- Email: [email protected]
Purification of “colony-forming” cells from immature rat testis
Experimental
PURIFICATION
Cell Research 106 (1977) 253-260
OF “COLONY-FORMING”
FROM
IMMATURE
J. C. DAVIS’ The Johns School
Hopkins
of Ilygiene
RAT
TESTIS
and A. W. SCHUETZ
University,
Deparrment
und
Health,
Public
CELLS
of Population Bnlrimore.
MI1
Dynrrmics, 21205.
USA
SUMMARY Follicle stimulating hormone (FSH) enhances colony formation (as a result of reaggregation) by dissociated IO-day-old rat testis cells in primary culture. The purpose of this study was to examine various cytological characteristics of the FSH-responsive cells and develop techniques for their purification. The ability of testis cells to form colonies in response to FSH (5 pg/ml) was tested at various ages and was found to be maximal at I5 days of age. No colony formation occurred at ages greater than 20 days. Using colony formation as an assay for the FSH-responsive cells. techniques were developed for their purification. Colony cells were purified on a continuous bovine serum albumin (BSA) step gradient (I ml/step). In fractions purified in this manner and subsequently cultured 24 h with FSH (5 pg/ml) 99.4% of attached cells were colony cells. Light and electron microscopy indicated that the colony cells (I) were one cell type; (2) were not germinal cells; (3) ultrastructurally resembled in situ Sertoli cell of the immature rat testis; and (4) contained a nucleolus with satellite karyosomes, structures which are characteristic of rat Sertoli cells. The mitotic index of the purified cells was 0.014% following 24 h in IO-J M colchicine. Based on these data, it was concluded that the FSH responsive cells in culture are Sertoli cells.
It has been reported that follicle stimulating hormone (FSH) enhances attachment and reaggregation of dissociated cells from 10 day old rat testes in culture [l]. This response can be monitored by the presence of cell colonies following 24 h of culture. These groups (colonies) of attached cells are formed from a single cell suspension presumably by cell-specific aggregation. Identity of the cells involved in this response has not been unequivocally resolved. FSH stimulation of colony formation occurs in a preparation which contains several types of somatic cells (Leydig, Sertoli, and myoid cells), and early germinal
ceils (spermatogonia and early spermatocytes). To further establish the identity of the FSH-responsive cells we have examined their ultrastructure, lipid content, nucleolar structure, and mitotic index. Colony formation provided a bioassay which was used to monitor purification of cells during isolation. Using this criterion, techniques were developed to further purify the hormone responsive cells. Results of these studies suggest that the FSH-responsive cells are Sertoli cells.
’ Present address: Department of Physiology and Cell Biology, Snow Hall, The University of Kansas. Lawrence, KS 66045, USA.
Testes (6-20) from 8, IO, 13, 15, 18, 20 and 25 day old Sprague-Dawley rats (Flow Laboratories) were removed and decapsulated. The teased tubules were in-
MATERIALS
AND
METHODS
Cell dissociation
Exp Cd
HP.\ loo
C/977)
254
Davis and Schuetz
cubated in 15 ml of 0.1% trypsin (GIBCo 1: 250) in Ca-, Mg-free phosphate-buffered saline (pH 7.35) in a 50 ml trypsinizing flask [2]. Trypsinization was carried out at room temperature with a magnetic stirrer at 50 rpm. The tissue-free supernatant was collected every 10 min, diluted with 8 % fetal calf serum (1: l), and filtered through 28 p Nitex cloth (TET-Kresslik) [12]. After 50 min of dissociation, pooled cells were collected by centrifugation at 1000 g for 10 min.
Culture and colony density determinations Single cell suspensions were cultured in 2 ml NCTC135 media (GIBCo) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 pg/ml streptomycin in 35 mm plastic dishes at 32.5”C under an air: CO2 atmosnhere (95 % : 5 %). Cells were cultured for 24 h, the media removed, and dishes washed twice in 2 ml saline to remove non-attached cells. Dishes were fixed 24 h with 4% formalin, stained 4 min with hematoxylin, and the number of colonies in five randomly chosen 0.25 cm2 areas counted at 6~ magnification [2]. This procedure allows the visualization of cell groups greater than 4-5 cells/group. Counts of suspended cells were made on a cytograf (Biophysics Instruments); the instrument was set to count only those particles greater than the size of red blood cells.
Cytological
Cell identification Identification of cells attached to the dish bottom were made by morphology and growth pattern. Colony cells: rounded cells growing in close association with one another in colonies [l]. Leydig cells: flattened, non-spindle-shaped, cells, oriented at random on the dish bottom. Fibroblast: spindle-shaped cells oriented at random on the dish bottom.
procedures
For electron microscopy, monolayers from l&day old testis cells, cultured 24 h with FSH (5 pg/ml), were fixed and embedded by the method of Brinkley et al. 131. For Feulgen staining, cells were fixed 30 min in acetic acid : eihanol (1: 3), hydrolyzed 60 min at 25°C in 5 N HCl. and stained with Schiffs reagent. To detect lipids,‘24 h cultures were fixed in 10 %Iformalin and stained in 0.7% Sudan Black B dissolved in ethylene glycol [4]. To determine the relative mitotic index of whole testis and isolated cells, cells were incubated 24 h followed by 24 h in low5 M colchicine. Cultures in three separate experiments were fixed in acetic acid/ethanol (1: 3), stained with aceto-orcein, and the number of mitotic figures in 10000 cells, chosen at random on the dish bottom, determined. The number of attached cells was not affected under these conditions.
BSA gradients Continuous bovine serum albumin (BSA) solutions were prepared as described by Snyder et al. [5]. Gradients were generated as described by Leif et al. [6]. Gradients containing 13-24% BSA and 1.5~10~ cells of a single cell suspension from 15-day-old rat testes were centrifuged at 4 000 g for 40 min in the HG4L head in an International model RC3 centrifuge at 4C and fractions collected by topflow by gradient displacement with 80 % sucrose. Each fraction was then counted, 0.1 ml placed in media containing 5 pg/ml FSH and 0.1 ml in control media. Following 24 h, cultures were stained for colonv counts as oreviouslv described. Densities of bovine serum albumin fraitions were determined with a Bausch & Lomb hand refractometer. Exp CellRes
For isolation on discontinuous gradients 5~ 106 cells of a sinale cell susnension were lavered onto a BSA step gradient (14%; 17%, 25 %, BSA, 1 ml/step) and centrifuged 40 min at 4 000 g (4C), in 13.5 ml centrifuge tubes (Beckman). Following centrifugation, fractions were collected from the gradient (1.05, 0.82,0.84, and 0.87 ml) and 0.1 ml of each fraction placed in either control or FSH (5 pg/ml) media. Following 24 h of incubation, dishes were processed for colony counts. Several precautions were taken in preparing albumin gradients. Extensive dialvsis of the albumin was req&ed to remove traces of-salt and secondly, with different “batches” of albumin, some differences in sedimentation may be noted. This necessitated monitoring gradient fractions when new “batches” are used.
106 (1977)
RESULTS Age specificity In the immature rat, the cellular population of the testis undergoes rapid changes [7]. In order to choose the optimal age for purification of colony-forming cells, the effect of age on. FSH-induced colony formation was tested. To investigate the age specificity of this response, cells from 8, 10, 13, 15, 18, 20 and 25 days old rat testes were cultured in 24 h in FSH (5 pg/ml) or control media and determination of colony densities performed [ 11. Five determinations were performed at each point in two experiments. The incidence of FSH responsiveness was highest between 8 and 15 days of age (fig. 1) while relatively few colonies were seen in cultures of cells taken from testes of 18, 20, and 25 days old animals. Because of this age effect, testes of 15dayold animals were subsequently used for puritication of colony-forming cells.
Purification
1. Abscissa: time (days); ordinate: no. colonies/ cm*X 10-l. CO, FSH; O---O, control. Effect of age on the FSH induction of colony formation by rat testicular cells in primary culture. Each point is the mean +_S.E.M. of ten determinations.
Fig.
Isolation procedures The technique employed for cell purification was equilibrium centrifugation on 1324 % BSA gradients as described. As can be seen in fig. 2a, a major peak of cells appeared at a density of 1.053 g/cc; also, at this density, a peak of FSH-responsive cells was noted (fig. 2h) following 24 h of culture. When the percentage of cell types on the dish bottom was scored on the basis of their attachment and morphology, the fraction at 3.4 ml vol contained over 95 % colony cells. Fibroblasts appeared at the top and Leydig cells at the bottom of the gradient. These data indicate that further purification of colony cells can be accomplished with the combined techniques of density gradient centrifugation and culture. However, because of problems in maintaining sterility and in obtaining large numbers of cells, a step gradient was designed. Using the characteristic density of colony cells found with the continuous gradient, a step gradient was designed for purification of these cells from the 15day-old rat testis. Cells (5x 10” in 0.5 ml) were layered onto a 14%, 17%, 25% BSA step gradient as described. Following centrifugation, a major peak of cells was seen at the 14%, 17%: BSA interface (fig. 3). In subsequent experiments, volumes were drawn by pipet from
of rat testis colony-forming
cells
25.5
the gradient volumes O-l .05, i .05-! .87, 1.87-2.76 and 2.76-3.63 ml. These four fractions were then monitored for cell number and 0.1 ml for each fraction (see table 1) cultured 24 h in control or FSH media (5 ,ug/ ml). Following culture, the number cf unattached cells was counted and the attached cells removed by treatment for 10 min with 5 mM EDTA. Counts of the number of attached cells allowed then a calculation of percentage of the cell population attached (table 1). Separate dishes were fixed and stained with hematoxylin and counted for
60 40 20 0
z
4
6
B
10
(ml);
ordinare:
12
(a, icj>) no. of cells X !Om5(O-O); (a, right) density (g/cm”) (O- --0); (b) co!ony/cm2x IO-?: (c) 5%cell type. Separation of Sertoli cells by equilibrium centrifugation. (a) Distribution of cells on a 13-24s bovine serum albumin gradient; 0. cell number; 0, density of fraction: (b) distribution of colonies on the dish bottom following 24 h culture of 0.1 ml of each gradient fraction in FSH (O-O) (5 p&m!), or control media (O---O); (c) distribution of cell types on the dish bottom following 24 h cultures of each gradient fraction. I 000 cells were counted at each point (c). ill- - -0, Fibroblast; O-O, Sertoli; l -- -0. Leydig. Fig.
2. Abscissa:
vol.
256
Davis and Schuett
Table 1. Distribution, attachment, and FSH responsiveness centrifuged on a 14, 17 and 25 70 albumin step gradient Gradient distribution Gradient fraction
Vol. (ml)
Total no. cells X 10m5 Media 8.kk1.4
0.82kO.02
13.9kO.5
0.89f0.04
8.4+ 1.8
0.87+0.10
6.1f0.2
N
Control 3 FSH 3 Control 3 FSH 3 Control 3 FSH 3 Control 3 FSH 3
No. % No. floating attached Attached cells X lo-* cells X lo-* cells
% Colony cells
8.1+1.4 7.4+_ 1.o 23 f0.7 22 kO.9 8.9f0.9 8.2kO.6 5.4kO.4 4.8kO.3
33 +17 20 t10 94.4f 3.2 99.4+ 0.2 16 k14 40 + 4.0 9 * 7.0 30 f18
percentage colony cells (2 000 cells counted/ dish) and for colony density (colonies/cm2). Gradient fraction 2 (I .05-l .87 ml) contained the highest density of colonies, 402+48 colonies/cm2 (n=3, S.E.M.) and colony cells (99.4+0.2%, n=3) following 24 h of culture; in addition, FSH stimulation of colony density and percentage attached cells following culture was greatest in this gradient volume. It should be noted that comparable purities were obtained with a two step (14%, 17 %) gradient (unpublished). To investigate the FSH responsiveness of the isolated cells, ce!‘ls were plated at various plating concentrations in FSH (5 pg/mI) or control cultures and colony density determined following 24 h of culture. Colony density was significantly affected by FSH and by plating density (analysis of variance). Interestingly, least square analysis indicates that the data are best fitted to an exponential (fig. 46) rather than a linear regression line (fig. 4a, SY/X=residual variance). This may indicate some kind of media conditioning effect or cell number limiting interaction involved in colony formation.
testis cells
Cell types after culture
Cell nos after culture
1.05io.05
Exp Cell .‘,‘a 106 (1977)
of IS-day-old
2.1kO.3 2.OkO.2 9.8kO.l 12 f0.03 4.9&O. 1 8.2kO.6 3.4kO.2 2.9kO.l
26f 1.0 28kO.5 37k 18 54k4.0 63k6.0 59f3.0 62f2.0 61f2.0
Colony/ cm2 5.2f 2.9 20.1+10 260 +55 402 _+48 51 +27 59 +27 7 i 6.0 12 +11
Mitotic index Mitotic index offers a basic measurement of cell function in cultured cells. Also, since cultures of whole testis cells contain fibroblasts, a cell with a high mitotic index, mitotic activity offers another monitor of cell purity during isolation procedures. To determine the relative mitotic activities of cells before and after purification, an aliquot of a whole testis cell preparation and an aliquot of that preparation after gradient purification were cultured as described. The mitotic index of the isolated colony cells was O.Ol?O.Ol% (n=3) versus 2.7+ 0.3 % (n=3) in the starting preparation.
vol. (ml); ordinate: no. cells X lo-‘. Distribution of testis cells on a 14, 17 and 25 % albumin step gradient. The 1.4-I .9 ml fraction is adjacent to the 14 and 17 % albumin interface.
Fig. 3. Abscissa:
Purification
Fig. 4. Abscissa: no. cells x IO+; ordinate: (colonies/ cm2)X 10-l. Effect of plating density and FSH on colony formation by isolated colony cells. The regression lines were calculated by least sauares. (a) Linear regression lines, y=Bx. m---O, Control: y=2.53+21.2~; S(y/x)=32.1; O-O, FSH: y=3.93+43.7x; S(y/x)=51.6; (b) exponential regression lines, y=(ax)B. Control: y= (16.6~)‘5 S(v/x)=O.6; FSH: y=(39.0X)‘.09; S(y/x)=O.5. (a, b) n=5.
To investigate the cytological properties of the colony cells, 5x 105 cells were cultured 24 h in FSH (5 pg/ml) and the monolayers fixed for light and electron microscopy. Fig. 5 is an electron micrograph of a colony cell from a lo-day-old rat testis; the cytoplasm contains osmophilic bodies and an abundance of free ribosomes. Examination of the ultrastructure of cells in different colonies and culture dishes suggests that they are of one cell type, are not germinal cells (Dr Don Fawcett, personal communication), and are similar to the Sertoli cell of the immature rat testis in situ (M. Dym, personal communication). Fig. 6 is a light micrograph of a colony from a lO-day-old rat fixed with formalin and stained with Sudan black B and hematoxylin. The sudanophilia suggests that the osmophilic bodies seen in the electron microscope are lipid. Essentially, all (99%) of the colony cells exhibited rounded, tight associations in primary culture while the fibroblasts were spindleshaped and the Leydig cells flattened in culture. Other staining methods indicated that the cells were of one cell type. All colony
of rat testis co~o~y-~o~~i~~
ceEEs 257
cells exhibited the finely dispersed nuclear chromatin seen in fig. 7. Also. with epon embedding, all colony cells appeared identical after toluidine blue staining (fig. 8). No differences in cytology were noted between colony cells in control and in FSH-treated cultures. Nucleolar morphology has been suggested to be unique in rat Sertoli cells [8,9]. Fig. 9 is a photomicrograph of a Feulgenstained culture of IS-day-old colony cells incubated 24 h in FSH (5 pg/ml) showing the presence of nucleolar satellite karyosomes. These structures resemble those described by Sapsford and contain a faintly Feulgenpositive central region and strongly staining structures adjacent to the main body of the Sertoli cell nucleolus [8]. The number of cells with karyosomes was quantitated by examining one cell in each of 200 colonies at 1250x magnification. In five dishes, 87+5 o/c, of the colony cells contained a nucleolus with recognizable karyosomes. This nucleolar morphology was not seen in the same number of fibroblasts or Leydig cells in the same dishes. DISCUSSION Problems of cell purification and cell identification are of major significance for the study of dissociated tissues in culture. The immature (lO-20-day-old) rat offers several advantages for the study of testis cells in culture. (1) Testes from immature rats contain only early germinal cells [7] and therefore a relative enrichment of somatic (Sertoli and Leydig) cells. (2) We have shown that FSH stimulates cell attachment in cultures of testis cells from the immature rat [I]. For purification, other techniques have been developed for isolation of Sertoli cells from rats of varying ages [13, 14, 151. These
258
Exp Cell
Davis and Schuetz
Res 106 (1977)
Purijkation
of
rat
testis
coio~y-fording
cells
Fig. 8. Photomicrograph of a colony from a 15day-oid testis cultured 24 h with FSH (5 &ml) fixed with glutaraldehyde, epon embedded, and stained with toluidine blue. x 1400. Fig. 9. Phase contrast photomicrograph of cells in a colony from a 15day-old testis cultured 24 h in FSH (5 pg/ml), fixed with acetic acid/ethanol (3: 1) and stained by the Feulgen procedure. Note satellite karyoX 1400. somes (orrows).
techniques employ differential enzymatic digestion of seminiferous tubules. While our technique employs different means, equilibrium centrifugation, both methods take advantage of the observation that somatic cells attach to the culture substratum while germinal cells do not [16]. The equilibrium method is rapid (less than 3 h), results in good purity (99&0.4% colony cells), and yields 8~ IV Sertoli cells from 10, 15 day
Fig. 5. Electron photomicrograph of a colony cell from a IO-day-old testis cultured 24 h with FSH (5 &ml). Note lipid droplets (arrows). x 10300. Frg. 6. Photomicrograph of a colony from a l@day-old testis cultured 24 h with FSH (5 &g/ml), stained-with Sudan black B and hemotoxylin. Note sudanophilia in cytoplasm (arrows). Xl OOO.Fig. 7. Photomicrograph of a colony from a &day-old testis cultured 24 h with FSH (5 &ml) fixed in Bouins and stained with hemotoxylin. X 1000.
old rat testes. The relative densities of fibroblast, Leydig, and Sertoli cells using this method agrees with the densities seen by Meistrich [ 171. The problem of cell identification is central to the study of differentiated cells in culture. Because of the loss of tissue topography used in the identification of cell types in situ, cell-specific cytological criteria are required for identification in culture. Only then can cell-specific biochemical activities be proven and used as criteria for identification. The present results strongly suggest that the colony-forming cells are primarily Sertoli cells. The ceils are responsive to FSH; FSH responsiveness has been noted in the Sertoli cell enriched testis in vivo [ 193 and Sertoli cells in vitro [I4, 15, 181. In addition, Means has isp
Cd/
NPF 106 (iYZ7)
260
Davis and Schuetz
shown a variety of other FSH actions on the testis of the immature rat [ 10, 11, 121. It is of interest that the age specificity of the colony-forming response to FSH is similar to that noted bv Means for other FSH responses. The age specificity of the response is similar to other FSH-stimulated Sertoli cell activities in vivo [lo]. Ultrastructural investigation of colony cell cytology indicates that they are (1) of one cell type; (2) are not germinal cells; and (3) resemble Sertoli cells of the immature rat testis in situ. The cytoplasmic sudanophilia has been suggested by one author to be indicative of Sertoli cells [13] and the low mitotic index of the isolated cells is similar to that noted for Sertoli cells in vivo [20]. Most importantly, the presence of nucleolar satellite karyosomes are considered to be specific for Sertoli cells [8, 91. These structures have been noted in several rodent Sertoli cells; in partitular, Sapsford indicates that the nucleolus of the rat Sertoli cell is weakly Feulgen positive while the karyosomes are strongly Feulgen-positive [8]. As these structures have been found in the colony cells from the 15day-old rat testis but not in fibroblasts or Leydig cells, the identification of the cells as Sertoli cells appears to be well grounded. We thank Drs Barry Zirkin and Ann help with the electron microscopy. Pat Welch and Dr Robert Mitchell staining is gratefully acknowledged.
Exp Cell
Res 106 (1977)
Belanger for their The help of Mr with the Feulgen The assistance of
Drs Don W. Fawcett and Martin Dym in examination of the colony cell ultrastructure was of considerable help We thank the National Institute of Arthritis and Metabolic Diseases for NIH-FSH-SlO. This research was supported by contract NOl-HD-3-2794, NICHD. Funds for some equinment were provided bv the Rockefeller Foundation.
REFERENCES 1. Davis, J C & Schuetz, A W, Nature 254 (1975) 611. - Exp cell res 91 (1975) 79. 3. Brinkley, B R & Hsu, T C, Cytologia 35 (1970) 424. 4. Humason, G L, Animal tissue techniques, 4th edn, p. 308. W H Freeman & Co., San Francisco, CA 2.
(1962). .__ _-,.
5. Snyder, G & Hymer, W C, Endocrinology 96 (1975) 792. 6. Leif. R C & Vinozrad. J, Proc natl acad sci US 51 (196i)
520.
-
7. 8. 9. 10.
Clermont, Y & Perey, B, Am j anat 100 (1957) 241. Sapsford, C S, J anat 97 (1962) 225. Bouin, P, Bibliog anat 7 (1899) 242. Means, A R, MacDougall, E, Soderling, T R & Corbin. J D. J biol them 249 (1974) 1231. 11. Means,‘A R; Endocrinology 89 (1971) 981. 12. Means, A R & Hall, P F, Endocrinology 81 (1967) 1151.
13. Welsh, M J & Wiebe, J P, Endocrinology 96 (1975) 618. 14. Tung, P S, Dorrington, H J & Fritz, I B, Proc natl acad sci US 72 (1975) 1838. 15. Steinberger, A, ‘Heindel, J J, Lindsey, J H, Elkington. J H. Sanborn. B M & Steinberger. E. Endocrine rks commun 2 (1975) 261. - ’ 16. Steinberger, A, Anat ret 151(1965) 420. 17. Meistrich, M L & Trostle, P K, Exp cell res 92 (1975) 231. 18. Dorrington, J & Armstrong, D T, Proc natl acad sci US 72 (1975) 2677. 19. Means, A R. Fakunding, J L & Tindall, D J, Biol reprod. (1976) 54. -’ 20. Nagy, F, J reprod fertil28 (1972) 389. Received December 1, 1976 Accepted December 6, 1976
PURIFICATION
Cell Research 106 (1977) 253-260
OF “COLONY-FORMING”
FROM
IMMATURE
J. C. DAVIS’ The Johns School
Hopkins
of Ilygiene
RAT
TESTIS
and A. W. SCHUETZ
University,
Deparrment
und
Health,
Public
CELLS
of Population Bnlrimore.
MI1
Dynrrmics, 21205.
USA
SUMMARY Follicle stimulating hormone (FSH) enhances colony formation (as a result of reaggregation) by dissociated IO-day-old rat testis cells in primary culture. The purpose of this study was to examine various cytological characteristics of the FSH-responsive cells and develop techniques for their purification. The ability of testis cells to form colonies in response to FSH (5 pg/ml) was tested at various ages and was found to be maximal at I5 days of age. No colony formation occurred at ages greater than 20 days. Using colony formation as an assay for the FSH-responsive cells. techniques were developed for their purification. Colony cells were purified on a continuous bovine serum albumin (BSA) step gradient (I ml/step). In fractions purified in this manner and subsequently cultured 24 h with FSH (5 pg/ml) 99.4% of attached cells were colony cells. Light and electron microscopy indicated that the colony cells (I) were one cell type; (2) were not germinal cells; (3) ultrastructurally resembled in situ Sertoli cell of the immature rat testis; and (4) contained a nucleolus with satellite karyosomes, structures which are characteristic of rat Sertoli cells. The mitotic index of the purified cells was 0.014% following 24 h in IO-J M colchicine. Based on these data, it was concluded that the FSH responsive cells in culture are Sertoli cells.
It has been reported that follicle stimulating hormone (FSH) enhances attachment and reaggregation of dissociated cells from 10 day old rat testes in culture [l]. This response can be monitored by the presence of cell colonies following 24 h of culture. These groups (colonies) of attached cells are formed from a single cell suspension presumably by cell-specific aggregation. Identity of the cells involved in this response has not been unequivocally resolved. FSH stimulation of colony formation occurs in a preparation which contains several types of somatic cells (Leydig, Sertoli, and myoid cells), and early germinal
ceils (spermatogonia and early spermatocytes). To further establish the identity of the FSH-responsive cells we have examined their ultrastructure, lipid content, nucleolar structure, and mitotic index. Colony formation provided a bioassay which was used to monitor purification of cells during isolation. Using this criterion, techniques were developed to further purify the hormone responsive cells. Results of these studies suggest that the FSH-responsive cells are Sertoli cells.
’ Present address: Department of Physiology and Cell Biology, Snow Hall, The University of Kansas. Lawrence, KS 66045, USA.
Testes (6-20) from 8, IO, 13, 15, 18, 20 and 25 day old Sprague-Dawley rats (Flow Laboratories) were removed and decapsulated. The teased tubules were in-
MATERIALS
AND
METHODS
Cell dissociation
Exp Cd
HP.\ loo
C/977)
254
Davis and Schuetz
cubated in 15 ml of 0.1% trypsin (GIBCo 1: 250) in Ca-, Mg-free phosphate-buffered saline (pH 7.35) in a 50 ml trypsinizing flask [2]. Trypsinization was carried out at room temperature with a magnetic stirrer at 50 rpm. The tissue-free supernatant was collected every 10 min, diluted with 8 % fetal calf serum (1: l), and filtered through 28 p Nitex cloth (TET-Kresslik) [12]. After 50 min of dissociation, pooled cells were collected by centrifugation at 1000 g for 10 min.
Culture and colony density determinations Single cell suspensions were cultured in 2 ml NCTC135 media (GIBCo) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 pg/ml streptomycin in 35 mm plastic dishes at 32.5”C under an air: CO2 atmosnhere (95 % : 5 %). Cells were cultured for 24 h, the media removed, and dishes washed twice in 2 ml saline to remove non-attached cells. Dishes were fixed 24 h with 4% formalin, stained 4 min with hematoxylin, and the number of colonies in five randomly chosen 0.25 cm2 areas counted at 6~ magnification [2]. This procedure allows the visualization of cell groups greater than 4-5 cells/group. Counts of suspended cells were made on a cytograf (Biophysics Instruments); the instrument was set to count only those particles greater than the size of red blood cells.
Cytological
Cell identification Identification of cells attached to the dish bottom were made by morphology and growth pattern. Colony cells: rounded cells growing in close association with one another in colonies [l]. Leydig cells: flattened, non-spindle-shaped, cells, oriented at random on the dish bottom. Fibroblast: spindle-shaped cells oriented at random on the dish bottom.
procedures
For electron microscopy, monolayers from l&day old testis cells, cultured 24 h with FSH (5 pg/ml), were fixed and embedded by the method of Brinkley et al. 131. For Feulgen staining, cells were fixed 30 min in acetic acid : eihanol (1: 3), hydrolyzed 60 min at 25°C in 5 N HCl. and stained with Schiffs reagent. To detect lipids,‘24 h cultures were fixed in 10 %Iformalin and stained in 0.7% Sudan Black B dissolved in ethylene glycol [4]. To determine the relative mitotic index of whole testis and isolated cells, cells were incubated 24 h followed by 24 h in low5 M colchicine. Cultures in three separate experiments were fixed in acetic acid/ethanol (1: 3), stained with aceto-orcein, and the number of mitotic figures in 10000 cells, chosen at random on the dish bottom, determined. The number of attached cells was not affected under these conditions.
BSA gradients Continuous bovine serum albumin (BSA) solutions were prepared as described by Snyder et al. [5]. Gradients were generated as described by Leif et al. [6]. Gradients containing 13-24% BSA and 1.5~10~ cells of a single cell suspension from 15-day-old rat testes were centrifuged at 4 000 g for 40 min in the HG4L head in an International model RC3 centrifuge at 4C and fractions collected by topflow by gradient displacement with 80 % sucrose. Each fraction was then counted, 0.1 ml placed in media containing 5 pg/ml FSH and 0.1 ml in control media. Following 24 h, cultures were stained for colonv counts as oreviouslv described. Densities of bovine serum albumin fraitions were determined with a Bausch & Lomb hand refractometer. Exp CellRes
For isolation on discontinuous gradients 5~ 106 cells of a sinale cell susnension were lavered onto a BSA step gradient (14%; 17%, 25 %, BSA, 1 ml/step) and centrifuged 40 min at 4 000 g (4C), in 13.5 ml centrifuge tubes (Beckman). Following centrifugation, fractions were collected from the gradient (1.05, 0.82,0.84, and 0.87 ml) and 0.1 ml of each fraction placed in either control or FSH (5 pg/ml) media. Following 24 h of incubation, dishes were processed for colony counts. Several precautions were taken in preparing albumin gradients. Extensive dialvsis of the albumin was req&ed to remove traces of-salt and secondly, with different “batches” of albumin, some differences in sedimentation may be noted. This necessitated monitoring gradient fractions when new “batches” are used.
106 (1977)
RESULTS Age specificity In the immature rat, the cellular population of the testis undergoes rapid changes [7]. In order to choose the optimal age for purification of colony-forming cells, the effect of age on. FSH-induced colony formation was tested. To investigate the age specificity of this response, cells from 8, 10, 13, 15, 18, 20 and 25 days old rat testes were cultured in 24 h in FSH (5 pg/ml) or control media and determination of colony densities performed [ 11. Five determinations were performed at each point in two experiments. The incidence of FSH responsiveness was highest between 8 and 15 days of age (fig. 1) while relatively few colonies were seen in cultures of cells taken from testes of 18, 20, and 25 days old animals. Because of this age effect, testes of 15dayold animals were subsequently used for puritication of colony-forming cells.
Purification
1. Abscissa: time (days); ordinate: no. colonies/ cm*X 10-l. CO, FSH; O---O, control. Effect of age on the FSH induction of colony formation by rat testicular cells in primary culture. Each point is the mean +_S.E.M. of ten determinations.
Fig.
Isolation procedures The technique employed for cell purification was equilibrium centrifugation on 1324 % BSA gradients as described. As can be seen in fig. 2a, a major peak of cells appeared at a density of 1.053 g/cc; also, at this density, a peak of FSH-responsive cells was noted (fig. 2h) following 24 h of culture. When the percentage of cell types on the dish bottom was scored on the basis of their attachment and morphology, the fraction at 3.4 ml vol contained over 95 % colony cells. Fibroblasts appeared at the top and Leydig cells at the bottom of the gradient. These data indicate that further purification of colony cells can be accomplished with the combined techniques of density gradient centrifugation and culture. However, because of problems in maintaining sterility and in obtaining large numbers of cells, a step gradient was designed. Using the characteristic density of colony cells found with the continuous gradient, a step gradient was designed for purification of these cells from the 15day-old rat testis. Cells (5x 10” in 0.5 ml) were layered onto a 14%, 17%, 25% BSA step gradient as described. Following centrifugation, a major peak of cells was seen at the 14%, 17%: BSA interface (fig. 3). In subsequent experiments, volumes were drawn by pipet from
of rat testis colony-forming
cells
25.5
the gradient volumes O-l .05, i .05-! .87, 1.87-2.76 and 2.76-3.63 ml. These four fractions were then monitored for cell number and 0.1 ml for each fraction (see table 1) cultured 24 h in control or FSH media (5 ,ug/ ml). Following culture, the number cf unattached cells was counted and the attached cells removed by treatment for 10 min with 5 mM EDTA. Counts of the number of attached cells allowed then a calculation of percentage of the cell population attached (table 1). Separate dishes were fixed and stained with hematoxylin and counted for
60 40 20 0
z
4
6
B
10
(ml);
ordinare:
12
(a, icj>) no. of cells X !Om5(O-O); (a, right) density (g/cm”) (O- --0); (b) co!ony/cm2x IO-?: (c) 5%cell type. Separation of Sertoli cells by equilibrium centrifugation. (a) Distribution of cells on a 13-24s bovine serum albumin gradient; 0. cell number; 0, density of fraction: (b) distribution of colonies on the dish bottom following 24 h culture of 0.1 ml of each gradient fraction in FSH (O-O) (5 p&m!), or control media (O---O); (c) distribution of cell types on the dish bottom following 24 h cultures of each gradient fraction. I 000 cells were counted at each point (c). ill- - -0, Fibroblast; O-O, Sertoli; l -- -0. Leydig. Fig.
2. Abscissa:
vol.
256
Davis and Schuett
Table 1. Distribution, attachment, and FSH responsiveness centrifuged on a 14, 17 and 25 70 albumin step gradient Gradient distribution Gradient fraction
Vol. (ml)
Total no. cells X 10m5 Media 8.kk1.4
0.82kO.02
13.9kO.5
0.89f0.04
8.4+ 1.8
0.87+0.10
6.1f0.2
N
Control 3 FSH 3 Control 3 FSH 3 Control 3 FSH 3 Control 3 FSH 3
No. % No. floating attached Attached cells X lo-* cells X lo-* cells
% Colony cells
8.1+1.4 7.4+_ 1.o 23 f0.7 22 kO.9 8.9f0.9 8.2kO.6 5.4kO.4 4.8kO.3
33 +17 20 t10 94.4f 3.2 99.4+ 0.2 16 k14 40 + 4.0 9 * 7.0 30 f18
percentage colony cells (2 000 cells counted/ dish) and for colony density (colonies/cm2). Gradient fraction 2 (I .05-l .87 ml) contained the highest density of colonies, 402+48 colonies/cm2 (n=3, S.E.M.) and colony cells (99.4+0.2%, n=3) following 24 h of culture; in addition, FSH stimulation of colony density and percentage attached cells following culture was greatest in this gradient volume. It should be noted that comparable purities were obtained with a two step (14%, 17 %) gradient (unpublished). To investigate the FSH responsiveness of the isolated cells, ce!‘ls were plated at various plating concentrations in FSH (5 pg/mI) or control cultures and colony density determined following 24 h of culture. Colony density was significantly affected by FSH and by plating density (analysis of variance). Interestingly, least square analysis indicates that the data are best fitted to an exponential (fig. 46) rather than a linear regression line (fig. 4a, SY/X=residual variance). This may indicate some kind of media conditioning effect or cell number limiting interaction involved in colony formation.
testis cells
Cell types after culture
Cell nos after culture
1.05io.05
Exp Cell .‘,‘a 106 (1977)
of IS-day-old
2.1kO.3 2.OkO.2 9.8kO.l 12 f0.03 4.9&O. 1 8.2kO.6 3.4kO.2 2.9kO.l
26f 1.0 28kO.5 37k 18 54k4.0 63k6.0 59f3.0 62f2.0 61f2.0
Colony/ cm2 5.2f 2.9 20.1+10 260 +55 402 _+48 51 +27 59 +27 7 i 6.0 12 +11
Mitotic index Mitotic index offers a basic measurement of cell function in cultured cells. Also, since cultures of whole testis cells contain fibroblasts, a cell with a high mitotic index, mitotic activity offers another monitor of cell purity during isolation procedures. To determine the relative mitotic activities of cells before and after purification, an aliquot of a whole testis cell preparation and an aliquot of that preparation after gradient purification were cultured as described. The mitotic index of the isolated colony cells was O.Ol?O.Ol% (n=3) versus 2.7+ 0.3 % (n=3) in the starting preparation.
vol. (ml); ordinate: no. cells X lo-‘. Distribution of testis cells on a 14, 17 and 25 % albumin step gradient. The 1.4-I .9 ml fraction is adjacent to the 14 and 17 % albumin interface.
Fig. 3. Abscissa:
Purification
Fig. 4. Abscissa: no. cells x IO+; ordinate: (colonies/ cm2)X 10-l. Effect of plating density and FSH on colony formation by isolated colony cells. The regression lines were calculated by least sauares. (a) Linear regression lines, y=Bx. m---O, Control: y=2.53+21.2~; S(y/x)=32.1; O-O, FSH: y=3.93+43.7x; S(y/x)=51.6; (b) exponential regression lines, y=(ax)B. Control: y= (16.6~)‘5 S(v/x)=O.6; FSH: y=(39.0X)‘.09; S(y/x)=O.5. (a, b) n=5.
To investigate the cytological properties of the colony cells, 5x 105 cells were cultured 24 h in FSH (5 pg/ml) and the monolayers fixed for light and electron microscopy. Fig. 5 is an electron micrograph of a colony cell from a lo-day-old rat testis; the cytoplasm contains osmophilic bodies and an abundance of free ribosomes. Examination of the ultrastructure of cells in different colonies and culture dishes suggests that they are of one cell type, are not germinal cells (Dr Don Fawcett, personal communication), and are similar to the Sertoli cell of the immature rat testis in situ (M. Dym, personal communication). Fig. 6 is a light micrograph of a colony from a lO-day-old rat fixed with formalin and stained with Sudan black B and hematoxylin. The sudanophilia suggests that the osmophilic bodies seen in the electron microscope are lipid. Essentially, all (99%) of the colony cells exhibited rounded, tight associations in primary culture while the fibroblasts were spindleshaped and the Leydig cells flattened in culture. Other staining methods indicated that the cells were of one cell type. All colony
of rat testis co~o~y-~o~~i~~
ceEEs 257
cells exhibited the finely dispersed nuclear chromatin seen in fig. 7. Also. with epon embedding, all colony cells appeared identical after toluidine blue staining (fig. 8). No differences in cytology were noted between colony cells in control and in FSH-treated cultures. Nucleolar morphology has been suggested to be unique in rat Sertoli cells [8,9]. Fig. 9 is a photomicrograph of a Feulgenstained culture of IS-day-old colony cells incubated 24 h in FSH (5 pg/ml) showing the presence of nucleolar satellite karyosomes. These structures resemble those described by Sapsford and contain a faintly Feulgenpositive central region and strongly staining structures adjacent to the main body of the Sertoli cell nucleolus [8]. The number of cells with karyosomes was quantitated by examining one cell in each of 200 colonies at 1250x magnification. In five dishes, 87+5 o/c, of the colony cells contained a nucleolus with recognizable karyosomes. This nucleolar morphology was not seen in the same number of fibroblasts or Leydig cells in the same dishes. DISCUSSION Problems of cell purification and cell identification are of major significance for the study of dissociated tissues in culture. The immature (lO-20-day-old) rat offers several advantages for the study of testis cells in culture. (1) Testes from immature rats contain only early germinal cells [7] and therefore a relative enrichment of somatic (Sertoli and Leydig) cells. (2) We have shown that FSH stimulates cell attachment in cultures of testis cells from the immature rat [I]. For purification, other techniques have been developed for isolation of Sertoli cells from rats of varying ages [13, 14, 151. These
258
Exp Cell
Davis and Schuetz
Res 106 (1977)
Purijkation
of
rat
testis
coio~y-fording
cells
Fig. 8. Photomicrograph of a colony from a 15day-oid testis cultured 24 h with FSH (5 &ml) fixed with glutaraldehyde, epon embedded, and stained with toluidine blue. x 1400. Fig. 9. Phase contrast photomicrograph of cells in a colony from a 15day-old testis cultured 24 h in FSH (5 pg/ml), fixed with acetic acid/ethanol (3: 1) and stained by the Feulgen procedure. Note satellite karyoX 1400. somes (orrows).
techniques employ differential enzymatic digestion of seminiferous tubules. While our technique employs different means, equilibrium centrifugation, both methods take advantage of the observation that somatic cells attach to the culture substratum while germinal cells do not [16]. The equilibrium method is rapid (less than 3 h), results in good purity (99&0.4% colony cells), and yields 8~ IV Sertoli cells from 10, 15 day
Fig. 5. Electron photomicrograph of a colony cell from a IO-day-old testis cultured 24 h with FSH (5 &ml). Note lipid droplets (arrows). x 10300. Frg. 6. Photomicrograph of a colony from a l@day-old testis cultured 24 h with FSH (5 &g/ml), stained-with Sudan black B and hemotoxylin. Note sudanophilia in cytoplasm (arrows). Xl OOO.Fig. 7. Photomicrograph of a colony from a &day-old testis cultured 24 h with FSH (5 &ml) fixed in Bouins and stained with hemotoxylin. X 1000.
old rat testes. The relative densities of fibroblast, Leydig, and Sertoli cells using this method agrees with the densities seen by Meistrich [ 171. The problem of cell identification is central to the study of differentiated cells in culture. Because of the loss of tissue topography used in the identification of cell types in situ, cell-specific cytological criteria are required for identification in culture. Only then can cell-specific biochemical activities be proven and used as criteria for identification. The present results strongly suggest that the colony-forming cells are primarily Sertoli cells. The ceils are responsive to FSH; FSH responsiveness has been noted in the Sertoli cell enriched testis in vivo [ 193 and Sertoli cells in vitro [I4, 15, 181. In addition, Means has isp
Cd/
NPF 106 (iYZ7)
260
Davis and Schuetz
shown a variety of other FSH actions on the testis of the immature rat [ 10, 11, 121. It is of interest that the age specificity of the colony-forming response to FSH is similar to that noted bv Means for other FSH responses. The age specificity of the response is similar to other FSH-stimulated Sertoli cell activities in vivo [lo]. Ultrastructural investigation of colony cell cytology indicates that they are (1) of one cell type; (2) are not germinal cells; and (3) resemble Sertoli cells of the immature rat testis in situ. The cytoplasmic sudanophilia has been suggested by one author to be indicative of Sertoli cells [13] and the low mitotic index of the isolated cells is similar to that noted for Sertoli cells in vivo [20]. Most importantly, the presence of nucleolar satellite karyosomes are considered to be specific for Sertoli cells [8, 91. These structures have been noted in several rodent Sertoli cells; in partitular, Sapsford indicates that the nucleolus of the rat Sertoli cell is weakly Feulgen positive while the karyosomes are strongly Feulgen-positive [8]. As these structures have been found in the colony cells from the 15day-old rat testis but not in fibroblasts or Leydig cells, the identification of the cells as Sertoli cells appears to be well grounded. We thank Drs Barry Zirkin and Ann help with the electron microscopy. Pat Welch and Dr Robert Mitchell staining is gratefully acknowledged.
Exp Cell
Res 106 (1977)
Belanger for their The help of Mr with the Feulgen The assistance of
Drs Don W. Fawcett and Martin Dym in examination of the colony cell ultrastructure was of considerable help We thank the National Institute of Arthritis and Metabolic Diseases for NIH-FSH-SlO. This research was supported by contract NOl-HD-3-2794, NICHD. Funds for some equinment were provided bv the Rockefeller Foundation.
REFERENCES 1. Davis, J C & Schuetz, A W, Nature 254 (1975) 611. - Exp cell res 91 (1975) 79. 3. Brinkley, B R & Hsu, T C, Cytologia 35 (1970) 424. 4. Humason, G L, Animal tissue techniques, 4th edn, p. 308. W H Freeman & Co., San Francisco, CA 2.
(1962). .__ _-,.
5. Snyder, G & Hymer, W C, Endocrinology 96 (1975) 792. 6. Leif. R C & Vinozrad. J, Proc natl acad sci US 51 (196i)
520.
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7. 8. 9. 10.
Clermont, Y & Perey, B, Am j anat 100 (1957) 241. Sapsford, C S, J anat 97 (1962) 225. Bouin, P, Bibliog anat 7 (1899) 242. Means, A R, MacDougall, E, Soderling, T R & Corbin. J D. J biol them 249 (1974) 1231. 11. Means,‘A R; Endocrinology 89 (1971) 981. 12. Means, A R & Hall, P F, Endocrinology 81 (1967) 1151.
13. Welsh, M J & Wiebe, J P, Endocrinology 96 (1975) 618. 14. Tung, P S, Dorrington, H J & Fritz, I B, Proc natl acad sci US 72 (1975) 1838. 15. Steinberger, A, ‘Heindel, J J, Lindsey, J H, Elkington. J H. Sanborn. B M & Steinberger. E. Endocrine rks commun 2 (1975) 261. - ’ 16. Steinberger, A, Anat ret 151(1965) 420. 17. Meistrich, M L & Trostle, P K, Exp cell res 92 (1975) 231. 18. Dorrington, J & Armstrong, D T, Proc natl acad sci US 72 (1975) 2677. 19. Means, A R. Fakunding, J L & Tindall, D J, Biol reprod. (1976) 54. -’ 20. Nagy, F, J reprod fertil28 (1972) 389. Received December 1, 1976 Accepted December 6, 1976