Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Printed in Sweden Copyright 0 197.5 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental

Cell Research 92 (1975) 231-244

SEPARATION OF MOUSE TESTIS CELLS BY EQUILIBRIUM DENSITY CENTRIFUGATION IN RENOGRAFIN GRADIENTS M. L. MEISTRICH

and PATRICIA

K. TROSTLE

Section of Experimental Radiotherapy, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Tex. 77025, USA

SUMMARY Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by 8H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11-13 elongating spermatids, 1Zfold; Leydig cells, ‘IO-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.

The assignment of specific macromolecular events to particular cells in a differentiation sequence requires relatively homogeneous populations of cells at well defined stages of maturation. In the case of mammalian spermatogenesis, the great variety of cell types present in the adult testis has restricted biochemical analysis of the sequence of events during differentiation. Hence, physical means for separating these cells into homogeneous populations must be employed. Methods have been developed for separation of intact testis cells by velocity sedimentation [l-4] and isolated nuclei by velocity sedimentation [5, 61 and by equilibrium density centrifugation [7]. We have now extended the basis for separation to include the density of the intact cell. In this report we describe

the separation obtained using isopycnic centrifugation in gradients of Renografin [8].

MATERIALS

AND METHODS

Testis cell suspensions Cells were prepared from testes of 9-1%week-old male mice of strain C3Hf/Bu maintained in a specificpathogen-free colony. To obtain radioactively labelled cells the mice were injected intraperitoneally with 1 &i/g body weight of tritiated thvmidine (3H-TdR: ‘AmemhamSea&, spec. act. 5 Ci/mM) at theindicated times prior to sacrifice. Unless otherwise noted, cell suspensions were prepared using trypsin, essentially as described previously [2]. Following trypsin and DNase treatment. fetal calf serum was added to 8 % concentration .and the suspension was filtered through an 80 pm screen. The suspension was centrifuged (130 g, 5 min) and the supernatant withdrawn and recentrifuged (260 g, 10 min). The two pellets were pooled and resuspended at a concentration of approx. 1 x 10’ cells/ml in Ringer’s containing 20 ,ug/ml DNase, Exptl Cell Res 92 (1975)

232

Me&rich

and Trostle

chilled on ice, and 1 ml of this cell suspension was then layered on top of the gradient. A new method for oreoaration of susoensions employing EDTA was also used, as suggested by Davis & Schuetz 191. in order to obtain higher vields of spermatogonia. The testis tissue was -incubated (with stirring) in 7 ml calcium-magnesium-free PBS (8.0 g NaCl, 5.2 g KCI, 1.15 g Na,HPO,, 0.2 g KH,PO, per liter, pH 7.3) containing 5 mM EDTA, for 10 min. Then 0.7 ml of trypsin (GIBCO, 2.5 % trypsin in saline) was added. Ten min later 0.7 ml of 100 mM MgCl% and 0.07 ml of 2 mg/ml DNase (Sigma, crude pancreatic DN-25) were added and the suspension was stirred for an additional 10 min. Subsequent steps were the same as with the above method.

Cell counting, viability and identification Cell concentrations were determined by counting in a hemacytometer using phase contrast optic: The radioactivity in acid-insoluble intracellular material was determined by filtering onto Whatman GF/C filters. washing the filters. and liquid scintillation counting with- 34% efficiency as described previously [l]. Cell integrity was determined by adding trypan blue (0.1 % final concentration) to aliquots of cell suspensions. Cells were identified on smears, fixed in Bouins and stained with PAS-hematoxylin, according to the criteria developed by Meistrich, Bruce & Clermont [3]. Cell differential counts, based on at least 500 cells/fraction and 1 000 cells for each total cell suspension, were performed on randomly selected area of the smears. Correction for the possible deviation of the differential counts on these smears from the actual cellular content of the suspensions was not made; small deviations as observed previously are expected [3].

DNA recovery in cell suspensions Cell suspensions were prepared from two testes, one from each of two mice, centrifuged (500 g, 15 min) and resuspended in 5 ml of cold 5 mM EDTA (pH 7.2). The other two testes from the two mice were decapsulated and placed in 5 ml of EDTA. Both samoles were then homogenized in a Sorvall Omnimixer (15 set at 70 V). Aiiquots were precipitated with equal volumes of cold 10% PCA (perchloric acid) for 30 min, washed with cold 5 % PCA and resusoended in 1.5 ml of 5 % PCA. The DNA was then’hydrolysed at 70°C for 30 min and the precipitate was removed by centrifugation in the cold. Aliquots of the supernatant were taken for DNA analysis by the diphenylamine method [lo] and for scintillation counting in Aquasol (New England Nuclear Corp.)

Gradient materials and formation Renografin-76 (E. R. Squibb & Sons, New York) is an aqueous solution of 66 % methylglucamine diatrizoate (N,N’-diacetyl-3,5-diamino-2,4,6-tri-iodobenzoate) and 10% sodium diatrizoate containing Exptl

Cell Res 92 (1975)

the chelating buffers: 10.9 mM sodium citrate and 1.4 mM disodium EDTA. The osmolality of the solution is 1700 mOsm (data provided by manufacturer). The stock of Renografin-76 was diluted to the desired concentration with Ringer’s solution (147 mM NaCl, 4 mM KCI, 2.5 mM CaCI,). DNase was added to a concentration of 100 pg/ml to disperse DNA released from lysed cells. To enhance the action of DNase, additional CaCl, was included in the solutions to yield a calcium concentration slightly greater than twice the ionic strength of EDTA plus citrate. Gradients were generated into 18 ml cellulose nitrate centrifuge tubes as described previously [8]. Unless otherwise stated, gradients consisted of 1.9 ml of a 24 % Renografin underlayer followed by a 14.6 ml linear gradient from 22 to 8% Rencgrafin, and are referred to as 8-24 % gradients. The density of the Renografin solutions at 4°C (~0 in g/cm* was related to the refractive index measured at 24°C (&) by the equation: e4 = 3.5419N,,-3.7198.

Centrifugation and collection of gradients Gradients were centrifuged in an SW27.1 rotor in a Beckman L5-50 ultracentrifuge. Unless otherwise indicated, the centrifugation was for 20 min at 10 000 rpm (13 000 g&. Deceleration occurred with the brake off. Fractions of about 1.2 ml were pumped from the top of gradient using an Auto-Densi-Flow collector (Buchler Instruments, Ft Lee. N.J.). In a few experiments, however, fractions were collected through a hole punched in the bottom of the centrifuge tube. All operations were performed at WC.

Concentration of fractions Several steps were required to wash the cells free of the hypertonic Renografin solutions without causing cell lysis. The fractions were diluted with 3 ml of hypertonic Ringer’s containing 0.5 % bovine serum albumin (BSA), pH 7.0, and 10 % sucrose. The cells were centrifuaed (230 g. 10 min) in oolvstvrene tubes (Falcon no. 5057) anTresuspended in- 1 ml of cold hypertonic PBSG [2] containing 10% sucrose. The fractions were warmed to 31°C and diluted slowly by droowise addition of 3 ml of isotonic PBSG containing-0.5 % BSA over a 20 min period. The cells were then centrifuged at room temperature, resuspended in 4 ml of PBSG-BSA and recentrifuged. The pellet of cells was resuspended in a small amount of residual supernatant for smearing.

Expression of density profiles Comparison between gradients was facilitated by olottina results as described previously 1111. Cell cork&rations were converted to the p&c&&ge of total recovered cells found in a density increment and plotted using density as the abscissa. The density increment was arbitrarily taken as 0.0067 g/cm* which was the density change across a fraction of the 8-24 % gradient. The-cells f&nd at the top of the gradient (T), at the 22-24 % interface (S) and in the last fraction including the pellet (P) were plotted as histograms with a width of one fraction. In this procedure, the

Density separation of testis cells

Table 1. Yields of cells, total DNA and 3H-labelled DNA

in mouse testis cell suspensionf EDTA-trypsin preparation

Trypsin preparation Total cells per testis Sperm’ (percentage of total cells) Percentage of total DNA recovered in cell suspension (diphenylamine assay)’ Percentage of 3H-labelled DNA in spermatogonia and preleptotene spermatocytes recovered in cell suspension (1 h after SH-TdR injection) Percentage of 3H-labelled DNA in round spermatids recovered in cell suspension (15 days after injection) a ’ ’ ’

233

3.4 (fO.l) 1311 35*2

x 10’d

2.7 (kO.3) x 10’ 7fl 30*3

24&8

51k12

52k6

44+8

All values given as mean k SE. Late spermatids with an elongated nucleus and an intact flagellum were counted as sperm. Total DNA per 80 mg (wet weight) testis was 320 ,ug. Lower than previous values [2] because of smaller testes from C3H mice.

areas under all the curves were normalized to equal values. Counts of radioactivity were also expressed in this manner. The average density of a peak, & was determined graphically by averaging densities at the two half-maximum points of the peak.

Expression of enrichment of cell types A method for calculating the enrichment of cells was developed to account for both the increase in the percentage of the cell type under consideration and the decrease in contaminating cells. The enrichment is given by the expression: (az/aI) x (b,/b,), where a2 and a, represent the final and initial percentages of the cell of interest and b, and 6, the same for contaminating cells. This enrichment factor is characteristic only of the separation process and the nature of the contaminating cells, and is independent of the initial concentration of the cell being considered.

Staput cell separation The “Staput” method of testis cell separation by velocity sedimentation at unit gravity [l] was used with minor modifications. Staput chambers of 12.5 or 28.1 cm diameter were employed and the cells were introduced into these chambers in 20 or 100 ml of buffer solution, respectively. Then a nonlinear l-4% BSA gradient was formed under the cell suspension as described previously [12]. The separation was run for 3 h before starting to unload.

RESULTS The methods used for preparation of cell suspensions were first compared. The yields of total testicular DNA and DNA of specific cell types carrying a 3H-label in the cell

suspensions were measured. Although the standard trypsin method provided higher yields of cells, total DNA and intact sperm, the EDTA-trypsin method yielded higher numbers of spermatogonia and preleptotene spermatocytes (table l), as well as leptotene, zygotene and early pachytene spermatocytes (data not shown). The preferential recovery of germinal cell DNA, than of total DNA, is partly attributable to the lower percentages of non-germinal cells in suspensions than in the intact testis [3]. Cytological observations by phase contrast microscopy showed that with the EDTA-trypsin method more Sertoli cells were usually obtained and these Sertoli cells had no attached spermatogonia or early spermatocytes as had been observed with the trypsin method. Sedimentation velocity analysis of the spermatogonia and preleptotene spermatocytes prepared with EDTA-trypsin demonstrated that these cells sedimented as a more homogeneous population at about 4.4 mm/h as opposed to the broad distribution observed with the trypsin method [2]. Next, the effects of the Renografin solutions on testis cells were examined. Examination of the cells by phase contrast microscopy revealed that for most cells, no morphological Expti Cell Res 92 (1975)

234

Me&rich

and Trostle

Table 2. Effect of storage in Renograjin mouse testis cells

Total cells, n Cells excluding Trypan blue, % Intact cells, 12 Dead cells, n

solutions for 90 min on intact and already damaged

Mechanical preparation’

Trypsin preparation

Cells in Ringer’s

Cells in Renografin

Cells in Ringer’s

Cells in Renografin

100C

71 i-P

100

104&7

57kl% 57+1 43fl

93&l% 66+5 5+1

93?1% 93+1

96fl% lOOk

7+1

5*1

a Testes homogenized in Ringer’s with glass-Teflon homogenizer. b Solutions contained Renografin, DNase and CaCl, in Ringer’s as employed for the gradients. Results were averages of data obtained with 8, 16 and 24 % Renografin, which were similar at the three Renografin concentrations. The only difference was that at 24 % Renografin, all dead cells with round nuclei lysed; however, the swollen spermatozoan nuclei now stained with the dye. ’ Cell concentrations were approx. 10B/ml. For comparison of samples, this value was normalized to 100, and all other numbers are relative to this value. d S.E. determined from between 3 and 12 independent measurements on each sample. occurred except for the hypertonic shrinkage. Coulter volume measurements confirmed a decrease in the average cell volume. The only exceptions were the sperm

changes

25

t

a

t

1.06

1.08

1.10

1.12

heads and spermatozoa, some of which were noticeably swollen at Renografin concentrations greater than 20 %. The viability of cells following density gradient centrifugation

b

1.06

1.08

I.10

I.12

Fig. I. Abscissa: density (g/cm”); ordinate: % cells/density increment. Distribution of mouse testis cells following centrifugation in 8-24 % Renografin gradients. T, top of gradient; S, interface between 22 and 24 %; P, pellet. (a) Effect of centrifugation time at 13 000 g: ---, 10 mm; -, 30 min; . . +, 60 mm. (b) Effect of centrifugal force: ---, 3 300 g for 30 min; -, 3 300 g for 10 mm + 13 000 g for20min; *a*, 3 300 g for IO min + 30 000 g for 20 mm. (c) Effect of position of loading: -, cells, layered . . . . cells dispersed throughout gradient; ---, cells in bottom layer, centrifuged at 13 000 g for 20 mm. ~~).t~~bandbg of fractions from first gradient on identical gradients: -, first gradient; ---, . . . , rebanding of fractions from first gradient marked with horizontal bars, centrifuged at 13 000 g for 20 mm. Exptl Cell Res 92 (1975)

Density separation of testis cells

was 93 % compared with 98 % in the initial cell suspension, as measured by the trypan blue test. Most of the dead cells were sperm and sperm heads which stained more intensely as a result of swelling. However, the following experiment indicates that the high cell viabilities might be a result of selective lysis of damaged cells by the Renografin solutions. Suspensions, prepared by mechanical means to produce a high percentage of damaged cells, were stored for 90 min in either Ringer’s or various concentrations of Renografin containing DNase. The concentration of cells was determined and cells were mixed with trypan blue to measure viability. The results (table 2) indicated that the number of intact cells was unchanged by storage in Renografin-DNase, but that the damaged cells were lysed by the combined action of the Renografin and DNase, with only partial loss observed in the presence of either agent alone. In preliminary experiments, various ionic compositions of the diluent for Renografin were tested. In some gradients, the hypertonicity was reduced by first diluting the Renografin to 20% with water [13] followed by further dilution with Ringer’s. However, the bands of cells observed upon visual examination of these gradient tubes were not as sharp as when Ringer’s solution alone was used as diluent. The centrifugation conditions necessary to achieve equilibrium were examined. Variation of centrifugal force and time of centrifugation (fig. 1 a, b) demonstrated little change in the cell distributions indicating that equilibrium was attained. Therefore, centrifugation at 13 000 g for 20 min was adopted in all subsequent experiments. To demonstrate that the observed bands were formed by isopychic separation, the cells were introduced into tubes either at the top, bottom, or throughout the gradient. Follow16-751807

235

Fig. 2. Abscissa: density (g/cm”); ordinate: % total cells which are of a given type per density increment. Distribution of specific classes of mouse testis cells following centrifugation in g-24 % Renografin gradients. The cells were prepared by trypsin method. The gradient was collected from the top. (a) Total cell distribution; (f~) spermatogonia; (c) -, leptotene-zygotene-early pachytene spermatocytes; ---, pachytene spermatocytes; (d) -, dividing spermatocytes; ---, secondary spermatocytes; (e) spermatids: -, steps l-g; ---, steps 14-16; (f) spermatids: steps 9-10; ---, steps 11-13; (g) cytoplasmic fragments and residual bodies; (/z) -, Sertoli cells; ---, Leydig cells.

ing centrifugation, bands were observed at approximately the same positions in all three cases (fig. 1 c). There was, however, an increase in mean density as the initial position of the cells was lowered, which may reflect wall effects or an effect of the high Renografin concentration on the cells. To demonstrate that the ‘distribution of cells truly reflects the distribution of cell densities, we performed a rebanding experiment in which a single fraction, rerun on a second gradient, should form a sharp band at the same density [14]. Two fractions from separate areas of a gradient were concentrated and rebanded in subsequent gradients (fig. Exptl Cell Res 92 (1975)

236

Me&rich

and Trostle

2c IC

2C IC

2C IC

3c 2C IC

ld). A fraction taken from near the peak of distribution rebanded more sharply at the same position. A fraction from the lower part of the gradient rebanded as two peaks of cells, one at the original position and one at the density of early spermatids which were present as contaminants because of the method employed for gradient collection. The positions of the major bands of cells were highly reproducible with the peak of cell counts centered at 1.076 g/cm3 with a standard deviation between experiments of 0.002 g/cm3. The average recoveries of cells and of radioactivity loaded on the gradients were 80 and 100 %, respectively. The distributions of various classes of testis cells along the gradient were determined. Differential counts were performed on each fraction and the percentages of each cell type were multiplied by the cell concentrations. Two complete gradients were counted with similar results; one set of profiles is presented (fig, 2). The spermatogonia were distributed near the bottom of the gradient (,@= 1.101~0.003 g/cm”). Because of their Exptl Cell Res 92 (1975)

Fig. 3. Abscissa: density (g/cm”); ordinate: % radioactivity/density increment. Density profiles of radioactively labelled mouse spermatogenie cells. Cell suspensions were prepared with trypsin 1 h to 26 days after injection of $H-TdR. 10’ cells were layered on tou of Renografin gradients and centrifuged at 13 000 g for 20 min. For most experiments (-) 8-24% gradients were used. Other gradient Conditions were as follows: . . . . .. 1 h, lO-30% Renografin; 2 days, 12-32 % Renografin; ---, 10.519.5 % Renografin.

low numbers, the various types were pooled. The average density of the A spermatogonia was, however, greater than that of the class of intermediate and type B spermatogonia and preleptotene spermatocytes. Young primary spermatocytes in the early stages of the meiotic prophase were less dense than spermatogonia (c = 1.0895 t- 0.0007 g/cm”). Because the maturation of pachytene spermatocytes is a continuous and gradual process, the classification of cells as young primary spermatocytes vs pachytene spermatocytes was sometimes arbitrary. Comparing our results [2, 31 with kinetic data obtained microscopically [15, 16, 171, we conclude that the young primary spermatocytes identified in smears included leptotene, zygotene and early pachytene cells prior to stage V of the cycle of the seminiferous epithelium. The later pachytene cells had significantly lower density (c = 1.0847 + 0.0012 g/cm3), and secondary spermatocytes (Q = 1.0775 k 0.0002 g/cm”) were even lighter. Cells in the process of meitotic division, between metaphase and early telophase, had the lowest

Density separation of testis cells

density of any testicular cell type (@= 1.068 rh 0.003 g/cm”). The round spermatids @= 1.0781~0.0003 g/cm3) had the same density as the secondary spermatocytes. The distribution of step 9-10 spermatids was skewed towards greater density as a result of cytoplasmic loss from some cells. Spermatids at steps 11-13 were found in three density g/cm3 regions: a peak at 1.0801+0.0004 with their full cytoplasmic complement; a broad distribution at about 1.lO g/cm3 corresponding to those which have lost part of their cytoplasm during the preparation of the cell suspensions and finally a pellet containing those with no cytoplasm, some of which had intact flagella. Mature spermatids (steps 14-16) were found in the pellet either as free sperm heads or as spermatozoa with flagella, but with little or no cytoplasm. The average density of Sertoli cells was 1.081 -tO.O07 g/cmS while Leydig cells had a density of 1.119 k 0.003 g/cm3. Numerous cytoplasmic fragments were observed in a band at a density of 1.0667* 0.0008 g/cm3. In this density region, most of the fragments were the size of the step 9-13 spermatid cytoplasm with no evidence of formation of the residual body, but there were also a few large cytoplasmic fragments, apparently detached from Sertoli cells. In the denser regions (Q > 1.10 g/cm3), however, most of the cytoplasmic fragments were residual bodies [ 181. The densities of cells at various stages of spermatogenesis were also determined by relating the density profiles of radioactively labelled cells obtained from mice at various times after injection of 3H-TdR (fig. 3) to the kinetics of spermatogenesis. The radioactivity profiles (fig. 3) correspond well to the density profiles of the various cells measured microscopically (fig. 2). The density variations with cell maturation are graphically summarized in fig. 4. To calculate

237

Fig. 4. Abscissa: average density of cells (g/cm*); ordinate: (left) davs between ink&ion of *H-TdR and sacrifkcd; ‘(right) cell type. Average densities of testis cells in Renografin gradients as a function of time after preleptotene spermatocyte stage. q , Densities of cell types as determined from microscopic analysis; circles indicate densities of cells as calculated from radioactivity profiles (fig. 3) after correction for radioactivity in less mature cells; l , experiments performed using 8-24 % Renografin gradients; o @, use of shallower or steeper gradients, respectively. The solid line drawn through the points represents the proposed differentiation pathway of spermatogenic cells with respectto the parameter of cell density.

the densities of the cell types from the radioactivity profiles obtained in fig. 3, we must consider that only about half of the 3H-TdR incorporated was into the preleptotene spermatocytes; with appreciable amounts also incorporated into spermatogonia. At any time after injection about half of the radioactivity will be in the most advanced wave of labelled cells [17]. To correct for radioactivity in less mature cells, we note that the radioactivity profile of less mature cells will be proportional to the profile of all cells obtained from mice sacrificed at a shorter post-injection interval. Therefore, the profiles obtained one (or two) days earlier are multiplied by a proportionality factor and subtracted from the profile being analysed, to yield the density distribution of the most advanced wave of labelled cells. The proportionality factor is 2-tlT, where t is the time interval between obtaining the two profiles and T is the average spacing Exptl Cell Res 92 (197.5)

238

Me&rich

and Trostle

Table 3. Maximum purity of mouse testicular cell types obtained by equilibrium density centrifugation in 8-24 % Renografin gradients Data are presented for trypsin prepared cell suspensions; however, where greater purity was obtained from EDTA-trypsin prepared suspensions the corresponding results are given in parentheses

Cell type Spermatogonia Spermatocytes Young primary Pachytene Meiotic Secondary Spermatids Steps l-8 Steps 9-10 Steps 11-13 Steps 14-16 Cytoplasmic fragments (including residual bodies) Residual bodies Sertoli cells Leydig cells

% in initial suspension 0.6 (2.3) 1.1 4.0 0.08 0.3

Maximum purity (X) 8 (19)

Enrichment factorb 15 (10)

9 2Sa 1.4 3.9

9 9 18 13

22 2.2 12 12

66 7= a 2:

7 3 9 12

38 7 0.2 (0.3) 0.5

ii:

19 Y (1.4)

: (5) 15

a Gradient unloaded by puncturing bottom of tube. In all other cases, top unloading was used. b (~/a~) x (b&J where a, and a, represent the final and initial percentages of the cell type of interest and be and bl, the same for all contaminating cells.

between the S phases of the spermatogonia and the preleptotene spermatocytes [32]. The shifts in density with cell development were, in general, consistent with morphological changes. Cells with condensed nuclei such as the leptotene and zygotene spermatocytes and elongated spermatids, and those with little cytoplasm such as the spermatogonia and later spermatids displayed the

higher densities. The low density of cytoplasmic fragments and the increased density with the formation of the residual body were expected from the lack of nuclear material and the subsequent formation of a dense ribonucleoprotein sphere [ 181. Differential counts on each fraction from four gradient separations of cells were performed. The two methods for preparation of cell suspensions were employed as were the two modes of collection of the gradient (collection of a gradient containing EDTAtrypsin prepared cells from the bottom was not tested). The maximum purities in which the various cell types were obtained are presented in table 3. When the gradients were collected from the top, contamination of the lower fractions with cells which banded higher in the gradient was observed. Collection from the bottom eliminated this, but the lighter fractions were then contaminated with cells which banded lower in the gradient. In an attempt to obtain better separation, experiments were run using shallower gradients of 10.5 to 19.5 %, 11.5 to 19 %, or 10.5 to 16% Renografin. Sharper peaks and reduced skewness towards higher densities can be obtained by using shallower gradients (fig. 3). There was also an increased homogeneity of cell types, based on differential cell counts, with shallower gradients. Greater enrichment of specific cell types was achieved by density gradient centrifugation of fractions of cells obtained from Staput separations (fig. 5). Two Staput separations were performed. In the first run, 2.8 X lo* cells were separated in the smaller chamber

Fig. 5. Photomicrographs of fractions of mouse testicular cells purified by a combination of Staput velocity sedimentation and Renografin equilibrium centrifugation. Magnification 320 x . (a) Unseparated testis cell suspension; (b) fraction E-l, spermatogonia (arrows);(c) fraction B-2, late pachytene spermatocytes; (d) fraction B-l cells in first meiotic division; (e) fraction F-l, round spermatids; cf) fraction I-2, step 11-13 spermatids; (g) fraciion I-l, cytoplasmic fragments (residual bodies in early stage of condensation, arrows); (h) fraction A-l, Leydig cells (arrows), readily identified by PAS+ cytoplasm. Exptl Cell Res 92 (1975)

Density separation of testis cells

Exptl

239

Cell Res 92 (1975)

240

Me&rich

and Trostle

Table 4. Percentage of cellular composition of fractions Staput method (slowly sedimenting fractions) jugation in Renografin gradients

and further

of mouse testis cells obtained by the enriched by equilibrium density centri-

Cell type

Total cell suspension

Ea s = 5.gb

E-l ~=1.107’

F s=4.8

F-l g=1.078

Spermatogonia Spermatid l-8

1.4 23

7 57

29d 46

1.7 74

0.1 90

0 1.3

0 1.1

Spermatid 9-10 11-13 Cyto. fragments and residual bodies

163.0

102.9

2.3 6

192.0

0.2 1.5

8

1.9

73

34

2.4 7 15

0.1 7 4

I s=2.4

I-l n=1.066 c

97

I-2 e=l.lll 0.1 0.4 7k5 24

a Fractions designated by a single letter were obtained by Staput sedimentation of trypsin prepared mouse testis cells. Letters were chosen to conform to the notation used in this laboratory and in a previous publication [3]. The fractions with numbers after the letter indicate the Staput fractions further purified by equilibrium density centrifugation in Renografin gradients. b The average sedimentation velocity of cells in each fraction, s, is given in mm/h. ’ The average density of cells in each fraction, Q, is given in g/ems. ’ Cell types most enriched in a particular fraction are indicated.

and three fractions designated E, F and I were obtained (table 4). In the second experiment, 9.2 x lo* cells were separated in the larger chamber, yielding three fractions designated A, B and C (table 5). About 10’ cells from each of the six fractions obtained by Staput separation were centrifuged to

equilibrium in Renografin gradients and differential counts were performed. The slowest sedimenting fraction, I, contained cytoplasmic fragments and step 1 l-1 3 elongated spermatids with a small amount of residual cytoplasm and no flagella. This mixture was further separated into a light

Table 5. Percentage cellular composition of fractions of mousetestis cells obtained by the Staput method (rapidly sedimentingfractions) and further enriched by equilibrium density centrijugation in Renograjin gradients Cell type Spermatogonia Young primary spermatocyte Pachytene spermatocyte Meiotic spermatocyte Spermatid l-8 Leydig cell

Total cell suspension

Aa ~=12.4~

1.5

5.4

2.3

4.3

4.8 0.3 24 0.7

49 1.2 12 5

A-l P=1.122’ 21d 4.7 1.5 0.3 0.3 33

B s=9.8

B-l e=1.073

2.7

1.3

2.0

1.2

65 0.9 14 2.6

7.6 34 16 0.2

B-2 ~=1.083

C s=7.6

C-l e=1.094

0.1

2.9

1.5

0.5

7

9

71

15

47

0.4 21 0.2

2.0 46 1.8

0 36 0.2

a Fractions designated by a single letter were obtained by Staput sedimentation of trypsin prepared mouse testis cells. Letters were chosen to conform to the notation used in this laboratory and in a previous publication [3]. The fractions with numbers after the letter indicate the Staput fractions further purified by equilibrium density centrifugation in Renografin gradients. b The average sedimentation velocity of cells in each fraction, s, is given in mm/h. ’ The average density of cells in each fraction, Q, is given in g/cm8. ’ Cell types most enriched in a particular fraction are indicated. Exptl Cell Res 92 (1975)

Density separation of testis cells

fraction, I-l, which consisted almost exclusively of cytoplasmic fragments and a dense fraction, I-2, enriched in elongated spermatids. The contamination of the latter by cytoplasmic fragments was a result of the presence of dense residual bodies in the fragments and the collection of the gradient from the top. A second Staput fraction, F, containing round spermatids (steps 1-8) was further enriched in these cells by density gradient centrifugation (fraction F-l). A third fraction, E, represented the maximum enrichment we have obtained by the Staput method of spermatogonia from trypsinprepared suspensions. The spermatogonia were denser than the contaminating spermatids and cytoplasmic fragments and were enriched to 29 Y0in fraction E-l. Staput fraction C was enriched in pachytene primary spermatocytes sedimenting at 7.6 mm/h. 3H-TdR labelling data had shown that spermatocytes sedimenting at 7.6 mm/h became labelled at 6.5 days after injection [2] and, hence, correspond to pachytene cells in stages V and VI [15]. Further enrichment of these mid-pachytene spermatocytes was obtained in fraction C-l by equilibrium density centrifugation. Fraction B was most enriched in late pachytene spermatocytes in stages IX to XII. This fraction also contains dividing cells which, on the basis of their size and sedimentation rate, must be in the first meiotic division. Although density gradient centrifugation yielded a fraction, B-2, only slightly more enriched in pachytene cells than B, the contamination by cells, other than secondary spermatocytes and round spermatids, was reduced from 18 % to less than 3 %. A fraction of lighter density, B-l, was also obtained which was markedly enriched in spermatocytes in the first meiotic division. This stage is infrequently seen in testis sections or in cell suspensions and the

241

overall enrichment obtained is calculated to be 170-fold. A most rapidly sedimenting fraction, A, contained large multinucleate cells, pachytene spermatocytes, Leydig cells, Sertoli cells and spermatogonia. Density gradient centrifugation of this mixture of cells yielded a dense fraction, A-l, which was enriched in both Leydig cells and spermatogonia, with the nearly total removal of pachytene spermatocytes. No alterations in cell morphology were observed following recovery from the density gradients (fig. 5), indicating that testis cells can withstand the two-step purification method employed here. Unexpected results were obtained when cells prepared by the EDTA-trypsin method were centrifuged on Renografin gradients. Although the distributions of total cells, late pachytene spermatocytes and round spermatids were similar to those obtained with cells prepared by the trypsin method, the spermatogonia were nearly uniformly distributed throughout the gradient and the young primary spermatocytes displayed a major peak at a density of 1.08 g/cm3 with a second peak near the top of the gradient. These findings were obtained both by radioactive labelling and cytological examination. We do not yet understand the factors responsible for the decreased densities of the EDTA-trypsin prepared spermatogonia and spermatocytes which appeared morphologically normal. However, it is apparent that cell density is a sensitive parameter for measurement of cellular alterations. DISCUSSION A variety of supporting media have been used for equilibrium density separation of mammalian cells [4, 8, 11, 13, 14, 19-241. Renografin has the advantages of high Exptl Cell Res 92 (1975)

242

Me&rich

and Trostle

density, low viscosity, good cell dispersing properties, low cost and availability in commercially prepared sterile solutions. After separation, cell viability with respect to proliferative capacity, macromolecular synthesis and dye exclusion were preserved for blood [8, 131, tumor [20] and cultured mammalian cells [l I]. The separation of different cell types with Renografin was at least as good as with BSA gradients [l 11. The most apparent disadvantage of Renografin is its osmotic effect. True cell density cannot be measured as the hypertonicity causes loss of water and increased density. Testis cells are more sensitive than other mammalian cell types to lysis upon subsequent dilution from Renografin into isotonic solutions. Lysis appears to result from osmotic effects and not from the chemical properties of Renografin, and can be prevented by first washing the cells in a hypertonic solution followed by slow dilution. In general, intact testis cells appear undamaged in Renografin even with storage times of 5 h. The maximum number of testis cells which could be loaded onto these 17 ml Renografin gradients without lowering the quality of the separation was about 3 x 10’. However, with BSA as a density medium, separation of larger numbers of cells (5 x 108) has been reported [24]. Our preliminary experiments have demonstrated that separation of testicular cells can also be obtained on BSA density gradients. However, because of the cost, high viscosity and laborious procedure for preparing concentrated BSA solutions we have performed most of our studies with Renografin. Previous reports of density centrifugation of testis cells have indicated only separation of round testis cells from sperm [4, 251. However, we have shown that a significant separation of many different classes of testis ExptI

Cell Res 92 (1975)

cells can be obtained. The high density of viable mature spermatozoa (Q = 1.17 g/cm3) in various media, including one compound similar to Renografin, has been shown previously [22]. The separation of residual cytoplasmic fragments on the basis of their low density has also been reported [26]. The decreased density of cells in meiotic division is analogous to results obtained with mitotic HeLa cells in isotonic gradients of colloidal silica [ 191. It is apparent that complete separation of different cell types was not achieved. The factors limiting this resolution fall into three categories: (1) density shifts caused by morphologically detectible alterations within a cell class; (2) density differences within a class of morphologically identical cells; and (3) technical limitations of the physical separation procedure. As has been observed with the sedimentation velocity [3], the cell density is also affected by loss of cytoplasm, as occurs with the late spermatids. In contrast to the sedimentation velocity, the cell density is not measurably affected by the formation of multinucleate cells. Significant differences in density do occur within certain morphological classes of cells, as demonstrated by the density decrease of the pachytene spermatocytes during maturation. The widths of other peaks may either be a result of true density variations or technical limitations. One such technical factor may be the mixing of layers of the gradient during deceleration or handling, but this was ruled out by experiments in which we generated gradients containing sharp layers of blue Dextran dye. Centrifugation of these gradients did not affect the width of the band of dye as measured by optical absorption profiles. Another factor may be the broadening of the bands caused by cell clumping, but this is unlikely because lower cell concentrations

Density separation of testis cells

did not yield better resolution and previous experiments had shown that cell interactions were negligible (fig. 3b, ref. [ll]). Several lines of evidence do indicate that the width and the skewness of the bands of cells were a result of the collection method and possibly wall effects. The narrower peaks observed in shallow gradients (fig. 3) indicated that some factor other than a spread of cell densities contributed to the widths observed on the 8-24% gradients. The direction of skewness of the cell bands depended on the direction of unloading the gradients and, therefore, must be an artifact of the unloading [23]. The band widths and skewness were less when the blue Dextran dye was used than with the cells. A mechanism, such as gradual elution of cells adhering to the centrifuge tube walls, might cause the additional broadening of cell bands [23]. Although wall effects could be eliminated by introduction of the cells into the lower Part of the gradient [24], with our Renografin gradients this modification resulted in lower cell recovery and increased release of DNA. Comparison of the Staput method of velocity sedimentation at unit gravity with the present technique indicates that the number of bands of testis cells, the purity of most cell types and the number of cells loaded are greater with the Staput method. Density centrifugation does, however, have the advantages of speed and greatly reduced cost of materials. It is important to note that the physical parameters underlying velocity sedimentation and equilibrium density separation are different, resulting in different crosscontamination between separated fractions. Therefore, the technique of equilibrium density centrifugation will be most useful to further enrich fractions of cells obtained by velocity sedimentation. The development of faster methods of velocity sedimentation would make such

243

two-step separation procedures more practical. Rapid preparative scale separation of mouse and hamster testicular cells has been achieved by centrifugal elutriation in the (Beckman) Elutriator Rotor [27] with the same resolution of cell classes as at unit gravity [28]. Further improvement in cell separation methods will facilitate biochemical analysis of events at specific stages of spermatogenesis. Differential synthesis of basic nucleoproteins by various cell types separated by velocity sedimentation has been demonstrated [29, 30, 311. Further enrichment of specific cells by equilibrium density centrifugation should resolve any ambiguities resulting from the presence of several cell types in Staput fractions. We wish to thank Dr D. J. Grdina for many valuable discussions regarding the use of Renografin for density gradient centrifugation. The technical assistance of MS B. 0. Reid and the assistance of Mr J. Kuykendall with the photomicrography are greatly appreciated. This work was supported by FDA contract number 73-204 from the NCTR, a grant f rom the Population Council, New York and NIH grant number CA-06294 from the NCI.

REFERENCES 1. Lam, D M K, Furrer, R & Bruce, W R, Proc natl acad sci US 65 (1970) 192. 2. Me&rich, M L, J cell physiol 80 (1972) 299. 3. Meistrich, M L, Bruce, W R & Clermont, Y, Exptl cell res 79 (1973) 213. 4. Pretlow, T G, Scalise, M M & Weir, E E, Am j path01 74 (1974) 83. 5. Meistrich, M L & Eng, V W S, Exptl cell res 70 (1972) 237. 6. Utakoji, T, Muramatsu, M & Sugano, H, Exptl cell res 53 (1968) 447. 7. Loir, M & Wyrobek, A, Exptl cell res 75 (1972) 261. 8. Grdina, D J, Milas, L, Hewitt, R R & Withers, H R, Exptl cell res 81 (1973) 250. 9. Davis, J & Schuetz, A, J cell biol 59 (1973) 72a. 10. Burton, K, B&hem j 62 (1956) 315. 11. Grdina, D J, Meistrich, M L & Withers, H R, Exptl cell res 85 (1974) 15. 12. Miller, R G & Phillips, R, J cell physiol 73 (1969) 191. 13. DeSimone, J, Kleve, L & Shaeffer, J, J lab clin med 84 (1974) 517. 14. Leif, R C, Automated cell identification and cell Exptl Cell Res 92 (1975)

244

15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Me&rich

and Trestle

sorting (ed G L Wied & G F Bahr) pp 21-96. Academic Press, New York (1970). Oakberg, E F, Am j anat 99 (1956) 391. - Ibid 99 (1956) 507. Clermont, Y & Trott, M, Fert steril20 (1969) 805. Firlit. C F & Davis. J R. Ouart i microscon sci 106 (1965) 93. ’ ’ _ Wolff, D A & Pertoft, H, J cell biol 55 (1972) 579. Grdina, D J, Milas, L, Mason, K A & Withers, H R, J natl cancer inst 52 (1974) 253. Loos, J A & Roos, D, Exptl cell res 86 (1974) 333. Benedict, R C, Schumaker, V N & Davies, R E, J reprod fert 13 (1967) 237. Zucker, R M & Cassen, B, Blood 34 (1969) 591. Shortman, K, Aust j exptl biol med sci 46 (1968) 375

Exptl Cell Res 92 (1975)

25. Moav, B, Goldberg, A & Avivi, Y, Exptl cell res 83 (1974) 37. 26. Nyquist, S E, Acuff, K & Mollenhauer, H H, Biol reprod 8 (1973) 119. 27. Glick, D, von Redlich, D, Juhos, E Th & McEwen, C R, Exptl cell res 65 (1971) 23. 28. Grabske, R J, Lake, S, Gledhill, B L & Meistrich, M L, J cell physiol (1975). In press. 29. Lam, D M K & Bruce, W R, J cell physiol 78 (1971) 13. 30. Platz, R D, Grimes, S R, Meistrich, M L, Hord, G R & Hnilica, L S, Fed proc 33 (1974) 1411. 31. Goldberg, R B, Geremia, R & Bruce, W R, Proc Canad fed biol sci 17 (1974) 48. 32. Meistrich, M. In preparation. Received October 28, 1974