- Email: [email protected]
Separation of germinal cells from immature rat testes by sedimentation at unit gravity
Printed in Sweden Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reseroed
Experimental
SEPARATION TESTES
Cell Research 91 (1975) 79-86
OF GERMINAL
CELLS
BY SEDIMENTATION
FROM AT UNIT
IMMATURE
RAT
GRAVITY
J. C. DAVIS and A. W. SCHUETZ Department
of Population Dynamics, The Johns Hopkins University,
School of Hygiene and Public Baltimore, Md 21205, USA
Health,
SUMMARY Early spermatogenic cells from the testes of lo-, 13-, 15-, 18-, 20- and 25-day-old rats were purified by sedimentation at unit gravity. Cell dissociation was accomplished in 5 mM EDTA or 0.1 % trypsin in Ca-Mg-free phosphate-buffered saline (pH 7.35). Dissociation with trypsin resulted in more viable cells than with EDTA, while EDTA was more efficient for the dissociation of spermatogonia. The differential effects of the two dissociation media were particularly evident in cell preparations from the lo-day-old animal. Maximum purity of different cell types was obtained in different aged animals (spermatogonia, 98 %, 10 days; preleptotene spermatocytes, 98 %, 10 days; leptotene spermatocytes, 75 %, 13 days; zygotene spermatocytes, 68 %, 18 days; pachytene spermatocytes, 75 %, 25 days). Purity of particular types was correlated with the age of the animal. Earlier stages were purified to a greater extent in younger animals and later stages to a greater extent in older animals. Later stages exhibited increasing sedimentation at unit gravity in correlation with the increase in cell size as differentiation proceeded to pachytene spermatocyte. Two early germinal cell types, spermatogonia and preleptotene spermatocytes, were greatly purified with this technique.
Spermatogenesis in the mammalian testes produces millions of mature cells each day (1 x 10sspermatozoa/day in the rat) [l]. However, little is known about the specific cellular functions involved in this extremely prolific process. A major difficulty in studying these events is the existence of at least 27 distinguishable cell types or stages between the most immature (spermatqgonia A) and most mature (spermatozoa) spermatogenie cell. Because of this complexity, most studies of spermatogenesishave been indirect and rely on statistical analysis of cell counts in histological sections to delineate the cellular processesinvolved in spermatogenesis [2, 3, 41. Thus, in order to study the cellular aspects of spermatogenesis, it is important that the individual cells at different stages of differentiation be separated. 6-751805
One striking feature of the spermatogenic process is that during the early phase of differentiation (spermatogonia to diplotene spermatocytes) the cells increase considerably in size. A technique, unit gravity sedimentation, is available that separates cells on the basis of size [5]. This method has been used to partially purify spermatogenic cells from the adult mouse testis [6-91. However, because the early increase in cell size during spermatogenesisis followed by a later decrease in cell size at the time of the meiotic divisions, purification of most cell types in the adult testis by unit gravity sedimentation is not feasible. In immature animals (age lo25 days) differentiation has not progressed farther than diplotene spermatocyte (the largest germinal cell present) and thus the smaller post-meiotic stages are not present. These Exptl
Cell Res 91 (1975)
80
Davis and Schuetz
considerations suggest that separation of the early cell types prior to diakinesis should be possible in the immature animal. In this report, we have explored the possibility of purifying isolated testicular cells from the immature rat testis by sedimentation at unit gravity. METHODS Cell dissociation Testes from lo-, 13-, 15-, 18-, 20- or 25-day-old Holtzman rats (Madison, Wise.) were removed, decapsulated, and the tubules teased apart in calciummagnesium-free phosphate-buffered saline (PBS-free, pH 7.35). Four to twenty testes were used, depending on the age of the animals to yield a wet weight of 0.45-1.2 g of tissue for dissociation. Cell suspensions were prepared by continuous agitation of teased tubules at room temperature in 20 ml of medium in a 50 ml trvosinizing flask. The tubules were disoersed in either a-5 mM EDTA-PBS-free solution or in 0.1 % trypsin (Difco 1:250), PBS-free. The tissue-free supernatant was decanted every 10 min and filtered through Nitex cloth (28 pm, TET-Kresilk). The filtering proce.dure removed’ most of the cell clumps but did-not retain the large pachytene spermatocytes. Following trypsin dissociation, 8 % fetal calf serum was added (1 : 1) to the supernatant prior to filtration through Nitex. Dissociation with EDTA was accomplished in 70 min, and with trypsin in 50 min. With both treatments, filtered cells were centrifuged at 1 000 g for 15 min and resuspended in phosphate-buffered saline containing calcium and magnesium (PBS) prior to sedimentation. Cell viability was determined by the exclusion of trypan blue dye [lo].
Cell separation The procedure used was similar to that described by Peterson & Evans 151 as modified by Hymer et al. [ll]. Experiments were carried out at-room temperature. The gradient was generated with the aid of two 250 ml beakers (containing 250 ml 1 % BSA in PBS and 250 ml 3 % BSA in PBS, pH 7.3) connected by Silastic tubing (see fia. 1). All gradient solutions contained peni&in (16 units/ml) streptomycin (100 pg/ml) (GIBCo) and were filtered through Millipore membranes (HA 0.45 pm) prior to use. Tubing connecting the beakers containing the 1 and 3 % BSA was filled with 3 % BSA; tubing between the beakers containing 1 % BSA and the flask containing 0.3 % BSA was filled with 1 % BSA; 1 and 0.3 % BSA solutions were mixed by magnetic stirrers. The BSA solution was pumped into the bottom of the separation chamber by gravity flow (85 cm pressure head). The lucite chamber (which was custom fabricated) was 10.8 cm OD, 11.0 cm high and had a 500 ml capacity. Cells were layered onto the baffle at the bottom of the chamber with a long-tipped Pasteur pipette. In the usual experiment, 1.8 x 10’ cells at a concentraExprl
Cell Res 91 (I975)
P=
i
7% Sucrose
1. Diagram of gradient aunaratus. The beaker containing the 1 % BSA was stirred and connected to the 3 % BSA and 0.3 % BSA by Silastic tubing. The gradient (80 cm head) flowed into the bottom of the chamber. Cells were layered onto the baffle with a Pasteur pipet. After sedimentation, 7 % sucrose was introduced and the gradient collected by topflow. Fin.
tion of 1.5 x lo6 cells/ml were placed in the chamber. The BSA solution was then allowed to flow into the bottom of the chamber (10 ml/min), and after 5 min the flow rate was decreased to 6 ml/min. Flow was stopped after 450 ml were added to the chamber. Seventy minutes were required for the generation of the gradient, and the cells were allowed to settle for an additional 140 min. Following sedimentation, successive 10 ml fractions were obtained by pumping the BSA solution upward through the top of the chamber with a solution of 7 % sucrose in 0.9 % NaCl.
Processingof gradient fractions Gradient fractions (10 ml) were centrifuged at I 000 g in a Sorvall RC3 centrifuge for 15 min and the supernatants drawn off to 0.2 ml. The cell pellet was resuspended and the cell concentration determined with a hemocytometer. The remainder of each preparation was centrifuged (900 rpm for 20 min) onto pre-cleaned glass microscope slides in a Shandon cytocentrifuge. This procedure produces a pellet of flattened cells which greatly facilitates differential counting. Slides were fixed 12 h in Zenker-form01 fixative, washed 30 min in running water, 5 min in Lugol’s solution, 2 min in 5 % Na,S03, and stained 35 min in Giemsa stain (pH 5.68 or 5.70), air dried, and mounted with Permount. This procedure gave better detail for routine cell identification than fixation in Carnoy’s, Bouin’s or 10% formalin or staining with hemotoxylin and eosin, periodic acid Schiff’s or aceto-orcein.
Identification of cell types For routine identification, staining of cells with Giemsa (pH 5.70) was found to be better than hema-
Separation of germinal cells toxylin staining. As the topographical distribution of cells within the tubule is absent in single-cell preparations, cellular details were used for cell identification. Red blood cells appearing between 100-140 ml were not counted. The followine. criteria were used in the identification of the various cell types encountered: Spermatogonia. Pale, blue or pink-staining, oval nucleus with no condensation of the chromatin, palestaining cytoplasm, irregular shape. Preleptotene spermatocytes. Chromatin clumping, light to heavy throughout the round nucleus, light blue cytoplasm, cells round. Leptotene spermatocytes. Wispy blue chromatin throughout nucleus, chromatin homogeneously dispersed, cytoplasm medium blue, cells round, larger than preleptotene spermatocytes. Zygotene spermatocytes. Chromatin with irregular clumping, cytoplasm dark blue, cells round and larger than leptotene spermatocytes. Pachytene spermatocytes. Distinct chromosomes apparent in the nucleus, dark blue cytoplasm, round cells larger than zygotene. Sertoli ceks. Pale-blue nucleus, large cytoplasmic to nuclear ratio, irregular shape, larger than pachytenes. Leydig cells. Nucleus with dark blue grid-like appearance, lipid in the cytoplasm, large cytoplasmic to nuclear ratio, small nucleus.
RESULTS Cell dissociation Attempts to dissociate testis cells by agitation in phosphate-buffered saline resulted in the appearance of numerous cell clumps. As dissociation in 5 mM EDTA or 0.1 % trypsin produced few clumps, the effects of these two media on cell yield and viability were tested. In all ages investigated (lo-25 days), yield and viability were better in trypsin than in EDTA (table I). The mean cell yield for all aged animals was 9.7&3 x lo6 cells/testis dissociated in EDTA and 56.3k23 X lo6 cells/ testis dissociated in trypsin (n= 11). The percentage of viable cells isolated by the two methods was 71&2 % (EDTA) and 93+2 % (trypsin). The percentage of individual cell classes isolated from the testis also varied between the two media. When cell counts were done on EDTA and trypsin preparations from the 20-day-old animal, 32k 11% spermatogonia and 18+ 3 % preleptotene spermatocytes were found in the EDTA prep-
81
Table 1. Comparisonof cell yield and viability in EDTA and trypsin preparations
Age 10 13 15 18 20 25 r,
Dissociation medium
n
Cells x 1O-B per testis % Dead (mean) (mean)
EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin
2 2 1 1 2 2 1 1 3 3 1 1 11 11
3.9 9.2 4.4 7.4 3.9 9.5 9.0 13.8 15.0 103.0 30.0 243.0 9.7 + 3a 56.3 ?23
23 4 36 4 39 4 33 9 33 7 20 15 29.2 & 3 7.0+2
a Means *SE.
aration while the trypsin preparation contained 2f 1% spermatogonia and 41& 1% preleptotene spermatocytes (n = 3). The relative decreasein percentage of spermatogonia in the trypsin preparation was noted at all ages and indicates that EDTA is more efficient for obtaining this particular cell. Separation by unit gravity sedimentation Cells from EDTA and trypsin preparations were layered onto the gradient and allowed to sediment 34 h. Following sedimentation, 10 ml fractions were collected and analysed for cell number and cell type. The position of individual cell types on the gradient was then plotted. Photographs of cell types found in these preparations are seen on figs 2-8. Results from a gradient of cells dissociated from a 20-day-old animal are presented in fig. 9. Dead cells appeared at the top of the gradient while cells in volumes greater than 80 ml were over 95 % viable based on trypan blue dye exclusion. Spermatogonia in the Expti
Cell Res 91 (1975)
82
Davis and Schuetz
Table 2. Purity of cell types in gradients of dissociated testis from 20-day-old rats
Cell type
?I
Spermatogonia
3
Preleptotene Spermatocyte Leptotene Spermatocyte Zygotene Spermatocyte Pachytene Spermatocyte
3 3 3 3 z 3 3
Dissociation medium
Peak ( %)
Gradient volume (ml>
Number cells x 1O-5 in % peak fraction
% in starting prep.
EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin
98kO.6 25*17 72k8 92k5 46k6 68k12 70?6 58+4 5527 50+11
(9&130) (140-160) (160-190) (190-220) (20&270) (240-260) (270-320) (350-380) (40(X420) (4W30)
5.1 kO.6 0.1 kO.01 3.6&l 5.1 kO.6 1.3kO.l 2.1 kO.6 2.4kO.02 0.4 2 0.08’ 0.9 & 0.2 0.7kO.3
32kll 2*1 18+3 41&l 11&5 22+5 25k3 21i3 10+3 6&3
a In this sample, unlike all other samples, the peak % did not coincide with the number peak, the sample at 270 ml contained 8.9 k 4 x lo5 zygotene spermatocytes and was 42 % pure.
EDTA preparation (90-130 ml) and preleptotene spermatocytes in the trypsin preparation (190-220 ml) were greater than 92 % pure. Leptotene, zygotene and pachytene spermatocytes were localized at 220-260 ml, 270-350 ml and 400430 ml, respectively, and exhibited lower purities. The position of the cells in the gradient was related to their size and, therefore, their stage of differentiation. Leydig cells appeared at 320 ml and Sertoli cells at 400 ml; neither of these cell types constituted more than 5 % of the total cells in most gradient fractions. A summary of the purities of the various cell types in these gradients from the 20-day-old testes is seenin table 2. There was a greater than two-
fold purification of all cell types following unit gravity sedimentation. Purity of the different types ranged from 25-98 %, depending on cell type and dissociation medium. A consistent observation was a faster sedimentation of all cell types following trypsin preparation than with EDTA preparation. The reason for the difference is not apparent. The testis of the lo-day-old rat has a large percentage of early cell types (spermatogonia and preleptotene spermatocytes) [2]. Because of this, there is a striking difference between the gradient profiles of the lo-day-old (fig. 10) and the 20-day-old rat testis cells (fig. 9). With the younger animal, the difference between EDTA and trypsin in the dissociation
2-8. Magnification x 1 200. 2. Spermatogonia isolated from testis of lo-day-old animals. These cells appear at 90-130 ml following sedimentation. Fig. 3. Preleptotene spermatocytes isolated from testes of lo-day-old animals. These cells appear at 190-220 ml following sedimentation. Fig. 4. Leptotene spermatocytes (L) isolated from testis of 13-day-old animals. These cells appear at 220-280 ml following sedimentation. Fig. 5. Zygotene spermatocytes (2) isolated from testes of 18-day-old animals. These cells appear at 300-330 ml following sedimentation. Fig. 6. Pachytene spermatocytes isolated from testes of 25-day-old animals. These cells appear at 37&390 ml following sedimentation. Fig. 7. Sertoli cells isolated from testes of 20-day-old animals. These cells appear at 400 ml following sedimentation. Fig. 8. Leydig cells isolated from an extratubular preparation [13] of adult testes. These cells appear at 30&330 ml following sedimentation. Figs Fig,
Exprl
Cell Res 91 (1975)
Separation of germinal c:ells
Exptl
83
Cell Res 91 (197.5)
84
Davis and Schuetz 21
21
14 7
7
14 0 l--2!2
0 I.-
#
L
45
45
30 30 / 15
/\_
15 0 i,
0 60
60
40
40
20
20
lc-
L--
01 36
‘I,
t :%
JL
12 0 L.-
Fig. 9. Abscissa: volume (ml); ordinate: (top) cells x lo-“; (bottom) % cell type. Distribution of cells in a gradient of testicular cells from a 20-day-old rat. (Left) Cells dissociated in trypsin; (right) cells dissociated in EDTA. The top row shows the distribution of total cells on both gradients. Rows 2-6 show the distribution of specific cell types on the gradients. Sedimentation is to theright.
21
21
14
14
7
7
0 r
0-
15t
in/-
/
IO 9
Fig. II. Abscissa: age (days); ordinate: % of total cells. The proportion of cells in selected gradient regions as a function of the animal. Rows l-6 represent the sedimentation volumes of (I) dead cells (O-90 ml); (2) spermatogonia (90-170 ml); (3) preleptotene(l70240 ml), (4) leptotene (240-300 ml); (5) zygotene (3%340 ml) and (6) pachytene (340-400 ml) spermatocytes, respectively. Each point indicates the percentage of total cells which was found in that gradient region. (Lefr) EDTA-dissociated preparations; (right) trypsin-dissociated preparations. Sedimentation is to the right. 8 7 6 5 4 3 2
b lLL 0
100
200 VOLUME
300
400
(ml)
Fig. 10. Abscissa: volume (ml); ordinate: cells x 10-5. Distribution of total cells in gradients after dissociation of testes from lo-day-aid rat in (a) trypsin or(b) EDTA. The top graph shows trypsin-dissociated cells; the bottom graph, EDTA-dissociated cells. Sedimentation is to the right. Spermatogonia appear at 10s160 ml in these gradients. Exptl Cell Res 91 (1975)
of spermatogonia (100-I 60 ml in the gradient) is particularly evident. To investigate the relationship between sedimentation and differentiation, cells from testes of different aged rats (lo-25 day) were sedimented at unit gravity. The total number of cells recovered from each gradient was determined and the percentage of this total in each 10 ml fraction was calculated. The total percentage of cells in gradient volumes corresponding to the sedimentation positions
Separation of germinal cells Table 3. Purification Cell type
85
of cell types from testis of immature rats by sedimentation at unit gravity Age (days)
Volume (ml) 90-l 30
% Cell type in fraction
% Cell type in starting prep.
9820.7
45kll
Spermatogonia Preleptotene Spermatocyte
10 10
1go-220
98 kO.6
54+7
Leptotene Spermatocyte
13
220-280
751-3
31+6
Zygotene Spermatocyte
18
300-330
68
19
Pachytene Spermatocyte
25
370-390
7514
34+10
of individual cell classeswas plotted at each age (fig. 11). The first row (O-90 ml) contains unidentifiable dead cells. The volume presentedin rows 2-6 in fig. 11 are the sedimentation volumes of spermatogonia (90-170 ml), preleptotene (170-240 ml), leptotene (240300 ml), zygotene (300-340 ml), and pachytene (340-440 ml) spermatocyte, respectively. The age of greatest percentage for a particular cell type, thus, is the best age for its purification. With increasing age, there was a diminution in the percentage of cells at the top of the gradient (90-240 ml, earlier types) and an increase in the percentage of cells at the bottom (240440 ml, more mature types). As previously noted, there were more dead cells in the EDTA preparations (O-90 ml) and a lower number of spermatogonia in the trypsin preparations (90-170 ml). A summarization of the best age, under our conditions, for the purification of the different early germinal cell types is presented in table 3. Purity of the cells varied from 68 to 98 % depending on cell type. All the values in the table are for EDTA-dissociated cells, Similar purities for trypsin-dissociated cells, with the exception of spermatogonia, were found. In younger animals, relatively pure preparations of certain cell types were obtained with trypsin dissociation prior to sedi-
mentation (i.e. 80 % preleptotene spermatocytes from lo-day-old animals). DISCUSSION The differentiation of germinal cells to diakinesis is an important aspect of spermatogenesis. Data presented here demonstrate that early germinal cells can be separated by unit gravity sedimentation. To study the composition of these cell populations, definitive criteria for the routine identification of isolated cell types are required. Hematoxylin is routinely used for the identification of germinal cell types in tissue section [4]; however, it was not adequate for identifying certain dissociated cell types in our preparations. Giemsa stain was used and allowed a clear identification of all but the early meiotic stages. Admittedly, classification of meiotic cells on the basisof chromatin condensation, as was done with the Giemsa-stained preparation, gives only a relative position of the cells in prophase I of meiosis. Unequivocal identification of these cells awaits chromosomal analysis. However, the Giemsa-staining did allow the clear identification of all types except leptotene and zygotene. The sedimentation and purity of one cell type, pachytene spermatocyte, agrees with Exptl
Cell Res 91 (1975)
86
Davis and Schuetz
the work of Meistrich in the adult mouse [8]. The sedimentation of the other cell classes studied also agrees in general with the work in the adult. However, purity of the other cell classes is greatly diminished when adult preparations are used. For instance, in the adult, the percent spermatogonia falls to 4 vs 98 % in the immature; preleptotene spermatocytes are 2 y0 pure in the adult vs 98 y0 pure in the lo-day-old. These differences indicate that use of immature animals greatly enhances purification of early germinal cells at unit gravity. Meistrich reported that trypsin dissociation is the best method for dissociation of testis cells from the adult mouse [8]. Also, in our hands, trypsin produces a better yield of viable cells than does EDTA. However, in the trypsin preparations, a smaller number of spermatogonia are found. The reason for this appears to be destruction by trypsin as spermatogonia dissociated with EDTA are destroyed when placed in 0.1 % trypsin. There is a good correlation, as predicted, between maturational stages and sedimentation at unit gravity. Clearly, as the cell volume increases during differentiation, there is an increase in sedimentation. Also, because of the changes occurring during maturation in the prepuberal animal, best purification of particular cell types is accomplished at different ages. In fact, an excellent preparation of preleptotene spermatocytes (80 % pure) can be obtained from the IO-day-old animal with
Exptl
Cell Res 91 (1975)
trypsin treatment without sedimentation. It is clear that two cell types, spermatogonia and preleptotene spermatocyte, can be obtained essentially pure (98 %, table 3) by sedimentation at unit gravity. Also, absolute numbers of particular cells vary with age of the animal used. With the ability to isolate purified classes of spermatogenic cells, new approaches to the study of this process become feasible. This research was supported by Grants SPOI-HD 5594-02, STOl-HD 00109-07 and Contract NOl-HD3-2794 from NICHD. The assistance of Dr Claude Desjardins is gratefully acknowledged.
REFERENCES 1. Verma, M C, Sharma, U D & Singh, G, Ind j vet sci 36 (1966) 109. 2. Clermont, Y & Perey, B, Am j anat 100 (1957) 241. 3. Leblond, C P & Clermont, Y, Am j anat 90 (1952) 167. 4. - Ann NY acad sci 55 (1952) 548. 5. Peterson, E A, Elbert, A & Evans, W E, Nature 214 (1964) 824. 6. Lam, D M K, Furrer, R & Bruce, W R, Proc natl acad sci US 65 (1970) 192. 7. Meistrich, L, J cell physiol 80 (1972) 295. 8. Meistrich, M L, Bruce, W R & Clermont, Y, Exptl cell res 79 (1973) 213. 9. Go, V L W, Vernon, R G & Fritz, I B, Can j biochem 49 (1971) 213. 10. Parker, R Ci Methods of tissue culture, p. 282. Hoeber. New York (1970). 11. Hymer,’ W C, Evans, W’E, Kracier, J, Mastro, A, Davis, J & Griswold, E, Endocrinology 92 (1973) 275. Received June 24, 1974 Revised version received September 3, 1974
Experimental
SEPARATION TESTES
Cell Research 91 (1975) 79-86
OF GERMINAL
CELLS
BY SEDIMENTATION
FROM AT UNIT
IMMATURE
RAT
GRAVITY
J. C. DAVIS and A. W. SCHUETZ Department
of Population Dynamics, The Johns Hopkins University,
School of Hygiene and Public Baltimore, Md 21205, USA
Health,
SUMMARY Early spermatogenic cells from the testes of lo-, 13-, 15-, 18-, 20- and 25-day-old rats were purified by sedimentation at unit gravity. Cell dissociation was accomplished in 5 mM EDTA or 0.1 % trypsin in Ca-Mg-free phosphate-buffered saline (pH 7.35). Dissociation with trypsin resulted in more viable cells than with EDTA, while EDTA was more efficient for the dissociation of spermatogonia. The differential effects of the two dissociation media were particularly evident in cell preparations from the lo-day-old animal. Maximum purity of different cell types was obtained in different aged animals (spermatogonia, 98 %, 10 days; preleptotene spermatocytes, 98 %, 10 days; leptotene spermatocytes, 75 %, 13 days; zygotene spermatocytes, 68 %, 18 days; pachytene spermatocytes, 75 %, 25 days). Purity of particular types was correlated with the age of the animal. Earlier stages were purified to a greater extent in younger animals and later stages to a greater extent in older animals. Later stages exhibited increasing sedimentation at unit gravity in correlation with the increase in cell size as differentiation proceeded to pachytene spermatocyte. Two early germinal cell types, spermatogonia and preleptotene spermatocytes, were greatly purified with this technique.
Spermatogenesis in the mammalian testes produces millions of mature cells each day (1 x 10sspermatozoa/day in the rat) [l]. However, little is known about the specific cellular functions involved in this extremely prolific process. A major difficulty in studying these events is the existence of at least 27 distinguishable cell types or stages between the most immature (spermatqgonia A) and most mature (spermatozoa) spermatogenie cell. Because of this complexity, most studies of spermatogenesishave been indirect and rely on statistical analysis of cell counts in histological sections to delineate the cellular processesinvolved in spermatogenesis [2, 3, 41. Thus, in order to study the cellular aspects of spermatogenesis, it is important that the individual cells at different stages of differentiation be separated. 6-751805
One striking feature of the spermatogenic process is that during the early phase of differentiation (spermatogonia to diplotene spermatocytes) the cells increase considerably in size. A technique, unit gravity sedimentation, is available that separates cells on the basis of size [5]. This method has been used to partially purify spermatogenic cells from the adult mouse testis [6-91. However, because the early increase in cell size during spermatogenesisis followed by a later decrease in cell size at the time of the meiotic divisions, purification of most cell types in the adult testis by unit gravity sedimentation is not feasible. In immature animals (age lo25 days) differentiation has not progressed farther than diplotene spermatocyte (the largest germinal cell present) and thus the smaller post-meiotic stages are not present. These Exptl
Cell Res 91 (1975)
80
Davis and Schuetz
considerations suggest that separation of the early cell types prior to diakinesis should be possible in the immature animal. In this report, we have explored the possibility of purifying isolated testicular cells from the immature rat testis by sedimentation at unit gravity. METHODS Cell dissociation Testes from lo-, 13-, 15-, 18-, 20- or 25-day-old Holtzman rats (Madison, Wise.) were removed, decapsulated, and the tubules teased apart in calciummagnesium-free phosphate-buffered saline (PBS-free, pH 7.35). Four to twenty testes were used, depending on the age of the animals to yield a wet weight of 0.45-1.2 g of tissue for dissociation. Cell suspensions were prepared by continuous agitation of teased tubules at room temperature in 20 ml of medium in a 50 ml trvosinizing flask. The tubules were disoersed in either a-5 mM EDTA-PBS-free solution or in 0.1 % trypsin (Difco 1:250), PBS-free. The tissue-free supernatant was decanted every 10 min and filtered through Nitex cloth (28 pm, TET-Kresilk). The filtering proce.dure removed’ most of the cell clumps but did-not retain the large pachytene spermatocytes. Following trypsin dissociation, 8 % fetal calf serum was added (1 : 1) to the supernatant prior to filtration through Nitex. Dissociation with EDTA was accomplished in 70 min, and with trypsin in 50 min. With both treatments, filtered cells were centrifuged at 1 000 g for 15 min and resuspended in phosphate-buffered saline containing calcium and magnesium (PBS) prior to sedimentation. Cell viability was determined by the exclusion of trypan blue dye [lo].
Cell separation The procedure used was similar to that described by Peterson & Evans 151 as modified by Hymer et al. [ll]. Experiments were carried out at-room temperature. The gradient was generated with the aid of two 250 ml beakers (containing 250 ml 1 % BSA in PBS and 250 ml 3 % BSA in PBS, pH 7.3) connected by Silastic tubing (see fia. 1). All gradient solutions contained peni&in (16 units/ml) streptomycin (100 pg/ml) (GIBCo) and were filtered through Millipore membranes (HA 0.45 pm) prior to use. Tubing connecting the beakers containing the 1 and 3 % BSA was filled with 3 % BSA; tubing between the beakers containing 1 % BSA and the flask containing 0.3 % BSA was filled with 1 % BSA; 1 and 0.3 % BSA solutions were mixed by magnetic stirrers. The BSA solution was pumped into the bottom of the separation chamber by gravity flow (85 cm pressure head). The lucite chamber (which was custom fabricated) was 10.8 cm OD, 11.0 cm high and had a 500 ml capacity. Cells were layered onto the baffle at the bottom of the chamber with a long-tipped Pasteur pipette. In the usual experiment, 1.8 x 10’ cells at a concentraExprl
Cell Res 91 (I975)
P=
i
7% Sucrose
1. Diagram of gradient aunaratus. The beaker containing the 1 % BSA was stirred and connected to the 3 % BSA and 0.3 % BSA by Silastic tubing. The gradient (80 cm head) flowed into the bottom of the chamber. Cells were layered onto the baffle with a Pasteur pipet. After sedimentation, 7 % sucrose was introduced and the gradient collected by topflow. Fin.
tion of 1.5 x lo6 cells/ml were placed in the chamber. The BSA solution was then allowed to flow into the bottom of the chamber (10 ml/min), and after 5 min the flow rate was decreased to 6 ml/min. Flow was stopped after 450 ml were added to the chamber. Seventy minutes were required for the generation of the gradient, and the cells were allowed to settle for an additional 140 min. Following sedimentation, successive 10 ml fractions were obtained by pumping the BSA solution upward through the top of the chamber with a solution of 7 % sucrose in 0.9 % NaCl.
Processingof gradient fractions Gradient fractions (10 ml) were centrifuged at I 000 g in a Sorvall RC3 centrifuge for 15 min and the supernatants drawn off to 0.2 ml. The cell pellet was resuspended and the cell concentration determined with a hemocytometer. The remainder of each preparation was centrifuged (900 rpm for 20 min) onto pre-cleaned glass microscope slides in a Shandon cytocentrifuge. This procedure produces a pellet of flattened cells which greatly facilitates differential counting. Slides were fixed 12 h in Zenker-form01 fixative, washed 30 min in running water, 5 min in Lugol’s solution, 2 min in 5 % Na,S03, and stained 35 min in Giemsa stain (pH 5.68 or 5.70), air dried, and mounted with Permount. This procedure gave better detail for routine cell identification than fixation in Carnoy’s, Bouin’s or 10% formalin or staining with hemotoxylin and eosin, periodic acid Schiff’s or aceto-orcein.
Identification of cell types For routine identification, staining of cells with Giemsa (pH 5.70) was found to be better than hema-
Separation of germinal cells toxylin staining. As the topographical distribution of cells within the tubule is absent in single-cell preparations, cellular details were used for cell identification. Red blood cells appearing between 100-140 ml were not counted. The followine. criteria were used in the identification of the various cell types encountered: Spermatogonia. Pale, blue or pink-staining, oval nucleus with no condensation of the chromatin, palestaining cytoplasm, irregular shape. Preleptotene spermatocytes. Chromatin clumping, light to heavy throughout the round nucleus, light blue cytoplasm, cells round. Leptotene spermatocytes. Wispy blue chromatin throughout nucleus, chromatin homogeneously dispersed, cytoplasm medium blue, cells round, larger than preleptotene spermatocytes. Zygotene spermatocytes. Chromatin with irregular clumping, cytoplasm dark blue, cells round and larger than leptotene spermatocytes. Pachytene spermatocytes. Distinct chromosomes apparent in the nucleus, dark blue cytoplasm, round cells larger than zygotene. Sertoli ceks. Pale-blue nucleus, large cytoplasmic to nuclear ratio, irregular shape, larger than pachytenes. Leydig cells. Nucleus with dark blue grid-like appearance, lipid in the cytoplasm, large cytoplasmic to nuclear ratio, small nucleus.
RESULTS Cell dissociation Attempts to dissociate testis cells by agitation in phosphate-buffered saline resulted in the appearance of numerous cell clumps. As dissociation in 5 mM EDTA or 0.1 % trypsin produced few clumps, the effects of these two media on cell yield and viability were tested. In all ages investigated (lo-25 days), yield and viability were better in trypsin than in EDTA (table I). The mean cell yield for all aged animals was 9.7&3 x lo6 cells/testis dissociated in EDTA and 56.3k23 X lo6 cells/ testis dissociated in trypsin (n= 11). The percentage of viable cells isolated by the two methods was 71&2 % (EDTA) and 93+2 % (trypsin). The percentage of individual cell classes isolated from the testis also varied between the two media. When cell counts were done on EDTA and trypsin preparations from the 20-day-old animal, 32k 11% spermatogonia and 18+ 3 % preleptotene spermatocytes were found in the EDTA prep-
81
Table 1. Comparisonof cell yield and viability in EDTA and trypsin preparations
Age 10 13 15 18 20 25 r,
Dissociation medium
n
Cells x 1O-B per testis % Dead (mean) (mean)
EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin
2 2 1 1 2 2 1 1 3 3 1 1 11 11
3.9 9.2 4.4 7.4 3.9 9.5 9.0 13.8 15.0 103.0 30.0 243.0 9.7 + 3a 56.3 ?23
23 4 36 4 39 4 33 9 33 7 20 15 29.2 & 3 7.0+2
a Means *SE.
aration while the trypsin preparation contained 2f 1% spermatogonia and 41& 1% preleptotene spermatocytes (n = 3). The relative decreasein percentage of spermatogonia in the trypsin preparation was noted at all ages and indicates that EDTA is more efficient for obtaining this particular cell. Separation by unit gravity sedimentation Cells from EDTA and trypsin preparations were layered onto the gradient and allowed to sediment 34 h. Following sedimentation, 10 ml fractions were collected and analysed for cell number and cell type. The position of individual cell types on the gradient was then plotted. Photographs of cell types found in these preparations are seen on figs 2-8. Results from a gradient of cells dissociated from a 20-day-old animal are presented in fig. 9. Dead cells appeared at the top of the gradient while cells in volumes greater than 80 ml were over 95 % viable based on trypan blue dye exclusion. Spermatogonia in the Expti
Cell Res 91 (1975)
82
Davis and Schuetz
Table 2. Purity of cell types in gradients of dissociated testis from 20-day-old rats
Cell type
?I
Spermatogonia
3
Preleptotene Spermatocyte Leptotene Spermatocyte Zygotene Spermatocyte Pachytene Spermatocyte
3 3 3 3 z 3 3
Dissociation medium
Peak ( %)
Gradient volume (ml>
Number cells x 1O-5 in % peak fraction
% in starting prep.
EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin EDTA Trypsin
98kO.6 25*17 72k8 92k5 46k6 68k12 70?6 58+4 5527 50+11
(9&130) (140-160) (160-190) (190-220) (20&270) (240-260) (270-320) (350-380) (40(X420) (4W30)
5.1 kO.6 0.1 kO.01 3.6&l 5.1 kO.6 1.3kO.l 2.1 kO.6 2.4kO.02 0.4 2 0.08’ 0.9 & 0.2 0.7kO.3
32kll 2*1 18+3 41&l 11&5 22+5 25k3 21i3 10+3 6&3
a In this sample, unlike all other samples, the peak % did not coincide with the number peak, the sample at 270 ml contained 8.9 k 4 x lo5 zygotene spermatocytes and was 42 % pure.
EDTA preparation (90-130 ml) and preleptotene spermatocytes in the trypsin preparation (190-220 ml) were greater than 92 % pure. Leptotene, zygotene and pachytene spermatocytes were localized at 220-260 ml, 270-350 ml and 400430 ml, respectively, and exhibited lower purities. The position of the cells in the gradient was related to their size and, therefore, their stage of differentiation. Leydig cells appeared at 320 ml and Sertoli cells at 400 ml; neither of these cell types constituted more than 5 % of the total cells in most gradient fractions. A summary of the purities of the various cell types in these gradients from the 20-day-old testes is seenin table 2. There was a greater than two-
fold purification of all cell types following unit gravity sedimentation. Purity of the different types ranged from 25-98 %, depending on cell type and dissociation medium. A consistent observation was a faster sedimentation of all cell types following trypsin preparation than with EDTA preparation. The reason for the difference is not apparent. The testis of the lo-day-old rat has a large percentage of early cell types (spermatogonia and preleptotene spermatocytes) [2]. Because of this, there is a striking difference between the gradient profiles of the lo-day-old (fig. 10) and the 20-day-old rat testis cells (fig. 9). With the younger animal, the difference between EDTA and trypsin in the dissociation
2-8. Magnification x 1 200. 2. Spermatogonia isolated from testis of lo-day-old animals. These cells appear at 90-130 ml following sedimentation. Fig. 3. Preleptotene spermatocytes isolated from testes of lo-day-old animals. These cells appear at 190-220 ml following sedimentation. Fig. 4. Leptotene spermatocytes (L) isolated from testis of 13-day-old animals. These cells appear at 220-280 ml following sedimentation. Fig. 5. Zygotene spermatocytes (2) isolated from testes of 18-day-old animals. These cells appear at 300-330 ml following sedimentation. Fig. 6. Pachytene spermatocytes isolated from testes of 25-day-old animals. These cells appear at 37&390 ml following sedimentation. Fig. 7. Sertoli cells isolated from testes of 20-day-old animals. These cells appear at 400 ml following sedimentation. Fig. 8. Leydig cells isolated from an extratubular preparation [13] of adult testes. These cells appear at 30&330 ml following sedimentation. Figs Fig,
Exprl
Cell Res 91 (1975)
Separation of germinal c:ells
Exptl
83
Cell Res 91 (197.5)
84
Davis and Schuetz 21
21
14 7
7
14 0 l--2!2
0 I.-
#
L
45
45
30 30 / 15
/\_
15 0 i,
0 60
60
40
40
20
20
lc-
L--
01 36
‘I,
t :%
JL
12 0 L.-
Fig. 9. Abscissa: volume (ml); ordinate: (top) cells x lo-“; (bottom) % cell type. Distribution of cells in a gradient of testicular cells from a 20-day-old rat. (Left) Cells dissociated in trypsin; (right) cells dissociated in EDTA. The top row shows the distribution of total cells on both gradients. Rows 2-6 show the distribution of specific cell types on the gradients. Sedimentation is to theright.
21
21
14
14
7
7
0 r
0-
15t
in/-
/
IO 9
Fig. II. Abscissa: age (days); ordinate: % of total cells. The proportion of cells in selected gradient regions as a function of the animal. Rows l-6 represent the sedimentation volumes of (I) dead cells (O-90 ml); (2) spermatogonia (90-170 ml); (3) preleptotene(l70240 ml), (4) leptotene (240-300 ml); (5) zygotene (3%340 ml) and (6) pachytene (340-400 ml) spermatocytes, respectively. Each point indicates the percentage of total cells which was found in that gradient region. (Lefr) EDTA-dissociated preparations; (right) trypsin-dissociated preparations. Sedimentation is to the right. 8 7 6 5 4 3 2
b lLL 0
100
200 VOLUME
300
400
(ml)
Fig. 10. Abscissa: volume (ml); ordinate: cells x 10-5. Distribution of total cells in gradients after dissociation of testes from lo-day-aid rat in (a) trypsin or(b) EDTA. The top graph shows trypsin-dissociated cells; the bottom graph, EDTA-dissociated cells. Sedimentation is to the right. Spermatogonia appear at 10s160 ml in these gradients. Exptl Cell Res 91 (1975)
of spermatogonia (100-I 60 ml in the gradient) is particularly evident. To investigate the relationship between sedimentation and differentiation, cells from testes of different aged rats (lo-25 day) were sedimented at unit gravity. The total number of cells recovered from each gradient was determined and the percentage of this total in each 10 ml fraction was calculated. The total percentage of cells in gradient volumes corresponding to the sedimentation positions
Separation of germinal cells Table 3. Purification Cell type
85
of cell types from testis of immature rats by sedimentation at unit gravity Age (days)
Volume (ml) 90-l 30
% Cell type in fraction
% Cell type in starting prep.
9820.7
45kll
Spermatogonia Preleptotene Spermatocyte
10 10
1go-220
98 kO.6
54+7
Leptotene Spermatocyte
13
220-280
751-3
31+6
Zygotene Spermatocyte
18
300-330
68
19
Pachytene Spermatocyte
25
370-390
7514
34+10
of individual cell classeswas plotted at each age (fig. 11). The first row (O-90 ml) contains unidentifiable dead cells. The volume presentedin rows 2-6 in fig. 11 are the sedimentation volumes of spermatogonia (90-170 ml), preleptotene (170-240 ml), leptotene (240300 ml), zygotene (300-340 ml), and pachytene (340-440 ml) spermatocyte, respectively. The age of greatest percentage for a particular cell type, thus, is the best age for its purification. With increasing age, there was a diminution in the percentage of cells at the top of the gradient (90-240 ml, earlier types) and an increase in the percentage of cells at the bottom (240440 ml, more mature types). As previously noted, there were more dead cells in the EDTA preparations (O-90 ml) and a lower number of spermatogonia in the trypsin preparations (90-170 ml). A summarization of the best age, under our conditions, for the purification of the different early germinal cell types is presented in table 3. Purity of the cells varied from 68 to 98 % depending on cell type. All the values in the table are for EDTA-dissociated cells, Similar purities for trypsin-dissociated cells, with the exception of spermatogonia, were found. In younger animals, relatively pure preparations of certain cell types were obtained with trypsin dissociation prior to sedi-
mentation (i.e. 80 % preleptotene spermatocytes from lo-day-old animals). DISCUSSION The differentiation of germinal cells to diakinesis is an important aspect of spermatogenesis. Data presented here demonstrate that early germinal cells can be separated by unit gravity sedimentation. To study the composition of these cell populations, definitive criteria for the routine identification of isolated cell types are required. Hematoxylin is routinely used for the identification of germinal cell types in tissue section [4]; however, it was not adequate for identifying certain dissociated cell types in our preparations. Giemsa stain was used and allowed a clear identification of all but the early meiotic stages. Admittedly, classification of meiotic cells on the basisof chromatin condensation, as was done with the Giemsa-stained preparation, gives only a relative position of the cells in prophase I of meiosis. Unequivocal identification of these cells awaits chromosomal analysis. However, the Giemsa-staining did allow the clear identification of all types except leptotene and zygotene. The sedimentation and purity of one cell type, pachytene spermatocyte, agrees with Exptl
Cell Res 91 (1975)
86
Davis and Schuetz
the work of Meistrich in the adult mouse [8]. The sedimentation of the other cell classes studied also agrees in general with the work in the adult. However, purity of the other cell classes is greatly diminished when adult preparations are used. For instance, in the adult, the percent spermatogonia falls to 4 vs 98 % in the immature; preleptotene spermatocytes are 2 y0 pure in the adult vs 98 y0 pure in the lo-day-old. These differences indicate that use of immature animals greatly enhances purification of early germinal cells at unit gravity. Meistrich reported that trypsin dissociation is the best method for dissociation of testis cells from the adult mouse [8]. Also, in our hands, trypsin produces a better yield of viable cells than does EDTA. However, in the trypsin preparations, a smaller number of spermatogonia are found. The reason for this appears to be destruction by trypsin as spermatogonia dissociated with EDTA are destroyed when placed in 0.1 % trypsin. There is a good correlation, as predicted, between maturational stages and sedimentation at unit gravity. Clearly, as the cell volume increases during differentiation, there is an increase in sedimentation. Also, because of the changes occurring during maturation in the prepuberal animal, best purification of particular cell types is accomplished at different ages. In fact, an excellent preparation of preleptotene spermatocytes (80 % pure) can be obtained from the IO-day-old animal with
Exptl
Cell Res 91 (1975)
trypsin treatment without sedimentation. It is clear that two cell types, spermatogonia and preleptotene spermatocyte, can be obtained essentially pure (98 %, table 3) by sedimentation at unit gravity. Also, absolute numbers of particular cells vary with age of the animal used. With the ability to isolate purified classes of spermatogenic cells, new approaches to the study of this process become feasible. This research was supported by Grants SPOI-HD 5594-02, STOl-HD 00109-07 and Contract NOl-HD3-2794 from NICHD. The assistance of Dr Claude Desjardins is gratefully acknowledged.
REFERENCES 1. Verma, M C, Sharma, U D & Singh, G, Ind j vet sci 36 (1966) 109. 2. Clermont, Y & Perey, B, Am j anat 100 (1957) 241. 3. Leblond, C P & Clermont, Y, Am j anat 90 (1952) 167. 4. - Ann NY acad sci 55 (1952) 548. 5. Peterson, E A, Elbert, A & Evans, W E, Nature 214 (1964) 824. 6. Lam, D M K, Furrer, R & Bruce, W R, Proc natl acad sci US 65 (1970) 192. 7. Meistrich, L, J cell physiol 80 (1972) 295. 8. Meistrich, M L, Bruce, W R & Clermont, Y, Exptl cell res 79 (1973) 213. 9. Go, V L W, Vernon, R G & Fritz, I B, Can j biochem 49 (1971) 213. 10. Parker, R Ci Methods of tissue culture, p. 282. Hoeber. New York (1970). 11. Hymer,’ W C, Evans, W’E, Kracier, J, Mastro, A, Davis, J & Griswold, E, Endocrinology 92 (1973) 275. Received June 24, 1974 Revised version received September 3, 1974