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On stage single cell identification of rat spermatogenic cells
Biology of the Cell (1997) 89, 53-66 o Elsevier, Paris
53
Original article
On stage single cell identification
of rat spermatogenic
cells
Juan G Reyes, Alvaro Diaz, Nelson Osses, Carlos Opazo and Dale J Benos * hstituto de Quimica, Universidad Catolica de Valparaiso, Valparaiso; Depto Fisiologia y Biofisica, facultad de Medicina, U de Chile, Santiago, Chile; Department of Physiologyand Biophysics, H-IS6 704, the Universityof Alabama at Birmingham,1918 UniversityBoulevard,Birmingham,Al 35294-0005, USA
The study of spermatogenic cell physiology has been hindered by the absence of unbiased methods of identification of cells upon which single cell techniques are being applied. In this work, we have used histochemical techniques, digital videoimaging, quantification of chromatin-bound DNA probes, and measurements of cell diameter to identify single spermatogenic cells at different periods of development. Our criteria of identification permit the definition of four developmental stages of spermatogenesis on which to perform single cell analyses: spermatogonia B/preleptotene spermatocytes, lfptqtene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids, The use of voltage-sensitive dyes and Caz+-sensitive dyes does not interfere with the estimations of DNA content. The estimations of DNA content of spermatogenic cells can be performed both ) and long wavelength-excited with near-UV excited dyes (H= dyes (ethidium bromide), allowing the use of a wide range of physiological and immunocytochemical fluorescent probes to study the sperrnatogenic process.
testicle / seminiferous tubule / cell cycle / cell differentiation / male
INTRODUCTION Mammalian spermatogenesis takes place in the seminiferous tubule in the testis. This process begins on the serosal side of the seminiferous tubule with mitotic events of primordial cells and spermatogonia (A and B), followed by differentiation of spermatogonia B into preleptotene spermatocytes. These cells are characterized by a ploidy of 2 N and approximately 5 pm difference in cell diameter (Romrell et al, 1976; Bell&, 1977). Figure 1 shows the .schematiL; representation of the spermatogenie process in a plot of ploidy zwsus cell diameter. The progression of preleptotene spermatocytes into meiosis implies an S phase to reach a ploidy of 4 N, migration through the Sertoli cell tight junctions, and the entrapce into the prolonged meiotic prophase (leptoten&zygotene-pachytene sperma-
* Correspondence
and reprints
On stage single cell identification of rat spermatogenic cells
tocytes) with dramatic changes in cell size. Two successive meiotic divisions, generating secondary spermatocytes and round spermatids, are characterized by reductions of size and ploidies of 2 N and 1 N, respectively (Bellv4, 1979; deKretser and Kerr, 1988; Braun et al, 1995). Spermatogenesis is completed with remarkable changes in cell shape, organelle rearrangement, and cytoplasmic shading that accompany the spermiogenesis. In spite of the importance of the spermatogenic process, the study of the physiology of the spermatogenic cells and its regulatory aspects have only lately been approached more systematically (Hagiwara and Kawa, 1984; Reyes et al, 1990; Reyes et al, 1994; Lievano et al, 1996). Part of this delay can be understood because there are mainly two populations of spermatogenic cells that can be obtained in sufficient quantity and purity to be studied with averaging methods of cell physiology research: pachytene spermatocytes and round spermatids. Besides the fact that mainly the late meiotic stages can be studied in purified cell prepReyes et al
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Biology of the Cell (1997) 89, 53-66
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A spermatogonia
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Cell diameter (pm) Fig 1. Plot of ploidy versus cell diameter in the premeiotic and meiotic spermatogenic process. The values of cell diameters for each cell type were taken from the range of values published in the literature.
arations, the methodology to obtain pure enough populations of these and other spermatogenic cells require protease treatments (Lam et al, 1970; Grabske et al, 1975; Romrell et al, 1976) that are known to remove receptors and other proteins from cell membranes (D’Agostino et al, 1984; Reichter and Dattatreyamurty, 1989), or to mimic cell-cell interactions (Grotegoed et al, 1989; Aravindan ef al, 1996). In contrast to these shortcomings of the averaging methods to study spermatogenie cell physiology, powerful methodologies are available for single cell physiological and molecular biological studies of spermatogenic cells (eg video transmission and fluorescence microscopy, patch-clamp and single cell oligonucleotide amplification techniques). Because these methods do not require pure populations, they could avoid the use of extensive protease digestion of the seminiferous tubules. However, the use of these single cell methodologies requires, cell identification, and, even in ‘purified’ populations, ways to avoid the almost inevitable bias of choosing an inappropriate popuiation by their prominence or ease of manipulation. The usefulness of identification schemes for On stage single cell identification of rat spermatogenic cells
single cells requires them to be performed on the microscope stage simultaneously or subsequently to measurements of the physiological parameter of interest. The development of fluorescent probes to study quantitatively the cell content of macromolecules and of inexpensive video microscopy and image analysis techniques prompted our efforts to devise a strategy to identify, on the microscope stage, spermatogenic cells at different steps of development based on the differences in cell DNA structure and content, and the changes in cell size occurring during this differentiation process (Cowell and Franks, 1980; Grogan et al, 1981; Arndt-Jovin and Jovin, 1989; GottschalkSabag et al, 1995). We have also utilized cytochemical techniques to identify the presence of peritubular cells in the seminiferous tubule cell preparation. Vital staining with H,,,,, and a guessstimate of the respective cell type by the appearance of the chromatin, followed by a quantitation of the relative DNA content in the same cell shows that there is a close correspondence between both criteria of cell classification for rat spermatids, but not so for spermatogonia or primary spermatocytes. The use of a bivariable Reyes et al
Biology of the Cell (1997) 89, 53-66
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system of classification of seminiferous tubule cells allows us to define regions in a plot of relative DNA content versus cell diameter where there is a high probability of correctly classifying cells as spermatogonia B/preleptotene, leptotenezygotene primary spermatocytes, late pachytene primary spermatocytes or spermatids.
MATERIALS AND METHODS Rat spermatogenic
cell preparation
Rat seminiferous tubule cell populations were prepared from testicles of adult (60 days) or 12-14-day-old rats, as described by Romrell et al (1976) and Bellve et al (1977). The resulting cell suspensionwas filtered through a layer of cotton and 250 and 70 p nylon meshesand washed three times in Krebs-Henseleit10 mM Hepes, 10 mM (KH) lactate medium. The mixed seminiferoustubule cells were used either without purification, partially purified by passing the cells through a 2% BSA/15%/30% Perco11discontinuous gradient, or, the different cell populations were purified using sedimentation velocity in a 260mL 24 % BSA in K-H medium gradient at unit gravity and at 16 + 2°C (Bell&, 1977).The cells were allowed to settle for 3 II and then the BSA gradient was collected in 3 mL fractions at approximately 1 fraction per minute.
Measurements of cell diameter and DNA content using transmission and fluorescence digital video microscopy
Fig 2. A. Image of H,,,,, fluorescence in rat round spermatids and spermatozoa.The cells were exposedto 2 PM HSSSJ2 and 15 &mL digitonin in Krebs-Henseleitbuffer for 30 min and at room temperature (18 * 2°C). The imagewas taken with a cooled CCDvideo camera using a 40 x NA 0.85 objective and 0.1 s of exposure. The excitation and emissionwavelengthswere 330-380 and > 420, respectively. The bar represents 15 pm. B. Photomicrograph of a pachytene spermatocyte, a round spermatid and a trinucleated symplast. The ceils were exposed to 2 PM H,,,, in Krebs-Henseleitbuffer for 40 min at room temperature. The bar represents25 pm.
The studies of video microscopy were performed in an inverted Nikon Diaphot microscope with epifluorescence.A cooled CCD 12 bit video camera (Spectrasource, Los Angeles, CA) was attached to the videoport of the microscope. The image analyses were performed with the appropriate software using a 486 IBM compatible computer. Cell diameter measurementswere made by previously calibrating the optical and digital analysis systemin pixels/pm using a reticulated slide, and further calibrated using normal human red blood ceils. The within-assay variation coefficients for diameter determination of pachytene spermatocytesand round spermatids were 3 and 1 %, respectively. The non-intercalating bisbenzimide DNA probe H3= (excitation wavelengths 330-380 nm, emission wavelengths > 420 run) or ethidium bromide (excitation wavelengths 450490 run, emissionwavelength > 520nm), were used to estimate the DNA content of the cells.Becauseof the intrinsic difficulties of estimatingthe absolutevalues of cell DNA using fluorescentprobes(Amdt-Jovin and Jovin, 1989),we usedpermeabilized rat spermatozoaas an internal standard with constant ploidy (1 N) and an easily recognizable shapein image analysis(fig 2A). The spermatozoa were obtained from the cauda epididymis of rats by puncturing and gently pressing this organ. In order to remove the tails of the spermatozoaand to permeabilize them, we treated the spermatozoacell suspensionwith five sequencesof 10s sonicationpulses(BransonEl2 80 watts, Shelton, CO). To obtain a homogeneouslypermeabilized
spermatozoapopulation, treatment with 0.1% Triton X-100 saline, three washesand a further cycle of freezing and thawing was necessary.Thosecellswith two or more visible nuclei generatedduring the cell preparation were identified visually and either discarded from our analysis or utilized asinternal standardsfor quantitative DNA determinations(fig 2B).The within assayvariation coefficient of H,, fluorescenceof spermatozoa,spermatidsand pachytene spermatocyte determinations using a 0.85 NA 40 x objective,were 9,12, and 8%, respectively.The useof a 1.35 NA 100x oil immersion objective gave for the samecell types variation coefficientsof 8, 7, and lo%, respectively. Becauseeither objective gave comparablevariation coefficients in the HWZ fluorescence determination, but the 100x objective gave a limited number of cells in the field, we choseto usethe 0.85NA 40 x objective for all the quantitative estimationsof relative total cell DNA content.
On stage single cell identification of rat spermatogeniccellS
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Membrane potential and intracellular Ca*+ determinations in single rat spermatids In order to test the feasibility of performing physiological measurements and cell identification, we have successfully used the following protocol: 1) In dim light conditions, we let the cells (previously loaded with calcium sensitive dyes when appropriate) settle for 10 min in a microscope chamber (0.7 mL) having a coverslip as the bottom wall. Alternatively, the cells were adhered to a polylysine-covered coverslips which were attached to the bottom of the chamber with a minimal amount of silicone grease. Subsequently, ethidium bromide (5 PM final concentration) was added to the microscope chamber. 2) After 2-3 min of exposure to ethidium bromide, the unattached cells and the ethidium bromide solution were washed away by perfusing 10 mL (2 mL /mm) of K-H lactate buffer containing 2 @4 H33342. 3) 43342 was allowed to enter the cells for 30 min without perfusion. 4) The cells were selected for observation by taking a transmission image in order to estimate cell diameter, and by fluorescence observation of the chromatin stained with H33742. Those cells with a damaged membrane (stained violet by ethidium bromide and Ha& were easily identified and discarded. 5) The cells were exposed for 30 min in the dark to potential sensitive probes when membrane potential was to be determined. 6) At the appropriate excitation and emission wavelengths, a series of images were taken and stored, and calibrations of the fluorescent probes were performed. 7) The cells were perfused with 10 mL of K-H lactate buffer with 10 pgg/mL digitonin and 2 PM HsM2, and the dye was allowed to equilibrate for 30 min. Two images of H 33342stained cells were taken at 25 and 30 min to check for the effective equilibration of the dye. Subsequently, 5-10 H,,,,, images of spermatozoa were taken and stored. 8) The relative DNA content was calculated from the ratio of the cell DNA-H,, fluorescence and the average spermatozoa DNA-H-, fluorescence (interspermatozoa variation coefficient of lo-15%). The plasma membrane potential of rat spermatids was estimated at 18 + 3°C from the intra-extracellular distribution of bisoxonol determined in fluorescence video images taken with excitation wavelengths of 510-560 nm and emission wavelengths of > 590 nm (Ehrenberg et al, 1988). In order to estimate the out-of-focus extracellular fluorescence we utilized neutral tetramethylrhodaminedextran (M, 70 000) as an impermeant fluorescent probe.
Biology of the Cell (1997) 89, 53-66
The bisoxonol fluorescence distribution was calibrated for each cell at 0 mV and -100 mV by superfusion of a solution of bisoxonol with different Na+/K+ saline containing 10 $4 gramicidin (Reyes et al, 1994). Intracellular calcium was estimated after loading of the cells with Fluo-3 by incubating them for 30 mm at 33’C with 5 PM Fluo-3-AM. The cell fluorescence was determined at 18 * 3”C, from video images obtained with excitation wavelengths of 450-490 nm and emission wavelengths of > 520 run. The intracellular Ca2+ probe was calibrated with ionomycin, MnCl,, and digitonin as described by Kao et al (1989).
Thin section histological
techniques
The cells were pelleted in a micro centrifuge at 2000 g and fixed with Kamovsky’s reagent containing 2 % glutaraldehyde, 2 % formaldehyde, 5 % sucrose in 0.1 M cacodylate buffer (pH 7.3) (Kamosvky, 1965) for 2 h at 4°C. The cell pellet was washed twice for 4 h in 7% sucrose, 0.1 M cacodylate buffer (pH 7.3). Subsequently, the pellets were postfixed in 1% reduced osmium, dehydrated, and embedded in Epon. Thin sections (2 m) were stained with toluidine blue and examined by light microscopy.
Chemicals Hoechst 33342 (I&&, b&(1,3 dðyl) thiobarbiturate trimethine oxonol (bisoxonol), fluo-3, ethidium bromide and the substrate for alkaline phosphatase were obtained from Molecular Probes, Inc (Eugene, OR). Collagenase, DNAse, trypsin, Percoll, BSA, digitonin and the salts and buffers used were obtained from Sigma Chemical Co (St Louis, MO).
RESULTS Alkaline phosphatase activity in seminiferous tubule cells As described by Palombi and DiCarlo (1988), the peritubular (myoid) cells of the seminiferous tubule
have an alkaline phosphatase (AI?) activity (figs 3A, B, C). Using the Al’ substrate from Molecular Probes (Eugene, Or), the activity of the enzyme can be vitally determined and was localized only to peritubular cells and not to sub-adjacent seminiferous tubule cells (fig 3C) or spermatogenic cells (seen at the extruding ends of the tubule in figure 3B). In the mixed cell population obtained either by trypsin treatment or mechanical disruption of the seminiferous tubules, these alkaline phosphatase
Fig 3. A. Fluorescenceof the product of alkalinephosphatasereaction (performed at room temperature) in seminiferous ) tubulesisolated by collagenasetreatment. The excitation and emissionwavelengthswere 330-380 and > 420, respectively. The bar represents 100 pm. B. Optical transmissionphotomicrographof the seminiferoustubule depicted in A. Note the spermatogeniccells and spermatozoaextruding at the cut ends of the tubule. Other conditionswere similarto those of A. C. Fluorescencephotomicrographof a seminiferoustubulevitally stainedfor alkalinephosphataseactivity (yellow-green),and with H33342 to show the nuclear DNA (blue). Note that the layers of cells below the peritubular cells (in yellow green) are devoid of the alkalinephosphatasereaction product. The bar represents25 pm. Other conditionswere similarto those of A. On stage singlecell identification of rat spermatogeniccells
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On s;tage single cell identification of rat spermatogenic
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Biology of the Cell (1997) 89, 53-66
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positive cells were less than 0.5% of the total cell population (not shown), and hence they are unlikeiy to represent a significant contamination when using single cell physiological techniques.
Kinetics of H33342 binding to spermatogenic cell nucleus H 33342bound to nuclear material in intact spermatids with a half time of approximately 30 min On stage single cell identification of rat spermatogenic cells
(fig 4A). A similar time course of binding was observed with pachytene spermatocytes (not shown). The steady-state level of spermatid and pachytene H,,, fluorescence relative to permeabiIized spermatozoa fluorescence reached values of 0.5-0.7 and 2.3-2.8, respectively, when the cells were exposed to 2 ,uM H,,,,. In prepuberal spermatogenic cells (sperma3togonia-preleptotene spermatocytes and leptotene-zygotene spermatocytes), the time course of H 33342binding to the cell nucleus Reyes et al
Biology of the Cell (1997) 89, 53-66
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Time (min) Fig 4. a. Fractional increasein fluorescenceof H,,,,, in rat spermatidsas a function of time. The fluorescencereached at 70 min was arbitrarily given a value of 1.0. H3aaa2 concentrationwas 2 pm. The points and error bars represent the mean and standard deviation of six cells obtained in two different cell preparations.b. Changesin fluorescence of Ha,,, in rat prepuberal spermatogeniccells. Control data were obtained in a separate experiment with the same batch of cells. The HaaN concentration was 2 PM. The different symbolsrepresent individualcells in the same cell preparation. c. Fractional fluorescence in rat spermatidstreated with 10 pg/mL digitoninin Krebs-Henseleitbuffer. Ha,,, concenchangesin H33342 tration was 2 PM. The point and error bars represent the mean and standard deviation of six cells in three different cell preparations.
was characterized by a slow uptake. In these cells, the relative fluorescence (cell/spermatozoa) only reached steady values of 0.1-0.2 after 70 min of exposure to 2 w H,, (fig 4B). Treatment with digitonin (10 pg/mL) produced an increase in cell fluorescence that reached the expected values for diploid or tetraploid cells (fig 4B) in about 10 min. Exposure of rat spermatids or pachytene spermatocytes to digitonin, simultaneously with H,,,,, accelerated the appearance of nuclear fluorescence, reaching an equilibrium binding in about 10 min (fig 4C). In the experiments where the relative DNA content of the cells was estimated from the H,,,, fluorescence, this dye was allowed to equilibrate with the permeabilized cells for 30 min.
Relative DNA content of selected seminiferous
tubule cells
In order to validate the methodology to estimate the relative DNA content of the seminiferous tubule cells, we utilized cell populations that can be highly purified (> 90 % purity) such as pachytene spermatocytes and round spermatids. In these cells, the relative DNA contents obtained were 3.9 f 0.5 (N = 16) and 1.2 + 0.2 (N = 19), for On stage single cell identification of rat spermatogenic cells
pachytene spermatocytes and spermatids, respectively. The cell population obtained from fractions 20-25 of a BSA gradient of prepuberal (lZday-old) rat seminiferous tubule cells consisted of approximately 80 % spermatogonia A, judged from their prominent nucleolus and oval nucleus (fig SA, B; see also Bellve, 1977). The cells not presenting condensed chromosomes when stained with HW, had diameters of 14 f 1 m (N = 21, range 13-15 p). Those cells that are at M phase, and specifically at metaphase, can be easily recognized when stained with H,,,, (fig SC). These cells at metaphase were 14 + 1 p in diameter (N = 11, range 13-15 p) and, as expected for a 4 N cell at that stage of the cell cycle, their relative DNA content was 4.3 f 0.4 (N = 40). These data show that the relative DNA content of the cells estimated from the H,,,, fluorescence agreed with the expected ploidy of the seminiferous tubule cell populations, validating its use for relative DNA estimations in different spermatogenie cell populations. A similarly good agreement between expected ploidy and relative DNA content was found when we used polynucleated cells (symplasts) formed during the cell preparation (data not shown). Reyes et al
On stage single cell identification of rat spermatogenic cells
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Biology of the Cell (1997) 89, 5366
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Relative DNA content and cell diameter in purified spermatogenic cell populations In order to estimate the variance for the relative DNA determinations, we used purified populations of early primary spermatocytes (leptotenezygotene, 65-70 %), pachytene spermatocytes (85-90 %), and round spermatids (> 95 %) obtained by separation using velocity sedimentation at unit gravity in BSA gradients of rat spermatogenic cells obtained from prepuberal or adult rats (Romrell et al, 1976; Bellve, 1977). Besides using purified or enriched cell populations, the cells on which we performed relative DNA content determinations were chosen by their diameter (8-10, 16-20 and 11-13 m for leptotene/zigotene, pachytene and round spermatids, respectively), and by the appearance of the chromatin stained with H33342. After a transmitted light image was taken to measure cell diameter, and using the previously described permeabilization with digitonin and H33342 treatment, we determined the relative DNA content of single cells at different steps of the spermatogenesis. The estimated mean and standard deviations of DNA contents from at least three different cell preparations were 4.4 f 0.3 (N = 18), 3.9 + 0.5 (N = 16) and 1.2 f 0.2 (N = 19) for leptotene-zygotene spermatocytes, pachytene spermatocytes and round sperma tids, respectively. Use of ethidium bromide, instead of H,,,, for relative DNA determinations in spermatids and pachytene spermatocytes gave relative fluorescence values of 1.2. f 0.3 (N = 6), and 4.3 it 0.6 (N = 4), respectively. Thus, either dye yields comparable values of relative fluorescence.
The spermatogonia
A subpopulation
Our estimation of the relative DNA content of the spermatogonia A subpopulation not presenting condensed chromosomes (fig 5) showed that these cells had relative DNA contents from 2.5 to 4.6, suggesting that this population is composed of cells that were at different stages of the cell cycle. In fact, only lo-20 % of the cells appeared to be at G, as judged from their relative DNA content. The appearance of the cell chromatin stained with
H 33342showed a recognizable pattern, revealing an oval nuclei and a relatively homogenous chromatin distribution. Because they present a variable DNA content, we did not device a strategy for an independent confirmation of their identity in an unpurified cell population and on the microscope stage (see below).
Sertoli cell The size of Sertoli cells increases during development in rodents (eg Bellve, 1977). In adult rats, their reported diameter is in the range of 12-15 ,um (Floridi et al, 1983; Qian et al, 1985). In our experience, in adult rats, the cells that presented an irregular nucleus when stained with H,,, had diameters of 13.0 f 1.1 p (N = 13)(fig 6A, B). These cells had relative DNA contents of 2.3 + 0.3 (N = 5). In 12day-old rats, the cells with irregular nuclei and characteristic heterochromatin, showed diameters ranging from 9-12 p in cells stained with H,,,, or thin sections stained with toluidine blue (see also Bucci et al (1986)).
A bivariable distribution of seminiferous tubule cells obtained from prepuberal and adult rats The bivariable (relative cell DNA vs diameter) distribution of all the data obtained in purified, partially purified populations or non-purified cell populations (N = 299) is shown in figure 7. This figure also shows window zones where the abscissas were set using the diameters for isolated spermatogenic cells described in the literature where light or electron microscopy criteria of identification have been used (Romrell et al, 1976; Bell&, 1977; Meistrich et al, 1981; Bucci et al, 1986), as well as from our estimations of cell diameters from optic microscopy of thin sections of enriched fractions stained with toluidine blue or from transmission electron microscopy photomicrographs. The ordinate window widths were set using the mean and standard deviations established in our determinations of relative DNA content. The ordinate for the spermatogonia B/preleptotene subpopulation was established assuming a relative DNA content of 2 and using the average variation coefficient obtained in all the purified cell populations (11 + 4 %, N = 4). In
4 Fig 5. A. Spermatogonia A cell population isolated using cell sedimentation in a BSA gradient at unit gravity and stained with 4 PM Ha,,, for 35 min. The bar represents 10 pm. B. Transmission photomicrograph of a thin section stained with toluidine blue of the spermatogonia A population isolated using cell sedimentation in a BSA gradient at unit gravity. The bar represents 10 pm. C. Cells of the spermatogonia A BSA gradient fraction showing cells in prophase and metaphase. Other conditions were similar to A. The bar represents 10 pm. On stage single cell identification of rat spermatogenic
cells
Reyes et al
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Biology of the Cell (1997) 89, 53-66
Fig 6. A. Transmission photomicrograph of a thin section of a BSA gradient cell fraction stained with toluidine blue, showing Sertoli cells (arrow heads) with the typical irregular nucleus. The bar represents 10 pm. B. Fluorescence photomicrograph of non-purified seminiferous tubule cells stained with 4 PM H,,,,. Note the cell at the center and above (marked) showing a highly irregular nucleus typical of the Sertoli cell. The bar represents 10 pm.
these zones, there is a high probability of correctly classifying a cell as spermatogonia B/preleptotene, leptotene-zygotene primary spermatocytes, pachytene primary spermatocytes and round spermatids from their simultaneous diameter and relative DNA determination (see Discussion). On stage single cell identification of rat spermatogenic
cells
A guessing game for non-purified spermatogenic cell populations In order to estimate the confidence with which, in non-purified cell populations, a cell can be identified only by the appearance of the chromatin vitally Reyes et al
Biology of the Cell (1997) 89, 53-66
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Fig 7. Plot of relative H,,,,, fluorescence (relative DNA content) versus cell diameter for rat spermatogenic cells. This plot shows all the data obtained in purified, enriched and non-purified cell populations. The discriminating windows define four developmental steps in the spermatogenesis. The ranges in the ordinate of the windows were established as the mean f twice the standard deviation (except in leptotene-zygotene spermatocytes and spermatids where the upper ranges were set to f 1 S and + 1.25 S-2 S, respectively) of the relative DNA content measurements obtained in purified spermatogenie cells. The range of the window abscissa was set equal to the range of diameters described in the literature for each group of spermatogenic cells and from our estimations of the diameters in purified cell preparations and from thin sections stained with toluidine blue.
transmission stained with Ha,,,,, we acquired images of cells in the field of the microscope, labeled them as the guesstimated cell type according to their chromatin-H33342 appearance, and subsequently performed a relative DNA content estimation in the same cells. For ‘pachytene spermatocytes’ and ‘round spermatids’, 57 % (N = 44) and 80 % (N = 52) of the cells, respectively, relabeled as indicated, had relative DNA contents in the range of the mean + 2SD as estimated in purified cell populations (see above). In contrast, for preIabeled ‘spermatogonia B/preleptotene’ and ‘leptotene-zygotene spermatocytes’, 33 and 20 % of the cells, respectively, fell within the range mean -+ 2SD for relative DNA content estimated in purified cell populations. As described above, in the case of spermatogonia B and early primary spermatocytes in the absence of digitonin, there is also a low uptake of Hs=, making the guesstimate of these cell types less reliable. Thus, our data indicate that round spermatids can be identified by the appearance of their chromatin vitally stained with H,,. Instead, pachytene spermatocytes, classified only by their prophase chromatin appearance, seem to include also cell populations of either fused early primary spermatocytes or a likely spermatogonia A (secondary spermatocyOn stage single cell identification of rat spermatogenic
cells
tes?) subpopulation in the prophase stage of the cell cycle. Narrowing the guesstimate to cells that are 15-20 pm in diameter can increase the correspondence between chromatin appearance and relative DNA content of pachytene spermatocytes to 66% (N = 32). Type B spermatogonia and leptotenezygotene spermatocytes require a confirmation of their identity by determination of their relative DNA content and cell diameter.
Determinations of physiological parameters in conjunction with relative DNA content and cell diameter in rat spermatids We have utilized bisoxonol, a potential-sensitive dye, in conjunction with H33342to determine plasma membrane potentials in purified rat spermatids (-57 + 16 mV, N = 20). The identification protocol was performed by exposing the cells to KH-digitoafter the determination of the membrane ~-Hw4, potential. In these conditions, the bisoxonol concentration in the medium was decreased to 10 nh4 (not shown) and did not present interference with the use of bisbenzimide dyes for DNA determination. Under these conditions, the relative H33342 fluorescence was 1.1 ? 0.2 (N = 26), a value not signifiReyes et al
64
cantly different from that obtained in the absence of oxonol. A similar protocol was utilized to estimate (Ca2+)i with the fluorescent probe Fluo-3, giving a resting intracellular Ca2+ concentration of 87 + 20 nM (N = 10) and a relative HBX2 fluorescence value of 1.1 + 0.2.
DISCUSSION The study of the physiology and regulation of mammalian spermatogenesis, and some molecular approaches to this cell differentiation process have been hindered because of the difficulties of combining, at the single cell level and in the same cell, measurements of cell function and identification at different steps of spermatogenesis. Although the classical histology techniques used to identify spermatogenic stages are well worked out (eg deK.retser and Kerr, 1988), they are not generally applicable to studies where a single cell measurement has been made, and the same cell must be identified before removing it from the microscope field. Thus, it is difficult if not impossible to identify and functionally study the same cell using these techniques. In this work, we show that the labeling of the cell DNA with H,,,,, and the simultaneous measurement of cell diameter and relative DNA content of spermatogenic cells allows for the classification of the cells and the sequential use of other fluorescent probes to estimate physiological parameters or use of single cell electrophysiological and molecular biological techniques. In order to achieve equilibrium binding of the DNA probes and to decrease the time needed for estimation of cell relative DNA content, we found that the use of 10 pg/mL of digitonin was adequate. Higher than 20 pg/mL digitonin concentrations (not shown) gave values of relative fluorescence that differed substantially from the expected relative DNA content of spermatogenic cells. Lower digitonin concentrations did not permeabilize the entire cell population. As suggested for the effect of digitonin on nuclear Hsssa2 fluorescence, the low accessibility of Hsssd2 to premeiotic and early meiotic cell nuclei was limited at the level of the plasma membrane. Whether this restricted entry was due to a decreased membrane permeability for H,,,,,, or reflects the existence of active efflux system in these cells (eg Neya H33342 fakh, 1988) can not be elucidated from our data. The existence of a multidrug transport system is also suggested from the resistance of premeiotic spermatogenic cells to the action of extracellular hydrophobic drugs (Sjoblom et al, 1996). These authors found that premeiotic spermatogenic cells have low sensitivity to vinblastine, a drug which, On stage singlecell identification of rat spermatogeniccells
Biology of the Cell (1997) 89, 53-66
like H33342, is a substrate for the P-glycoprotein multidrug transporter. Ethidium bromide, an intercalating DNA probe, gave values of relative fluorescence that did not differ significantly from the values obtamed with H,,,,,. This result allows, in principle, to extend the use of the spermatogenie cell identification method proposed here in conjunction with the use of cell physiology fluorescent probes with excitation wavelengths in the near W range (eg SBFI, PBFI, fura-2, DPH). Visualization of nuclear material stained with H 33342in single cells also permits the identification of bi- or polynucleated cells formed during cell preparation. We have found that visibly polynucleated cells consist mainly of spermatid nuclei. Figure 1 shows some of the cell cycle transitions associated with the seminiferous tubule cells, such as preleptotene S phase (2 N -+ 4 N, 7-10 pm in diameter), type B spermatogonia mitotic S, G,, and M phases (2 N w 4 N cycling, 7-10 pm in diameter), or the mitotic S, G, and M phases of the cell cycle in spermatogonia A (2 N t) 4 N cycling, 12-15 ,um in diameter). This consideration can, in principle, limit the effective regions of interest in plot of ploidy ~1scell diameter, narrowing the late primary spermatocyte zone to 16-20 pm pachytene spermatocytes. Spermatogonia B are likely to spend around 30 % of its cell cycle in the DNA content region where it could be classified erroneously as leptotene-zygotene spermatocytes by cell diameter and relative DNA content (late S, G, phase and mitosis; (Bell&, 1979)). This spermatogonia B population in lo-14-day-old mice represents 30-40 % of a mixed leptotene/spermatogonia B population (Bell&, 1977). In these conditions, the probability of randomly finding a spermatogonia B in the region 4.4 + 0.3 relative DNA units and 7-12 pm area (leptotenelzygotene zone) is likely to be less than 15 % in non-purified prepuberal rat spermatogenie cells. In spite of the fact that Sertoli cells (cell diameters 9-12 pm and 12-15 pm for prepuberal and adult rats, respectively) could appear as interfering in the classification of spermatogenic cells in a plot of relative DNA content us cell diameter, their highly irregular nuclei allow to discard these cells (and any other irregularly nucleated cell) by simple appearance of their chromatin stained with H,,,,,. Thus, in a plot of relative DNA content us cell diameter (fig 7), we defined four regions of interest, representing different steps of spermatogenesis: 1) premeiotic diploid cells (7-11 ,um, spermatogonia B-preleptotene spermatocytes); 2) tetraploid primary spermatocytes (7-l 1 m, leptotene-zygotene spermatocytes); 3) advanced tetraploid priReyes et al
Biology of the Cell (1997) 89, 53-66
mary spermatocytes (16-20 pm, pachytene spermatocytes); and 4) haploid round spermatids (lo-14 pm). The range of the ordinate in each group was set to * 2 SD, except leptotene-zygotene spermatocytes where a f 1 SD window was chosen, and in spermatids, where an asymmetric range of -2 SD and +1.25 SD was utilized. These ranges allow > 95% confidence (except in the leptotene-zygotene spermatocyte region) to state that a random determination falling in those regions belongs to that cell subgroup and not to the other classes of spermatogenic cells. Acceptance of the cell classification presented here also establishes criteria to discard or accept data that, in part, could originate from being biased by the investigator in choosing a cell type by simple microscopic appearance or by easiness of manipulation in a certain single cell technique. The visualization of the cell nucleus stained reveals characteristic patterns of hetwith %,X2 ero- and euchromatin in spermatogenic cells that can orient the investigator to the cell type on which to perform the cell physiological measurement. However, these patterns of the chromatin stained with H33342 are relatively reliable only in the case of round spermatids. The classification method proposed in this paper provides an unbiased confirmation of the class of spermatogenie cell chosen. The cell classification presented here spans an important part of the spermatogenesis and thus permits a systematic approach to study the physiology and molecular biology of the differentiation process at the single cell level. This single cell classification can be used in unpurified cell populations, avoiding trypsinization that removes membrane proteins involved in spermatogenic cell physiology and cell-cell interactions (Jegou, 1993). Furthermore, this strategy for cell classification needs minute amounts of biological material allowing, in principle, the application of single cell physiological and cell molecular biological techniques to studies of the spermatogenesis in most species, using biopsy methods for tissue sampling.
ACKNOWLEDGMENTS We thank Dr E: Couve and Mr F Vargas for performing the histological techniques and TEM analysis of our preparations. This work was funded by DGI-UCV and Fondecyt 1960398/96
REFERENCES Aravindan GR, Pineau CP, Bardin CW and Cheng CY (1996) Ability of trypsin in mimicking germ cell factors that affect Sertoli cell secretory function. J Cell Physioll68,123-133 Arndt-Jovin DJ and Jovin TM (1989) Fluorescence labeling and
On stage single cell identification of rat spermatogenic cells
65
microscopy of DNA. In: Fluorescence Microscopy of Living Cells in Culture (Taylor DC, Wang Y, eds) Methods Cell Biol 3tJ417-448 Bell& AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar YM and Dym M (1977) Spermatogenic cells of the prepuberal mouse: Isolation and morphological characterization. 1 Cell Biol74,68-85 Bellve AR (1979) The molecular biology of mammalian spermatogenesis. In: Oxford Reuiews of Reproductive Biology (Finn CA, ed) Clarendon Press, Oxford, UK, 159-261 Braun RE, Lee K, Shumacher JM and Fajardo MA (1995) Molecular genetic analysis of mammalian spermatid differentiation. Ret Prog Horm Res 50,275286 Bucci LR, Brock WA, Johnson TS and Meistrich ML (1986) Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes. Biol Reprod 34,195-206 Cowell JK and Franks LM (1980) A rapid method for accurate DNA measurements in single cells in situ using a simple micro fluorimeter and Hoechst 33258 as a quantitative fluorochrome. J Histochem Cytochem 28,:206-210 D’Agostino A, Monaco L, Stefanini M and Geremia R (1984) Study of the interaction between germ cells and Sertoli cells in vitro. Exp Cell Res 150,430-435 de Kretser DM and Kerr JB (1988) The cytology of the testis. In: The Physiology of Reproduction (Knobil E, Neil1 J, eds) Raven Press, New York, 837-932 Ehrenberg B, Montana V, Wei MD, Wuskell JP and Loew LM (1988) Membrane potentials can be determined in individual cells from the Nernstian distribution of cationic dyes. Biophys 153‘785-794 Floridi A, Mercante ML, D’Atri S, Feriozzi R, Menichini R, Citro G, Cioli V and De Martin0 C (1983) Energy metabolism of normal and Lonidamine-treated Sertoli cells of rats. Exp Mel Path01 38,137-147 Gottschalk-Sabag SH, Weiss DB and Sherman Y (1995) Assessment of spermatogenic process by deoxyribonucleic acid image analysis. Fertil Steril64,403-407 Grabske RJ, Lake S, Gledhill BL and Meistrich ML (1975) Centrifugal elutriation: Separation of spermatogenic cells on the basis of sedimentation velocity. J Cell Physiol 86, 177-190 Grogan W MC, Farnham WF and Saban J (1981) DNA analysis and sorting of viable mouse testis cells. J Histochem Cytothem 29,738-746 Grotegoed JA, Den Boer PJ and Mackenbach P (1989) Sertoligerm cell communication. Ann NY Acad Sci 564,232-242 Hagiwara S and Kawa K (1984) Calcium and potassium currents in spermatogenic cells dissociated from rat seminiferous tubules. J Physiol356,135-149 Jegou B (1993) The Sertoli-germ cell communication network in mammals. lnt Rev Cytoll47,25-96 Kao JPY, Harootunian ATH and Tsien RY (1989) Photochemitally generated calcium pulses and their detection by Fluo3.1 Biol Chem 264,8179-8184 Karnovsky M J (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27,137a Lam DMK, Furrer R and Bruce WR (1970) The separation, physical characterization and differentiation kinetics of spermatogonial cells from mouse. Proc Natl Acad Sci LISA 65,192-199 Lievano A, Santi CM, Serrano CJ, Triviiio CL, Bellve AR, Hernandez-Cruz A and Darszon A (1996) T-type Ca2+ channels and alE expression in spermatogenic cells and their possible relevance to the sperm acrosome reaction. FEBS Lett 388,150-l.% Meistrich ML, Longtin J, Brock WA, Grmes SR and Mace ML (1981) Purification of rat spermatogenic cells and preliminary biochemical analysis of these cells. Biol Reprod 25, 1065-1077 Neyfakh AA (1988) Use of fluorescent dyes as molecular probes for the study of multidrug resistance. Exp Cell Res 174,168-176 Reyes et al
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Palombi F and DiCarlo C (1988) Alkaline phosphatase is a marker for myod cells in cultures of rat peritubular and tubular tissue. Biol Reprod 39,1101-1109 Qian Z, Tsai Y, Steinberger A, Lu M, Greenfield ARL and Haddox MK (1985) Localization of omithine decarboxylase in rat testicular cells and epididymal spermatozoa. Biol Reprod 33,1189-1195 Reichter Jr LE and Dattatreyamurty B (1989) The follicle-stimulating hormone (FSH) receptor in testis: Interaction with FSH, mechanisms of signal transduction and properties of the purified receptor. Biol Reprod 40,13-26 Reyes JG, Velarde MV, Ugarte R and Benos DJ (1990) The glycolytic component of rat spermatid energy and acid-base metabolism. Am J PhysioZ259, C66O-C667
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Reyes JG, Bacigalupo J, Araya R and Benos DJ (1994) Ion dependence of resting membrane potential of rat spermatids. J Repd Ferfil 102313-319 Romrell LJ, Bellve AR and Fawcett DW (1976) Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dar BioZ49,119-131 Sjoblom T, Parvinen M and Lahdetie J (1996) Stage-specific DNA synthesis of rat spermatogenesis as an indicator of genotoxic effect of vinblastine, mitomycin C and ionizing radiation on rat spermatogonia and spermatocytes. Mutat Res 331,181-190
Received 27 January 1997; accepted 31 March 1997
Reyes et al
53
Original article
On stage single cell identification
of rat spermatogenic
cells
Juan G Reyes, Alvaro Diaz, Nelson Osses, Carlos Opazo and Dale J Benos * hstituto de Quimica, Universidad Catolica de Valparaiso, Valparaiso; Depto Fisiologia y Biofisica, facultad de Medicina, U de Chile, Santiago, Chile; Department of Physiologyand Biophysics, H-IS6 704, the Universityof Alabama at Birmingham,1918 UniversityBoulevard,Birmingham,Al 35294-0005, USA
The study of spermatogenic cell physiology has been hindered by the absence of unbiased methods of identification of cells upon which single cell techniques are being applied. In this work, we have used histochemical techniques, digital videoimaging, quantification of chromatin-bound DNA probes, and measurements of cell diameter to identify single spermatogenic cells at different periods of development. Our criteria of identification permit the definition of four developmental stages of spermatogenesis on which to perform single cell analyses: spermatogonia B/preleptotene spermatocytes, lfptqtene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids, The use of voltage-sensitive dyes and Caz+-sensitive dyes does not interfere with the estimations of DNA content. The estimations of DNA content of spermatogenic cells can be performed both ) and long wavelength-excited with near-UV excited dyes (H= dyes (ethidium bromide), allowing the use of a wide range of physiological and immunocytochemical fluorescent probes to study the sperrnatogenic process.
testicle / seminiferous tubule / cell cycle / cell differentiation / male
INTRODUCTION Mammalian spermatogenesis takes place in the seminiferous tubule in the testis. This process begins on the serosal side of the seminiferous tubule with mitotic events of primordial cells and spermatogonia (A and B), followed by differentiation of spermatogonia B into preleptotene spermatocytes. These cells are characterized by a ploidy of 2 N and approximately 5 pm difference in cell diameter (Romrell et al, 1976; Bell&, 1977). Figure 1 shows the .schematiL; representation of the spermatogenie process in a plot of ploidy zwsus cell diameter. The progression of preleptotene spermatocytes into meiosis implies an S phase to reach a ploidy of 4 N, migration through the Sertoli cell tight junctions, and the entrapce into the prolonged meiotic prophase (leptoten&zygotene-pachytene sperma-
* Correspondence
and reprints
On stage single cell identification of rat spermatogenic cells
tocytes) with dramatic changes in cell size. Two successive meiotic divisions, generating secondary spermatocytes and round spermatids, are characterized by reductions of size and ploidies of 2 N and 1 N, respectively (Bellv4, 1979; deKretser and Kerr, 1988; Braun et al, 1995). Spermatogenesis is completed with remarkable changes in cell shape, organelle rearrangement, and cytoplasmic shading that accompany the spermiogenesis. In spite of the importance of the spermatogenic process, the study of the physiology of the spermatogenic cells and its regulatory aspects have only lately been approached more systematically (Hagiwara and Kawa, 1984; Reyes et al, 1990; Reyes et al, 1994; Lievano et al, 1996). Part of this delay can be understood because there are mainly two populations of spermatogenic cells that can be obtained in sufficient quantity and purity to be studied with averaging methods of cell physiology research: pachytene spermatocytes and round spermatids. Besides the fact that mainly the late meiotic stages can be studied in purified cell prepReyes et al
54
Biology of the Cell (1997) 89, 53-66
pachytene dyplotene
leptotene-zygotene 4
B spermatogonia
A spermatogonia
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Cell diameter (pm) Fig 1. Plot of ploidy versus cell diameter in the premeiotic and meiotic spermatogenic process. The values of cell diameters for each cell type were taken from the range of values published in the literature.
arations, the methodology to obtain pure enough populations of these and other spermatogenic cells require protease treatments (Lam et al, 1970; Grabske et al, 1975; Romrell et al, 1976) that are known to remove receptors and other proteins from cell membranes (D’Agostino et al, 1984; Reichter and Dattatreyamurty, 1989), or to mimic cell-cell interactions (Grotegoed et al, 1989; Aravindan ef al, 1996). In contrast to these shortcomings of the averaging methods to study spermatogenie cell physiology, powerful methodologies are available for single cell physiological and molecular biological studies of spermatogenic cells (eg video transmission and fluorescence microscopy, patch-clamp and single cell oligonucleotide amplification techniques). Because these methods do not require pure populations, they could avoid the use of extensive protease digestion of the seminiferous tubules. However, the use of these single cell methodologies requires, cell identification, and, even in ‘purified’ populations, ways to avoid the almost inevitable bias of choosing an inappropriate popuiation by their prominence or ease of manipulation. The usefulness of identification schemes for On stage single cell identification of rat spermatogenic cells
single cells requires them to be performed on the microscope stage simultaneously or subsequently to measurements of the physiological parameter of interest. The development of fluorescent probes to study quantitatively the cell content of macromolecules and of inexpensive video microscopy and image analysis techniques prompted our efforts to devise a strategy to identify, on the microscope stage, spermatogenic cells at different steps of development based on the differences in cell DNA structure and content, and the changes in cell size occurring during this differentiation process (Cowell and Franks, 1980; Grogan et al, 1981; Arndt-Jovin and Jovin, 1989; GottschalkSabag et al, 1995). We have also utilized cytochemical techniques to identify the presence of peritubular cells in the seminiferous tubule cell preparation. Vital staining with H,,,,, and a guessstimate of the respective cell type by the appearance of the chromatin, followed by a quantitation of the relative DNA content in the same cell shows that there is a close correspondence between both criteria of cell classification for rat spermatids, but not so for spermatogonia or primary spermatocytes. The use of a bivariable Reyes et al
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system of classification of seminiferous tubule cells allows us to define regions in a plot of relative DNA content versus cell diameter where there is a high probability of correctly classifying cells as spermatogonia B/preleptotene, leptotenezygotene primary spermatocytes, late pachytene primary spermatocytes or spermatids.
MATERIALS AND METHODS Rat spermatogenic
cell preparation
Rat seminiferous tubule cell populations were prepared from testicles of adult (60 days) or 12-14-day-old rats, as described by Romrell et al (1976) and Bellve et al (1977). The resulting cell suspensionwas filtered through a layer of cotton and 250 and 70 p nylon meshesand washed three times in Krebs-Henseleit10 mM Hepes, 10 mM (KH) lactate medium. The mixed seminiferoustubule cells were used either without purification, partially purified by passing the cells through a 2% BSA/15%/30% Perco11discontinuous gradient, or, the different cell populations were purified using sedimentation velocity in a 260mL 24 % BSA in K-H medium gradient at unit gravity and at 16 + 2°C (Bell&, 1977).The cells were allowed to settle for 3 II and then the BSA gradient was collected in 3 mL fractions at approximately 1 fraction per minute.
Measurements of cell diameter and DNA content using transmission and fluorescence digital video microscopy
Fig 2. A. Image of H,,,,, fluorescence in rat round spermatids and spermatozoa.The cells were exposedto 2 PM HSSSJ2 and 15 &mL digitonin in Krebs-Henseleitbuffer for 30 min and at room temperature (18 * 2°C). The imagewas taken with a cooled CCDvideo camera using a 40 x NA 0.85 objective and 0.1 s of exposure. The excitation and emissionwavelengthswere 330-380 and > 420, respectively. The bar represents 15 pm. B. Photomicrograph of a pachytene spermatocyte, a round spermatid and a trinucleated symplast. The ceils were exposed to 2 PM H,,,, in Krebs-Henseleitbuffer for 40 min at room temperature. The bar represents25 pm.
The studies of video microscopy were performed in an inverted Nikon Diaphot microscope with epifluorescence.A cooled CCD 12 bit video camera (Spectrasource, Los Angeles, CA) was attached to the videoport of the microscope. The image analyses were performed with the appropriate software using a 486 IBM compatible computer. Cell diameter measurementswere made by previously calibrating the optical and digital analysis systemin pixels/pm using a reticulated slide, and further calibrated using normal human red blood ceils. The within-assay variation coefficients for diameter determination of pachytene spermatocytesand round spermatids were 3 and 1 %, respectively. The non-intercalating bisbenzimide DNA probe H3= (excitation wavelengths 330-380 nm, emission wavelengths > 420 run) or ethidium bromide (excitation wavelengths 450490 run, emissionwavelength > 520nm), were used to estimate the DNA content of the cells.Becauseof the intrinsic difficulties of estimatingthe absolutevalues of cell DNA using fluorescentprobes(Amdt-Jovin and Jovin, 1989),we usedpermeabilized rat spermatozoaas an internal standard with constant ploidy (1 N) and an easily recognizable shapein image analysis(fig 2A). The spermatozoa were obtained from the cauda epididymis of rats by puncturing and gently pressing this organ. In order to remove the tails of the spermatozoaand to permeabilize them, we treated the spermatozoacell suspensionwith five sequencesof 10s sonicationpulses(BransonEl2 80 watts, Shelton, CO). To obtain a homogeneouslypermeabilized
spermatozoapopulation, treatment with 0.1% Triton X-100 saline, three washesand a further cycle of freezing and thawing was necessary.Thosecellswith two or more visible nuclei generatedduring the cell preparation were identified visually and either discarded from our analysis or utilized asinternal standardsfor quantitative DNA determinations(fig 2B).The within assayvariation coefficient of H,, fluorescenceof spermatozoa,spermatidsand pachytene spermatocyte determinations using a 0.85 NA 40 x objective,were 9,12, and 8%, respectively.The useof a 1.35 NA 100x oil immersion objective gave for the samecell types variation coefficientsof 8, 7, and lo%, respectively. Becauseeither objective gave comparablevariation coefficients in the HWZ fluorescence determination, but the 100x objective gave a limited number of cells in the field, we choseto usethe 0.85NA 40 x objective for all the quantitative estimationsof relative total cell DNA content.
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Membrane potential and intracellular Ca*+ determinations in single rat spermatids In order to test the feasibility of performing physiological measurements and cell identification, we have successfully used the following protocol: 1) In dim light conditions, we let the cells (previously loaded with calcium sensitive dyes when appropriate) settle for 10 min in a microscope chamber (0.7 mL) having a coverslip as the bottom wall. Alternatively, the cells were adhered to a polylysine-covered coverslips which were attached to the bottom of the chamber with a minimal amount of silicone grease. Subsequently, ethidium bromide (5 PM final concentration) was added to the microscope chamber. 2) After 2-3 min of exposure to ethidium bromide, the unattached cells and the ethidium bromide solution were washed away by perfusing 10 mL (2 mL /mm) of K-H lactate buffer containing 2 @4 H33342. 3) 43342 was allowed to enter the cells for 30 min without perfusion. 4) The cells were selected for observation by taking a transmission image in order to estimate cell diameter, and by fluorescence observation of the chromatin stained with H33742. Those cells with a damaged membrane (stained violet by ethidium bromide and Ha& were easily identified and discarded. 5) The cells were exposed for 30 min in the dark to potential sensitive probes when membrane potential was to be determined. 6) At the appropriate excitation and emission wavelengths, a series of images were taken and stored, and calibrations of the fluorescent probes were performed. 7) The cells were perfused with 10 mL of K-H lactate buffer with 10 pgg/mL digitonin and 2 PM HsM2, and the dye was allowed to equilibrate for 30 min. Two images of H 33342stained cells were taken at 25 and 30 min to check for the effective equilibration of the dye. Subsequently, 5-10 H,,,,, images of spermatozoa were taken and stored. 8) The relative DNA content was calculated from the ratio of the cell DNA-H,, fluorescence and the average spermatozoa DNA-H-, fluorescence (interspermatozoa variation coefficient of lo-15%). The plasma membrane potential of rat spermatids was estimated at 18 + 3°C from the intra-extracellular distribution of bisoxonol determined in fluorescence video images taken with excitation wavelengths of 510-560 nm and emission wavelengths of > 590 nm (Ehrenberg et al, 1988). In order to estimate the out-of-focus extracellular fluorescence we utilized neutral tetramethylrhodaminedextran (M, 70 000) as an impermeant fluorescent probe.
Biology of the Cell (1997) 89, 53-66
The bisoxonol fluorescence distribution was calibrated for each cell at 0 mV and -100 mV by superfusion of a solution of bisoxonol with different Na+/K+ saline containing 10 $4 gramicidin (Reyes et al, 1994). Intracellular calcium was estimated after loading of the cells with Fluo-3 by incubating them for 30 mm at 33’C with 5 PM Fluo-3-AM. The cell fluorescence was determined at 18 * 3”C, from video images obtained with excitation wavelengths of 450-490 nm and emission wavelengths of > 520 run. The intracellular Ca2+ probe was calibrated with ionomycin, MnCl,, and digitonin as described by Kao et al (1989).
Thin section histological
techniques
The cells were pelleted in a micro centrifuge at 2000 g and fixed with Kamovsky’s reagent containing 2 % glutaraldehyde, 2 % formaldehyde, 5 % sucrose in 0.1 M cacodylate buffer (pH 7.3) (Kamosvky, 1965) for 2 h at 4°C. The cell pellet was washed twice for 4 h in 7% sucrose, 0.1 M cacodylate buffer (pH 7.3). Subsequently, the pellets were postfixed in 1% reduced osmium, dehydrated, and embedded in Epon. Thin sections (2 m) were stained with toluidine blue and examined by light microscopy.
Chemicals Hoechst 33342 (I&&, b&(1,3 dðyl) thiobarbiturate trimethine oxonol (bisoxonol), fluo-3, ethidium bromide and the substrate for alkaline phosphatase were obtained from Molecular Probes, Inc (Eugene, OR). Collagenase, DNAse, trypsin, Percoll, BSA, digitonin and the salts and buffers used were obtained from Sigma Chemical Co (St Louis, MO).
RESULTS Alkaline phosphatase activity in seminiferous tubule cells As described by Palombi and DiCarlo (1988), the peritubular (myoid) cells of the seminiferous tubule
have an alkaline phosphatase (AI?) activity (figs 3A, B, C). Using the Al’ substrate from Molecular Probes (Eugene, Or), the activity of the enzyme can be vitally determined and was localized only to peritubular cells and not to sub-adjacent seminiferous tubule cells (fig 3C) or spermatogenic cells (seen at the extruding ends of the tubule in figure 3B). In the mixed cell population obtained either by trypsin treatment or mechanical disruption of the seminiferous tubules, these alkaline phosphatase
Fig 3. A. Fluorescenceof the product of alkalinephosphatasereaction (performed at room temperature) in seminiferous ) tubulesisolated by collagenasetreatment. The excitation and emissionwavelengthswere 330-380 and > 420, respectively. The bar represents 100 pm. B. Optical transmissionphotomicrographof the seminiferoustubule depicted in A. Note the spermatogeniccells and spermatozoaextruding at the cut ends of the tubule. Other conditionswere similarto those of A. C. Fluorescencephotomicrographof a seminiferoustubulevitally stainedfor alkalinephosphataseactivity (yellow-green),and with H33342 to show the nuclear DNA (blue). Note that the layers of cells below the peritubular cells (in yellow green) are devoid of the alkalinephosphatasereaction product. The bar represents25 pm. Other conditionswere similarto those of A. On stage singlecell identification of rat spermatogeniccells
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On s;tage single cell identification of rat spermatogenic
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positive cells were less than 0.5% of the total cell population (not shown), and hence they are unlikeiy to represent a significant contamination when using single cell physiological techniques.
Kinetics of H33342 binding to spermatogenic cell nucleus H 33342bound to nuclear material in intact spermatids with a half time of approximately 30 min On stage single cell identification of rat spermatogenic cells
(fig 4A). A similar time course of binding was observed with pachytene spermatocytes (not shown). The steady-state level of spermatid and pachytene H,,, fluorescence relative to permeabiIized spermatozoa fluorescence reached values of 0.5-0.7 and 2.3-2.8, respectively, when the cells were exposed to 2 ,uM H,,,,. In prepuberal spermatogenic cells (sperma3togonia-preleptotene spermatocytes and leptotene-zygotene spermatocytes), the time course of H 33342binding to the cell nucleus Reyes et al
Biology of the Cell (1997) 89, 53-66
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Time (min) Fig 4. a. Fractional increasein fluorescenceof H,,,,, in rat spermatidsas a function of time. The fluorescencereached at 70 min was arbitrarily given a value of 1.0. H3aaa2 concentrationwas 2 pm. The points and error bars represent the mean and standard deviation of six cells obtained in two different cell preparations.b. Changesin fluorescence of Ha,,, in rat prepuberal spermatogeniccells. Control data were obtained in a separate experiment with the same batch of cells. The HaaN concentration was 2 PM. The different symbolsrepresent individualcells in the same cell preparation. c. Fractional fluorescence in rat spermatidstreated with 10 pg/mL digitoninin Krebs-Henseleitbuffer. Ha,,, concenchangesin H33342 tration was 2 PM. The point and error bars represent the mean and standard deviation of six cells in three different cell preparations.
was characterized by a slow uptake. In these cells, the relative fluorescence (cell/spermatozoa) only reached steady values of 0.1-0.2 after 70 min of exposure to 2 w H,, (fig 4B). Treatment with digitonin (10 pg/mL) produced an increase in cell fluorescence that reached the expected values for diploid or tetraploid cells (fig 4B) in about 10 min. Exposure of rat spermatids or pachytene spermatocytes to digitonin, simultaneously with H,,,,, accelerated the appearance of nuclear fluorescence, reaching an equilibrium binding in about 10 min (fig 4C). In the experiments where the relative DNA content of the cells was estimated from the H,,,, fluorescence, this dye was allowed to equilibrate with the permeabilized cells for 30 min.
Relative DNA content of selected seminiferous
tubule cells
In order to validate the methodology to estimate the relative DNA content of the seminiferous tubule cells, we utilized cell populations that can be highly purified (> 90 % purity) such as pachytene spermatocytes and round spermatids. In these cells, the relative DNA contents obtained were 3.9 f 0.5 (N = 16) and 1.2 + 0.2 (N = 19), for On stage single cell identification of rat spermatogenic cells
pachytene spermatocytes and spermatids, respectively. The cell population obtained from fractions 20-25 of a BSA gradient of prepuberal (lZday-old) rat seminiferous tubule cells consisted of approximately 80 % spermatogonia A, judged from their prominent nucleolus and oval nucleus (fig SA, B; see also Bellve, 1977). The cells not presenting condensed chromosomes when stained with HW, had diameters of 14 f 1 m (N = 21, range 13-15 p). Those cells that are at M phase, and specifically at metaphase, can be easily recognized when stained with H,,,, (fig SC). These cells at metaphase were 14 + 1 p in diameter (N = 11, range 13-15 p) and, as expected for a 4 N cell at that stage of the cell cycle, their relative DNA content was 4.3 f 0.4 (N = 40). These data show that the relative DNA content of the cells estimated from the H,,,, fluorescence agreed with the expected ploidy of the seminiferous tubule cell populations, validating its use for relative DNA estimations in different spermatogenie cell populations. A similarly good agreement between expected ploidy and relative DNA content was found when we used polynucleated cells (symplasts) formed during the cell preparation (data not shown). Reyes et al
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Relative DNA content and cell diameter in purified spermatogenic cell populations In order to estimate the variance for the relative DNA determinations, we used purified populations of early primary spermatocytes (leptotenezygotene, 65-70 %), pachytene spermatocytes (85-90 %), and round spermatids (> 95 %) obtained by separation using velocity sedimentation at unit gravity in BSA gradients of rat spermatogenic cells obtained from prepuberal or adult rats (Romrell et al, 1976; Bellve, 1977). Besides using purified or enriched cell populations, the cells on which we performed relative DNA content determinations were chosen by their diameter (8-10, 16-20 and 11-13 m for leptotene/zigotene, pachytene and round spermatids, respectively), and by the appearance of the chromatin stained with H33342. After a transmitted light image was taken to measure cell diameter, and using the previously described permeabilization with digitonin and H33342 treatment, we determined the relative DNA content of single cells at different steps of the spermatogenesis. The estimated mean and standard deviations of DNA contents from at least three different cell preparations were 4.4 f 0.3 (N = 18), 3.9 + 0.5 (N = 16) and 1.2 f 0.2 (N = 19) for leptotene-zygotene spermatocytes, pachytene spermatocytes and round sperma tids, respectively. Use of ethidium bromide, instead of H,,,, for relative DNA determinations in spermatids and pachytene spermatocytes gave relative fluorescence values of 1.2. f 0.3 (N = 6), and 4.3 it 0.6 (N = 4), respectively. Thus, either dye yields comparable values of relative fluorescence.
The spermatogonia
A subpopulation
Our estimation of the relative DNA content of the spermatogonia A subpopulation not presenting condensed chromosomes (fig 5) showed that these cells had relative DNA contents from 2.5 to 4.6, suggesting that this population is composed of cells that were at different stages of the cell cycle. In fact, only lo-20 % of the cells appeared to be at G, as judged from their relative DNA content. The appearance of the cell chromatin stained with
H 33342showed a recognizable pattern, revealing an oval nuclei and a relatively homogenous chromatin distribution. Because they present a variable DNA content, we did not device a strategy for an independent confirmation of their identity in an unpurified cell population and on the microscope stage (see below).
Sertoli cell The size of Sertoli cells increases during development in rodents (eg Bellve, 1977). In adult rats, their reported diameter is in the range of 12-15 ,um (Floridi et al, 1983; Qian et al, 1985). In our experience, in adult rats, the cells that presented an irregular nucleus when stained with H,,, had diameters of 13.0 f 1.1 p (N = 13)(fig 6A, B). These cells had relative DNA contents of 2.3 + 0.3 (N = 5). In 12day-old rats, the cells with irregular nuclei and characteristic heterochromatin, showed diameters ranging from 9-12 p in cells stained with H,,,, or thin sections stained with toluidine blue (see also Bucci et al (1986)).
A bivariable distribution of seminiferous tubule cells obtained from prepuberal and adult rats The bivariable (relative cell DNA vs diameter) distribution of all the data obtained in purified, partially purified populations or non-purified cell populations (N = 299) is shown in figure 7. This figure also shows window zones where the abscissas were set using the diameters for isolated spermatogenic cells described in the literature where light or electron microscopy criteria of identification have been used (Romrell et al, 1976; Bell&, 1977; Meistrich et al, 1981; Bucci et al, 1986), as well as from our estimations of cell diameters from optic microscopy of thin sections of enriched fractions stained with toluidine blue or from transmission electron microscopy photomicrographs. The ordinate window widths were set using the mean and standard deviations established in our determinations of relative DNA content. The ordinate for the spermatogonia B/preleptotene subpopulation was established assuming a relative DNA content of 2 and using the average variation coefficient obtained in all the purified cell populations (11 + 4 %, N = 4). In
4 Fig 5. A. Spermatogonia A cell population isolated using cell sedimentation in a BSA gradient at unit gravity and stained with 4 PM Ha,,, for 35 min. The bar represents 10 pm. B. Transmission photomicrograph of a thin section stained with toluidine blue of the spermatogonia A population isolated using cell sedimentation in a BSA gradient at unit gravity. The bar represents 10 pm. C. Cells of the spermatogonia A BSA gradient fraction showing cells in prophase and metaphase. Other conditions were similar to A. The bar represents 10 pm. On stage single cell identification of rat spermatogenic
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Biology of the Cell (1997) 89, 53-66
Fig 6. A. Transmission photomicrograph of a thin section of a BSA gradient cell fraction stained with toluidine blue, showing Sertoli cells (arrow heads) with the typical irregular nucleus. The bar represents 10 pm. B. Fluorescence photomicrograph of non-purified seminiferous tubule cells stained with 4 PM H,,,,. Note the cell at the center and above (marked) showing a highly irregular nucleus typical of the Sertoli cell. The bar represents 10 pm.
these zones, there is a high probability of correctly classifying a cell as spermatogonia B/preleptotene, leptotene-zygotene primary spermatocytes, pachytene primary spermatocytes and round spermatids from their simultaneous diameter and relative DNA determination (see Discussion). On stage single cell identification of rat spermatogenic
cells
A guessing game for non-purified spermatogenic cell populations In order to estimate the confidence with which, in non-purified cell populations, a cell can be identified only by the appearance of the chromatin vitally Reyes et al
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63
,
6
00 -00
i
cell diameter (pm)
Fig 7. Plot of relative H,,,,, fluorescence (relative DNA content) versus cell diameter for rat spermatogenic cells. This plot shows all the data obtained in purified, enriched and non-purified cell populations. The discriminating windows define four developmental steps in the spermatogenesis. The ranges in the ordinate of the windows were established as the mean f twice the standard deviation (except in leptotene-zygotene spermatocytes and spermatids where the upper ranges were set to f 1 S and + 1.25 S-2 S, respectively) of the relative DNA content measurements obtained in purified spermatogenie cells. The range of the window abscissa was set equal to the range of diameters described in the literature for each group of spermatogenic cells and from our estimations of the diameters in purified cell preparations and from thin sections stained with toluidine blue.
transmission stained with Ha,,,,, we acquired images of cells in the field of the microscope, labeled them as the guesstimated cell type according to their chromatin-H33342 appearance, and subsequently performed a relative DNA content estimation in the same cells. For ‘pachytene spermatocytes’ and ‘round spermatids’, 57 % (N = 44) and 80 % (N = 52) of the cells, respectively, relabeled as indicated, had relative DNA contents in the range of the mean + 2SD as estimated in purified cell populations (see above). In contrast, for preIabeled ‘spermatogonia B/preleptotene’ and ‘leptotene-zygotene spermatocytes’, 33 and 20 % of the cells, respectively, fell within the range mean -+ 2SD for relative DNA content estimated in purified cell populations. As described above, in the case of spermatogonia B and early primary spermatocytes in the absence of digitonin, there is also a low uptake of Hs=, making the guesstimate of these cell types less reliable. Thus, our data indicate that round spermatids can be identified by the appearance of their chromatin vitally stained with H,,. Instead, pachytene spermatocytes, classified only by their prophase chromatin appearance, seem to include also cell populations of either fused early primary spermatocytes or a likely spermatogonia A (secondary spermatocyOn stage single cell identification of rat spermatogenic
cells
tes?) subpopulation in the prophase stage of the cell cycle. Narrowing the guesstimate to cells that are 15-20 pm in diameter can increase the correspondence between chromatin appearance and relative DNA content of pachytene spermatocytes to 66% (N = 32). Type B spermatogonia and leptotenezygotene spermatocytes require a confirmation of their identity by determination of their relative DNA content and cell diameter.
Determinations of physiological parameters in conjunction with relative DNA content and cell diameter in rat spermatids We have utilized bisoxonol, a potential-sensitive dye, in conjunction with H33342to determine plasma membrane potentials in purified rat spermatids (-57 + 16 mV, N = 20). The identification protocol was performed by exposing the cells to KH-digitoafter the determination of the membrane ~-Hw4, potential. In these conditions, the bisoxonol concentration in the medium was decreased to 10 nh4 (not shown) and did not present interference with the use of bisbenzimide dyes for DNA determination. Under these conditions, the relative H33342 fluorescence was 1.1 ? 0.2 (N = 26), a value not signifiReyes et al
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cantly different from that obtained in the absence of oxonol. A similar protocol was utilized to estimate (Ca2+)i with the fluorescent probe Fluo-3, giving a resting intracellular Ca2+ concentration of 87 + 20 nM (N = 10) and a relative HBX2 fluorescence value of 1.1 + 0.2.
DISCUSSION The study of the physiology and regulation of mammalian spermatogenesis, and some molecular approaches to this cell differentiation process have been hindered because of the difficulties of combining, at the single cell level and in the same cell, measurements of cell function and identification at different steps of spermatogenesis. Although the classical histology techniques used to identify spermatogenic stages are well worked out (eg deK.retser and Kerr, 1988), they are not generally applicable to studies where a single cell measurement has been made, and the same cell must be identified before removing it from the microscope field. Thus, it is difficult if not impossible to identify and functionally study the same cell using these techniques. In this work, we show that the labeling of the cell DNA with H,,,,, and the simultaneous measurement of cell diameter and relative DNA content of spermatogenic cells allows for the classification of the cells and the sequential use of other fluorescent probes to estimate physiological parameters or use of single cell electrophysiological and molecular biological techniques. In order to achieve equilibrium binding of the DNA probes and to decrease the time needed for estimation of cell relative DNA content, we found that the use of 10 pg/mL of digitonin was adequate. Higher than 20 pg/mL digitonin concentrations (not shown) gave values of relative fluorescence that differed substantially from the expected relative DNA content of spermatogenic cells. Lower digitonin concentrations did not permeabilize the entire cell population. As suggested for the effect of digitonin on nuclear Hsssa2 fluorescence, the low accessibility of Hsssd2 to premeiotic and early meiotic cell nuclei was limited at the level of the plasma membrane. Whether this restricted entry was due to a decreased membrane permeability for H,,,,,, or reflects the existence of active efflux system in these cells (eg Neya H33342 fakh, 1988) can not be elucidated from our data. The existence of a multidrug transport system is also suggested from the resistance of premeiotic spermatogenic cells to the action of extracellular hydrophobic drugs (Sjoblom et al, 1996). These authors found that premeiotic spermatogenic cells have low sensitivity to vinblastine, a drug which, On stage singlecell identification of rat spermatogeniccells
Biology of the Cell (1997) 89, 53-66
like H33342, is a substrate for the P-glycoprotein multidrug transporter. Ethidium bromide, an intercalating DNA probe, gave values of relative fluorescence that did not differ significantly from the values obtamed with H,,,,,. This result allows, in principle, to extend the use of the spermatogenie cell identification method proposed here in conjunction with the use of cell physiology fluorescent probes with excitation wavelengths in the near W range (eg SBFI, PBFI, fura-2, DPH). Visualization of nuclear material stained with H 33342in single cells also permits the identification of bi- or polynucleated cells formed during cell preparation. We have found that visibly polynucleated cells consist mainly of spermatid nuclei. Figure 1 shows some of the cell cycle transitions associated with the seminiferous tubule cells, such as preleptotene S phase (2 N -+ 4 N, 7-10 pm in diameter), type B spermatogonia mitotic S, G,, and M phases (2 N w 4 N cycling, 7-10 pm in diameter), or the mitotic S, G, and M phases of the cell cycle in spermatogonia A (2 N t) 4 N cycling, 12-15 ,um in diameter). This consideration can, in principle, limit the effective regions of interest in plot of ploidy ~1scell diameter, narrowing the late primary spermatocyte zone to 16-20 pm pachytene spermatocytes. Spermatogonia B are likely to spend around 30 % of its cell cycle in the DNA content region where it could be classified erroneously as leptotene-zygotene spermatocytes by cell diameter and relative DNA content (late S, G, phase and mitosis; (Bell&, 1979)). This spermatogonia B population in lo-14-day-old mice represents 30-40 % of a mixed leptotene/spermatogonia B population (Bell&, 1977). In these conditions, the probability of randomly finding a spermatogonia B in the region 4.4 + 0.3 relative DNA units and 7-12 pm area (leptotenelzygotene zone) is likely to be less than 15 % in non-purified prepuberal rat spermatogenie cells. In spite of the fact that Sertoli cells (cell diameters 9-12 pm and 12-15 pm for prepuberal and adult rats, respectively) could appear as interfering in the classification of spermatogenic cells in a plot of relative DNA content us cell diameter, their highly irregular nuclei allow to discard these cells (and any other irregularly nucleated cell) by simple appearance of their chromatin stained with H,,,,,. Thus, in a plot of relative DNA content us cell diameter (fig 7), we defined four regions of interest, representing different steps of spermatogenesis: 1) premeiotic diploid cells (7-11 ,um, spermatogonia B-preleptotene spermatocytes); 2) tetraploid primary spermatocytes (7-l 1 m, leptotene-zygotene spermatocytes); 3) advanced tetraploid priReyes et al
Biology of the Cell (1997) 89, 53-66
mary spermatocytes (16-20 pm, pachytene spermatocytes); and 4) haploid round spermatids (lo-14 pm). The range of the ordinate in each group was set to * 2 SD, except leptotene-zygotene spermatocytes where a f 1 SD window was chosen, and in spermatids, where an asymmetric range of -2 SD and +1.25 SD was utilized. These ranges allow > 95% confidence (except in the leptotene-zygotene spermatocyte region) to state that a random determination falling in those regions belongs to that cell subgroup and not to the other classes of spermatogenic cells. Acceptance of the cell classification presented here also establishes criteria to discard or accept data that, in part, could originate from being biased by the investigator in choosing a cell type by simple microscopic appearance or by easiness of manipulation in a certain single cell technique. The visualization of the cell nucleus stained reveals characteristic patterns of hetwith %,X2 ero- and euchromatin in spermatogenic cells that can orient the investigator to the cell type on which to perform the cell physiological measurement. However, these patterns of the chromatin stained with H33342 are relatively reliable only in the case of round spermatids. The classification method proposed in this paper provides an unbiased confirmation of the class of spermatogenie cell chosen. The cell classification presented here spans an important part of the spermatogenesis and thus permits a systematic approach to study the physiology and molecular biology of the differentiation process at the single cell level. This single cell classification can be used in unpurified cell populations, avoiding trypsinization that removes membrane proteins involved in spermatogenic cell physiology and cell-cell interactions (Jegou, 1993). Furthermore, this strategy for cell classification needs minute amounts of biological material allowing, in principle, the application of single cell physiological and cell molecular biological techniques to studies of the spermatogenesis in most species, using biopsy methods for tissue sampling.
ACKNOWLEDGMENTS We thank Dr E: Couve and Mr F Vargas for performing the histological techniques and TEM analysis of our preparations. This work was funded by DGI-UCV and Fondecyt 1960398/96
REFERENCES Aravindan GR, Pineau CP, Bardin CW and Cheng CY (1996) Ability of trypsin in mimicking germ cell factors that affect Sertoli cell secretory function. J Cell Physioll68,123-133 Arndt-Jovin DJ and Jovin TM (1989) Fluorescence labeling and
On stage single cell identification of rat spermatogenic cells
65
microscopy of DNA. In: Fluorescence Microscopy of Living Cells in Culture (Taylor DC, Wang Y, eds) Methods Cell Biol 3tJ417-448 Bell& AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar YM and Dym M (1977) Spermatogenic cells of the prepuberal mouse: Isolation and morphological characterization. 1 Cell Biol74,68-85 Bellve AR (1979) The molecular biology of mammalian spermatogenesis. In: Oxford Reuiews of Reproductive Biology (Finn CA, ed) Clarendon Press, Oxford, UK, 159-261 Braun RE, Lee K, Shumacher JM and Fajardo MA (1995) Molecular genetic analysis of mammalian spermatid differentiation. Ret Prog Horm Res 50,275286 Bucci LR, Brock WA, Johnson TS and Meistrich ML (1986) Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes. Biol Reprod 34,195-206 Cowell JK and Franks LM (1980) A rapid method for accurate DNA measurements in single cells in situ using a simple micro fluorimeter and Hoechst 33258 as a quantitative fluorochrome. J Histochem Cytochem 28,:206-210 D’Agostino A, Monaco L, Stefanini M and Geremia R (1984) Study of the interaction between germ cells and Sertoli cells in vitro. Exp Cell Res 150,430-435 de Kretser DM and Kerr JB (1988) The cytology of the testis. In: The Physiology of Reproduction (Knobil E, Neil1 J, eds) Raven Press, New York, 837-932 Ehrenberg B, Montana V, Wei MD, Wuskell JP and Loew LM (1988) Membrane potentials can be determined in individual cells from the Nernstian distribution of cationic dyes. Biophys 153‘785-794 Floridi A, Mercante ML, D’Atri S, Feriozzi R, Menichini R, Citro G, Cioli V and De Martin0 C (1983) Energy metabolism of normal and Lonidamine-treated Sertoli cells of rats. Exp Mel Path01 38,137-147 Gottschalk-Sabag SH, Weiss DB and Sherman Y (1995) Assessment of spermatogenic process by deoxyribonucleic acid image analysis. Fertil Steril64,403-407 Grabske RJ, Lake S, Gledhill BL and Meistrich ML (1975) Centrifugal elutriation: Separation of spermatogenic cells on the basis of sedimentation velocity. J Cell Physiol 86, 177-190 Grogan W MC, Farnham WF and Saban J (1981) DNA analysis and sorting of viable mouse testis cells. J Histochem Cytothem 29,738-746 Grotegoed JA, Den Boer PJ and Mackenbach P (1989) Sertoligerm cell communication. Ann NY Acad Sci 564,232-242 Hagiwara S and Kawa K (1984) Calcium and potassium currents in spermatogenic cells dissociated from rat seminiferous tubules. J Physiol356,135-149 Jegou B (1993) The Sertoli-germ cell communication network in mammals. lnt Rev Cytoll47,25-96 Kao JPY, Harootunian ATH and Tsien RY (1989) Photochemitally generated calcium pulses and their detection by Fluo3.1 Biol Chem 264,8179-8184 Karnovsky M J (1965) A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27,137a Lam DMK, Furrer R and Bruce WR (1970) The separation, physical characterization and differentiation kinetics of spermatogonial cells from mouse. Proc Natl Acad Sci LISA 65,192-199 Lievano A, Santi CM, Serrano CJ, Triviiio CL, Bellve AR, Hernandez-Cruz A and Darszon A (1996) T-type Ca2+ channels and alE expression in spermatogenic cells and their possible relevance to the sperm acrosome reaction. FEBS Lett 388,150-l.% Meistrich ML, Longtin J, Brock WA, Grmes SR and Mace ML (1981) Purification of rat spermatogenic cells and preliminary biochemical analysis of these cells. Biol Reprod 25, 1065-1077 Neyfakh AA (1988) Use of fluorescent dyes as molecular probes for the study of multidrug resistance. Exp Cell Res 174,168-176 Reyes et al
66
Palombi F and DiCarlo C (1988) Alkaline phosphatase is a marker for myod cells in cultures of rat peritubular and tubular tissue. Biol Reprod 39,1101-1109 Qian Z, Tsai Y, Steinberger A, Lu M, Greenfield ARL and Haddox MK (1985) Localization of omithine decarboxylase in rat testicular cells and epididymal spermatozoa. Biol Reprod 33,1189-1195 Reichter Jr LE and Dattatreyamurty B (1989) The follicle-stimulating hormone (FSH) receptor in testis: Interaction with FSH, mechanisms of signal transduction and properties of the purified receptor. Biol Reprod 40,13-26 Reyes JG, Velarde MV, Ugarte R and Benos DJ (1990) The glycolytic component of rat spermatid energy and acid-base metabolism. Am J PhysioZ259, C66O-C667
On stage single cell identification of rat spermatogenic cells
Biology of the Cell (1997) 89, 53-66
Reyes JG, Bacigalupo J, Araya R and Benos DJ (1994) Ion dependence of resting membrane potential of rat spermatids. J Repd Ferfil 102313-319 Romrell LJ, Bellve AR and Fawcett DW (1976) Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization. Dar BioZ49,119-131 Sjoblom T, Parvinen M and Lahdetie J (1996) Stage-specific DNA synthesis of rat spermatogenesis as an indicator of genotoxic effect of vinblastine, mitomycin C and ionizing radiation on rat spermatogonia and spermatocytes. Mutat Res 331,181-190
Received 27 January 1997; accepted 31 March 1997
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