1 day 0.6 (0.05) 22.8 (2.8) 8.9 (0.8) 57.6 (2.3) 14 days 3.1 (0.3) 3.5 (0.3) 11.7 (0.7) 67.3 (1.3) 29 days 3.8 (0.4) 56.6 (3.5) 6.9 (0.6) 17.3 (3.5) *Data are expressed as mean (standard deviation).

10 HOECHST CHARACTERIZATION OF MOUSE MEIOSIS 49 FIG. 7. Schematic diagram summarizes the Hoechst fluorescent profile of male germinal cells in adult spermatogenesis. [Color figure can be viewed in the online issue, which is available at wiley.com.] quency of binuclear cells in spermatocyte II population could reflect this phenomenon of abortive cytokinesis. Analysis of germinal cell populations by Ho staining allows information about the dynamic behavior of the differentiation process to be obtained (Fig. 7). The transition from the phase of proliferation and differentiation of spermatogonia (SP cells) to preleptotene spermatocyte can be observed. Meiotic process can be surveyed: prophase I progression followed by reduction in spermatocyte II, which will divide to produce haploid round spermatids. Spermatids will then differentiate in elongated spermatids, which will be discriminated according to FSC parameter. Hence, Ho staining constitutes a valuable tool to study normal and impaired spermatogenesis in adult mice or to sort viable germinal cells to study molecular mechanisms regulating adult spermatogenesis. ACKNOWLEDGMENTS We thank Dr. Okabe for the generous gift of the EGFP transgenic mice and Dr. C. Heyting for the generous supply of SCP3 antibody. We thank P. Flament for technical assistance in the animal facilities. LITERATURE CITED 1. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 1994;91: Chiarini-Garcia H, Russell LD. High-resolution light microscopic characterization of mouse spermatogonia. Biol Reprod 2001;65: Russell L, Ettlin A, Sinha Hikim A, Clegg E. Histological and histopathological evaluation of the testis. Clearwater, FL: Cache River Press; Bellve AR, Cavicchia JC, Millette CF, O Brien DA, Bhatnagar YM, Dym M. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol 1977;74: Meistrich ML. Separation of spermatogenic cells and nuclei from rodent testes. Methods Cell Biol 1977;15: van der Wee KS, Johnson EW, Dirami G, Dym TM, Hofmann MC. Immunomagnetic isolation and long-term culture of mouse type A spermatogonia. J Androl 2001;22: 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: Petit JM, Ratinaud MH, Cordelli E, Spano M, Julien R. Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis. Cytometry 1995;19: Suter L, Koch E, Bechter R, Bobadilla M. Three-parameter flow cytometric analysis of rat spermatogenesis. Cytometry 1997;27: Kubota H, Avarbock MR, Brinster RL. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci USA 2003;100: Falciatori I, Borsellino G, Haliassos N, Boitani C, Corallini S, Battistini L, Bernardi G, Stefanini M, Vicini E. Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis. FASEB J 2004;18: Lassalle B, Bastos H, Louis JP, Riou L, Testart J, Dutrillaux B, Fouchet P, Allemand I. Side population cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells. Development 2004;131: Lassalle B, Ziyyat A, Testart J, Finaz C, Lefevre A. Flow cytometric method to isolate round spermatids from mouse testis. Hum Reprod 1999;14: Watson JV, Nakeff A, Chambers SH, Smith PJ. Flow cytometric fluorescence emission spectrum analysis of Hoechst stained DNA in chicken thymocytes. Cytometry 1985;6: Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183: Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA 1999; 96: Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, Mulligan RC. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999;401: Hulspas R, Quesenberry PJ. Characterization of neurosphere cell phenotypes by flow cytometry. Cytometry 2000;40: Bunting KD. ABC transporters as phenotypic markers and functional regulators of stem cells. Stem Cells 2002;20: Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP. Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci USA 2002;99: Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. Green mice as a source of ubiquitous green cells. FEBS Lett 1997;407: Peters AH, Plug AW, van Vugt MJ, de Boer P. A drying-down technique for the spreading of mammalian meiocytes from the male and female germline. Chromosome Res 1997;5: Ventela S, Okabe M, Tanaka H, Nishimune Y, Toppari J, Parvinen M. Expression of green fluorescent protein under beta-actin promoter in living spermatogenic cells of the mouse: stage-specific regulation by FSH. Int J Androl 2000;23: Cohen PE, Pollard JW. Regulation of meiotic recombination and prophase I progression in mammals. Bioessays 2001;23: Hasegawa M, Zhang Y, Niibe H, Terry NH, Meistrich ML. Resistance of differentiating spermatogonia to radiation-induced apoptosis and loss in p53-deficient mice. Radiat Res 1998;149: van Pelt AM, van Dissel-Emiliani FM, Gaemers IC, van der Burg MJ, Tanke HJ, de Rooij DG. Characteristics of A spermatogonia and preleptotene spermatocytes in the vitamin A deficient rat testis. Biol Reprod 1995;53: Golan R, Lewin LM, Soffer Y, Lotan G, Shochat L, Vigodner M. Evaluation of spermatogenesis using flow cytometry and confocal microscopy. Isr Med Assoc J 2003;5: Ellwart JW, Dormer P. Vitality measurement using spectrum shift in Hoechst stained cells. Cytometry 1990;11: Manandhar G, Moreno RD, Simerly C, Toshimori K, Schatten G. Contractile apparatus of the normal and abortive cytokinetic cells during mouse male meiosis. J Cell Sci 2000;113: