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Reaggregation and reorganization of juvenile chicken testicular cellsin vitro
DEVELOPMENTAL.
BIOLOGY
24, 322-334
(1971)
Reaggregation and Reorganization of Juvenile Chicken Testicular Cells in Vitro H. LEE
HAROLD Department
of Biology,
The University Accepted
October
of Toledo,
Toledo,
Ohio 43606
27, 1970
INTRODUCTION
The physiology and endocrinology of the testis have been intensively investigated over the past several decades in a variety of domestic animals (Waites and Setchell, 1969; Young, 1961). However, developmental studies at the cellular level have been few (Bishop, 1968; Connell, 1966). Recently, several investigators have employed culture techniques to study the differentiation of testicular cells. Shin (1967) and Shin et al. (1968) used androgen-producing cells derived from interstitial cell tumors while organ cultures of testis have been used by Steinberger and Steinberg& (1966, 1967) and Steinberger et al. (1967). The development of the testis and the differentiation of the germ cells and spermatogenesis are either a cyclic or a continuous process, not confined to embryonic stages but also occurring during juvenile and adult development, depending on the species. In addition, the testis consists of several distinct cell types, as judged by both morphologic and physiologic criteria. The importance of cell-cell interactions during organogenesis of the testis and germ cell differentiation become apparent. Cell-cell interactions, such as adhesiveness with respect to histogenesis, have been under intensive investigation using embryonic vertebrate tissues and marine sponges (Lilien, 1969; Morris and Moscona, 1970; Moscona, 1962, 1968; Phillips and Steinberg, 1969; Steinberg, 1963). It is within this conceptual framework that the present study has been initiated employing juvenile chicken testicular cells in uitro. In this report, I describe the behavior of dissociated testicular cells in suspension and monolayer cultures. The ability of cells to reaggregate in oitro, the “sorting out” of different cell types in monolayer cultures, and the influence of sera on these phenomena will be described. At least one differentiated cell type has been defined by histochemical methods. The usefulness of this culture system in the study of 322
REAGGREGATION
cell interactions cussed.
AND
and related
MATERIALS
REORGANIZATION
phenomena
AND
OF
in the testis
CELLS
will
323 be dis-
METHODS
Chemicals. Dehydroisoandrosterone (DHA), 17a-hydroxypregnenolone, A5-pregnen-3/3-ol-20-one (pregnenolone), and nitro blue tetrazolium were obtained from Sigma Chemical Company. Trypsin (l-250) was purchased from Nutritional Biochemical Corporation. Tryptose phosphate broth (TPB) was obtained from Difco, Inc. Other chemicals were purchased from Fisher Chemical Company. Culture media. Horse serum was obtained from Hyland Laboratories, Los Angeles, California. Fetal calf and calf serum were obtained from Grand Island Biological Company, Grand Island, New York. The two synthetic media used were Ham’s F12 (Ham, 1965) modified to contain 2~ amino acids (Lee et al., 1968) and M-199 (Grand Island Biological Company). These two synthetic nutrient mixtures were supplemented with various sera (SC, serum containing). Some experiments were done with serum-free (SF) media (Tables 1 and 2). It was found later that F12 with 10% TPB and 1% horse serum gave identical results for monolayer cultures. This mixture was, therefore, routinely used unless otherwise stated. All culture media were also supplemented with penicillin, 50 units/ml; streptomycin sulfate, 50 pg/ml; amphotericin B, 2.5 pg/ml, and NaHCO,, 1.5 g/l. “Conditioned” medium was the medium in which monolayer cultures were cultivated for a week to 10 days. This medium was refiltered to ensure sterility. Cell cultures. Testes containing no mature sperm were obtained from juvenile chickens (lo-13 weeks posthatching) of White Leghorn varieties. In several experiments, Barred Plymouth Rock varieties were used and gave identical results. Testes were decapsulated, minced to small fragments, and washed with Puck’s saline-G. In order to obtain dissociated cells, tissue fragments from a testis of l-l.5 cm in length were first incubated in 10 ml of trypsin solution (0.125% in saline G) at 37°C with mild agitation. After 15 minutes, the suspension was drawn up-and-down several times in a pipette and 5 ml more of trypsin solution was added. This procedure was repeated once or twice depending on the yield of single cells. Cells which remained in this trypsin solution for as long as 1 hour did not appear to be damaged as judged by their ability to multiply. At the end of this period, 5
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ml of medium containing horse serum was added to stop the action of trypsin. Large tissue fragments were sedimented out by standing for a few minutes. The supernatant fraction, containing mostly single cells and loose cell clumps, were centrifuged in a clinical centrifuge. The cell pellet was resuspended in medium and the suspension was filtered through Nitex-102 nylon mesh (Tobler, Ernst and Traber, Inc., New York). This cell suspension, which consisted of more than 99% single cells, was washed twice with the appropriate culture medium and counted using a hemocytometer. Dissociated testicular cells obtained in this manner were cultivated at 37°C in an atmosphere of 5% CO, in air and 98% humidity. Each culture dish (Falcon, 60 mm) contained 2 x lo6 cells in 5 ml of medium. The medium was not changed until the cells had become attached to the bottom of the culture dish. Studies on cell reaggregation, mediated by mechanical agitation, were carried out at 37°C in a reciprocal shaker-bath (Thermo-shake, Forma Scientific Company) set at 80 strokes (4.0 cm per stroke) per minute in an atmosphere of 5% CO, in air. The cells were placed in the 30 mm Falcon tissue culture dishes at 3 X lo6 per ml, and 3 ml of cell suspension were used. At intervals, several random fields were photographed using an inverted microscope at low magnification. The sizes of the aggregates were measured from these photographs. Histochemical methods. Monolayer cultures were washed three times with saline G and frozen on crushed dry-ice. The frozen cultures can be stored at -70°C for at least 1 month before being used. The histochemical test which was used to determine the presence of A”-3/3hydroxysteroid dehydrogenase was that published by Levy et al. (1959). The quantities of substrates were, however, doubled. The optimal incubation duration was found to be 100 minutes at 37°C. Shorter incubation than 90 minutes gave weak or negative reactions, and longer incubation than 110 minutes gave nonspecific reactions. These nonspecific reactions may have been those already pointed out by Pearse (1960) and called “nothing-dehydrogenase.” After incubation, the cultures were washed thoroughly with phosphate-buffered saline and fixed in 10% formalin with 1% CaCI,. Cover glasseswere mounted with glycerin jelly. RESULTS
The modified Ham’s F12 nutrient mixture supplemented with horse serum was chosen initially because it has been used successfully to support differentiation of embryonic chick muscle (Lee et al., 1968)
REAGGREGATION
AND
REORGANIZATION
OF
CELLS
325
FIG. 1. Behavior of dissociated testicular cells in stationary cultures. (la) Eight hours after seeding in F12 with 15’;; horse serum. Phase contrast, x 132. (lb) Reorganization in monolayer. Five days after seeding in the same medium. F, fibroblastic cells; S, steroidogenic cells. Phase contrast, X 132.
and cartilage (Coon and Cahn, 1966) cells in clonal cultures. Eight hours after seeding in stationary cultures, dissociated testicular cells of juvenile chickens form small aggregates spontaneously as shown in Fig. la. The aggregates remain unattached and gradually increase in size for about 24 hours. Within 4 days after seeding, fibroblastic cells are observed to attached to the bottom of the culture dish while most of the aggregates still remain floating. A few of the aggregates do attach to the culture dish. This attachment is however very loose, and aggregates can be detached by gently shaking the dish. On the fifth day after seeding, the cultures have become monolayers. The monolayer shows an interesting and distinct pattern (Fig. lb) similar to those observed previously (Lee, 1969). At least two morphologically distinct cell types are present, fibroblastic (F) and epithelial-like (later shown to be steroidogenic, referred to as S cells). The latter are organized into discrete colonies surrounded by fibroblastic cells (Fig. lb). Furthermore, a refractile substance begins to appear in the intercellular spacesbetween the S cells. These phenomena, spontaneous reaggregation and reorganization, are exceedingly reproducible. So far
326
LEE TABLE BEHAVIOR
IN VARIOUS
1
Spontaneous Beaggregation 8 hours after seeding
Medium
F12 + 15% HS” F12 + 15% FCS F12 + 15% CS Ml99 + 15% HS Ml99 + 1% FCS 4% cs Ml99 + 10% TPB F12 + 10% TPB F12 + 10% TPB 1% HS M-199 F12 Saline G a Abbreviations: tryptose phosphate
HS, horse broth.
CELLS FROM
MEDIA OF DISSOCIATED TESTICULAR Z-MONTH-OLD CHICKENS
serum;
CS,
calf
serum;
Reorganization days after seeding
+ + -
+ + -
+ +
+ +
-
+ + +
FCS,
fetal
calf
serum;
5
TPB,
these results have been obtained in more than 40 experiments when chicken tests containing no mature spermatozoa have been used. To further examine the media suitable for the cultivation of juvenile chicken testicular cells, two synthetic media with different supplements were tested. The behavior of the dissociated testicular cells, which is illustrated in Fig. 1, is summarized in Table 1 for the various culture media. As shown in Table 1, M-199 or F-12 supplemented with either horse serum or fetal calf serum, can support aggregation as well as reorganization of the cells. Reaggregation is not found with either calf serum or SF media. Reorganization, however, is observed in media which has not been supplemented with sera. The experiments also show that it is necessary to supplement serum-free media with tryptose phosphate broth in order to obtain reorganization. Synthetic media without supplements and saline G are unable to support either the reaggregation or the reorganization of the cells. Figure 2 illustrates a colony of S cells cultivated for 8 days in M-199 supplemented only with 10% TPB. Note that the intercellular space is filled with refractile material. This material is also observed in cultures maintained in serum-containing (SC) media. The only difference between cells cultivated with SC and SF media is that the cells can
REAGGREGATION
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REORGANIZATION
OF
327
CELLS
reach confluency in the former whereas they begin to die in the latter roughly on the 9th to the 10th day after seeding. It has also been found that the behavior of the testicular cells, namely reorganization and the ability to reach confluency, is the same when cultivated in modified Ham’s F12 which is supplemented with only 1% horse serum and 10% TPB. We are now using this mixture as a routine medium for monolayer cultures. However, no spontaneous reaggregation was seen 8 hours after seeding (Table 1) in this medium. In order to study more efficiently the phenomena of aggregation in vitro, we employ the reciprocal-shaking-mediated procedure. The results of these experiments are presented in Table 2. As shown in Table
FIG. 2. Intercellular material between steroidogenic 199 with 10% TPB. Phase contrast, x 165. DIAMETERS
cells in serum-free
TABLE 2 (p) OF AGGREGATES IN DIFFERENT 15 minutes
medium,
M-
MEDIAO 45 minutes
Medium
F12, F12, Fl2, F12, Saline
15% HS 5% HS, conditioned serum-free serum-free, conditioned G
a Reciprocal
shaker,
80 strokes/minute,
Range
Average
124-214 94-111 118-159 69-156
169 102 139 111 -
37°C
5% CO,
Range 170-258 107-157 91-105 98-120 75-101 in air.
Average 214 132 98 108 88
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FIG. 3. Localized formazan deposits due to AS-3/SHSDH reactions in the testicular cell cultures. (3a) Phase contrast photograph of the cells after the reaction using DHA as the substrate. X 315 (3b) Same field as 3a but taken with ordinary optics. Formazan deposits (arrows) are clearly shown in the reorganized steroidogenic cells but not in the fibroblastic cells. (3c and 3d) These are similar to 3a and 3b, respectively, except that pregnenolone is the substrate. x 315.
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REORGANIZATION
OF
CELLS
329
2, within 15 minutes the size of the aggregates in SC medium is much greater than in other media. In fact, in SF medium, the size of the aggregates after 45 minutes is smaller than at 15 minutes. The average diameter of the aggregates in SC medium at 45 minutes is obviously much greater than in others. Such experiments demonstrated that the enhancement of reaggregation of testicular cells depends on some factor(s) present in the serum. Also, “conditioned” media do not enhance reaggregation. Although the dissociated testicular cells from juvenile chickens have the adhesive property of differentiating embryonic cells (Moscona, 1962; Steinberg, 1963), it was not clear whether or not they were functionally differentiated. That question was the next to be examined. The enzyme A5-3P-hydroxysteroid dehydrogenase (HSDH) oxidizes A5-3P-hydroxysteroids to A”-3-ketosteroids, a metabolic step involved in the synthesis of practically all steroids with hormonal activity (Baille et al., 1966). It has been successfully used to localize the steroidogenic cells in frozen sections of an adult and differentiating gonads of the chick (Narbaitz and Kolodny, 1964; Narbaitz and De Robertis, 1968; Woods and Domm, 1966). Using this enzymatic marker in conjunction with the formazan reaction, we have demonstrated that the epithelial-like cells, surrounded by fibroblastic cells, are steroidogenic. Figure 3a is a phase contrast photograph of the frozen culture after the enzymatic reactions with DHA used as the substrate. The formazan
FIG. 4. Control ture. No formazan
sister cultures of Fig. 3. No substrates deposits in any cell types. X 315.
added
to the incubation
mix-
330
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reaction granules are obscured by the cellular components. Figure 3b is the same field, but taken, however, with bright-field optics showing clearly the tetrazolium depositions only in the steroidogenic colonies, but not in the surrounding fibroblastic cells. Figures 3c and 3d are sister cultures of fig. 3a and 3b but with pregnenolone used as the substrate. Similar results were obtained when 17-a-hydroxypregnenolone was used as the substrate. Controls (Figs. 4a and 4b) for the cytochemical test are those in which no substrate was added to the incubation mixture. No tetrazolium deposit is observed in the control cultures. The cellular details are not clear because the cultures are not fixed with formalin until after the enzymatic reactions. DISCUSSION
Differentiating cells in suspensions from vertebrate embryos have been repeatedly demonstrated to have the ability to reaggregate and “sort out” according to cell type in uitro (Moscona, 1962; Steinberg, 1963). Studies on cell differentiation in monolayer cultures require the supplement of macromolecules, such as sera, embryo extracts, or collagen, in the culture medium (Coon and Cahn, 1966; Hauschka and Konigsberg, 1966). Cell interactions in the genesis and maintenance of organ-specific characteristics have been examined in monolayer cultures (Hilfer, 1968; Hilfer et al., 1968). It is within the conceptual framework of cell adhesion and the requirement for attaining a stable cellular organization that the present communication will be discussed with respect to juvenile chicken testis. Results presented here and in a previous study (Lee, 1969) indicate that the addition of horse or fetal calf sera, but not calf serum, supports cell reaggregation in cultures spontaneously, i.e., without mechanical agitation. Macromolecules present in the sera may not play any specific role in specific cell adhesion or in the phenomena of reorganization among testicular cells. This statement is supported by the fact that steroidogenic cells, cells having A5-3@hydroxysteroid dehydrogenase, form discrete colonies with fibroblastic cells around them in the absence of sera as seen in the monolayer cultures. To test the possibility that biogenesis of macromolecules, which are subsequently released to the environment, is necessary for reaggregation, “conditioned” medium was used. Results show that “conditioned” media, with or without sera, do not enhance cell reaggregation. The inability of “conditioned” medium containing serum to enhance reaggregation indicates that no specific factors are released by the testicular cells for reaggregation. The role of sera in this aspect is most likely nonspecific
REAGGREGATION
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REORGANIZATION
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CELLS
331
because cells in SF media did reaggregate although not to the same extent. The maintenance of a relatively stable organization of homotypic cells, however, may require cell surface-associated substances. The refractile substance observed only in the intercellular space of the steroidogenic cells, and studies by Khan and Overton (1969), tend to support this contention. Furthermore, the steroidogenic cells are those that form discrete colonies, not the fibroblastic cells. Whether or not these steroidogenic cells are in fact steroidogenic while in suspension as they undergo reaggregation has not yet been examined. Neuroblastoma cells do not show the structural characteristics of the differentiated neuron until their attachment to the substratum has been established (Schubert et al., 1969). Although the initial phase of specific cell adhesion may not require specific cell-surface factor, the attainment of morphogenetic stability may require cell-surface associated factors characteristic of different differentiating cell types. Another aspect of the present study concerns heterotypic cell interactions during the development and maturation of testicular tissue. Earlier reports (Steinberger and Steinberger, 1966; Steinberger, et al., 1967) indicated that rat testicular cells in vitro become fibroblastic. The present study clearly establishes the presence of at least two cell types with different morphology. One of these, the nonfibroblastic type, is steroidogenic even though the cells have been in culture for 10 days. Thus, the culture system does support differentiation of the steroidogenic cells. Although no cell counts were taken at intervals after seeding, the fact that the culture as a whole can reach confluency and still remain an organized pattern demonstrates the ability of the testicular cells to divide in the present culture conditions. One can ascertain, using the present system, whether or not the steroidogenic cells require the presence of other cell types to be functional and the growth potential of one cell type as compared to others. Recently, Lam et al. (1970) demonstrated that different cell types from rat testis can be separated by their difference in sedimentation velocities. These specific properties plus the biochemical markers, such as the ability to synthesize specific hormones, thus provide an excellent system to investigate heterotypic and homotypic cell interactions in uitro. A specific interaction has been demonstrated between secretory and capsule cells of the embryonic chick thyroid using monolayer cultures as one of the steps in the experimental procedures (Hilfer, 1968; Hilfer et al., 1968). The in vitro system employing juvenile chicken testis presented here may well prove to be equally useful to study cell-cell interactions
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in differentiation and morphogenesis as other systems (DeHaan, 1968; Miura and Wilt, 1970; Morris and Moscona, 1970; Steinberg, 1963). Interactions between the nongerminal and germinal populations of cells in relation to initiation of spermatogenesis could then be studied. The development of testicular cell culture represents an additional system to investigate the effects of oncogenic virus on differentiation at the cellular level (Ebert and Kaighn, 1966). SUMMARY
A culture system has been developed which maintains the characteristics of the androgen-producing cells together with fibroblastic cells dissociated from juvenile chicken testes. The behavior of these cells, including their ability to reaggregate in vitro and reorganize into distinct patterns in monolayer cultures, indicates that the maintenance of a stable arrangement of homotypic cells is achieved by intercellular materials holding the cells in discrete groupings. I am grateful to Dr. James D. Ebert, Carnegie Institution of Washington and Dr. Malcolm Steinberg, Princeton University, for their constructive criticism of the study. The techical assistance of Miss Suzanne Hinr is acknowledged. This investigation is aided by a grant (E-553) from The American Cancer Society, Inc. REFERENCES BAILLE, A. H., FERGUSON, M. M., and HART, D. M. (1966). “Development in Steroid Histochemistry.” Academic Press, New York. BISHOP, D. W., (1968). Testicular enzymes as fingerprints in the study of spermatogenesis. In “Reproduction and Sexual Behavior.” (M. Diamond, ed.), pp. 261-286. Indiana Univ. Press, Bloomington, Indiana. CONNELL, G. M., CONNELL, C. J., and EIK-NES, K. B. (1966). Testosterone synthesis by the two-day-old chick testis in uitro. Gen. Camp. Endocrinol. 7, 158-165. COON, H. G., and CHAN, R. D. (1966). Differentiation in uitro: Effects of Sephadex fractions of chick embryo extract. Science 153, 1116-1119. DEHAAN, R. H. (1968). Form and function in the embryonic heart. In “The Emergence of Order in Developing System” (Michael Locke, ed.), pp. 208-250. Academic Press, New York. EBERT, J. D., and KAIGHN, M. E. (1966). The key to change: Factors regulating differentiation. In “Major Problems in Developmental Biology” (Michael Locke, ed.), pp. 29-84. Academic Press, New York. HAM, R. G. (1965). Clonal growth of mammalian cells in a chemically defined synthetic medium. hoc. Nat. Acad. Sci. U. S. 53, 288-293. HAUSCHKA, S., and KONIGSBERG, I. R. (1966). The influence of collagen on the development of muscle clones. Proc. Nat. Acad. Sci. U. S. 55, 119-126. HILFER, S. R. (1968). Cellular interactions in the genesis and maintenance of thyroid characteristics. In “Epithelial’Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 177-199. William and Wilkins, Baltimore, Maryland.
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HILFER, S. R., LSZARD, L. B., and HILFER, E. K. (1968). Follicle formation in the embryonic chick thyroid. Z. Zellforsch. Mikrosk. Amt. 92, 256-269. KHAN, T., and OVERTON, T. (1969). Staining of intercellular material in reaggregating chick liver and cartilage cells. J. Exp. Zool. 171, 161-173. LAM, D. M. K., FURRER, R., and BRUCE, W. R. (1970). The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc. Nat. Acad. Sci. U. S. 65, 192-199. LEE, H. H. (1969). Aggregation and histogenesis in vitro of dissociated chick testicular cells. J. Cell Viol. 43, 76a. LEE, H. H., KAIGHN, M. E., and EBERT, J. D. (1968). Induction of thymidine-3H incorporation in multinucleated myotubes by Rous sarcoma virus. ht. J. Cancer 3, 126136. LEVY, H., DEANE, H. W., and RUBIN, B. L. (1959). Visualization of steroid 3B-01 dehydrogenase activity in tissues of intact and hypophysectomized rats. Endocrinology 65, 932-943. LILIEN, J. E. (1969). Toward a molecular explanation for specific cell adhesion. Curr. Top. Deuelop. Bio2. 4, 169-195. MIURA, Y., and WILT, F. H. (1970). The formations of blood islands in dissociated-reaggregated chick embryo yolk sac cells. Exp. Cell Res. 59, 217-226. MORRIS, J. E., and MOSCONA, A. A. (1970). Induction of glutamine synthetase in embryonic retina: Its dependence on cell interactions. Science 167, 1736-1738. MOSCONA, A. A. (1962). Analysis of cell recombination in experimental synthesis of tissues in uitro. J. Cell. Camp. Physiol. 60, Suppl. 1, 65-80. MOSCONA, A. A. (1968). Cell aggregation: Properties of specific cell-ligands and their role in the formation of multicellular system. Deoelop. Biol. 18, 250-277. NARBAITZ, R., and KOLODNY, L. (1964). A5-3P-hydroxysteroid dehydrogenase in differentiating chick gonads. Z. Zellforsch, Mikrosk, Amt. 63, 612-617. NARBAITZ, R., and DEROBERTIS, E. M., JR. (1968). Postnatal evolution of steroidogenic cells in chick ovary. Histochemie 15, 187-193. P!UFZjE, A. G. E. (1960). “Histochemistry.” Little, Brown, Boston. PHILLIPS, H. M., and STEINBERG, M. S. (1969). Equlibrium measurements of embryonic chick cell adhesiveness, I. Shape equilibrium in centrifugal fields. hoc. Nat. Acad. Sci. U. S. 64, 121-127. SHIN, S. (1967). Studies on interstitial cells in tissue culture: Steroid biosynthesis in monolayers of mouse testicular interstitial cells. Endocrinology 81, 440-448. SHIN, S., YASUMURA, Y., and SATO, G. H. (1968). Studies on interstitial cells in tissue culture. II. Steroid biosynthesis by a clonal line of rat testicular interstitial cells. Endocrinology 82, 614-616. SCHUBERT, D., HUMPHREYS, S., BARONI, C., and COHN, M. (1969). In vitro differentiation of a mouse neuroblastoma. Proc. Nat. Acad. Sci. U. S. 64, 316-323. STEINBERG, M. S., (1963). Reconstmction of tissues by dissociated cells. Science 141, 401-408. STEINBERGER, A., and STEINBERGER, E. (1966). In vitro culture of rat testicular cells. Exp. Cell Res. 44, 443-452. STEINBERGER, E., STEINBERGER, A., VILAR, O., SALAMON, I. I., and SUD, B. N. (1967). Microscopy, cytochemistry and steroid biosynthetic activity of Leydig cells in culture. Testis Ciba Found. Colloq. Endocrinol. 16, 56-78. Little, Brown, Boston. STEINBERGER, A., and STEINBERGER, E. (1967). Factors affecting spermatogenesis in organ cultures of a variety of mammalian testes. J. Reprod. Fertil., Suppl. 2, 117-124.
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G. M. H., and SETCHELL, B. P. (1969). Physiology of the testis, epididymis and scrotum. In “Advances in Reproduction Physiology” (Anne McLaren, ed.) Vol. 4, pp. l-63. Academic Press, New York. WOODS, J. E., and DOMM, L. V. (1966). A histochemical identification of the androgenproducing cells in the gonads of the domestic fowl and albino rat. Gen. Comp. Endocrinol. 7, 559-570. YOUNG, W. C. (Ed.) (1961). “Sex and Internal Secretions,” Vol. 1. Williams and Wilkins, Baltimore, Maryland. WAITES,
BIOLOGY
24, 322-334
(1971)
Reaggregation and Reorganization of Juvenile Chicken Testicular Cells in Vitro H. LEE
HAROLD Department
of Biology,
The University Accepted
October
of Toledo,
Toledo,
Ohio 43606
27, 1970
INTRODUCTION
The physiology and endocrinology of the testis have been intensively investigated over the past several decades in a variety of domestic animals (Waites and Setchell, 1969; Young, 1961). However, developmental studies at the cellular level have been few (Bishop, 1968; Connell, 1966). Recently, several investigators have employed culture techniques to study the differentiation of testicular cells. Shin (1967) and Shin et al. (1968) used androgen-producing cells derived from interstitial cell tumors while organ cultures of testis have been used by Steinberger and Steinberg& (1966, 1967) and Steinberger et al. (1967). The development of the testis and the differentiation of the germ cells and spermatogenesis are either a cyclic or a continuous process, not confined to embryonic stages but also occurring during juvenile and adult development, depending on the species. In addition, the testis consists of several distinct cell types, as judged by both morphologic and physiologic criteria. The importance of cell-cell interactions during organogenesis of the testis and germ cell differentiation become apparent. Cell-cell interactions, such as adhesiveness with respect to histogenesis, have been under intensive investigation using embryonic vertebrate tissues and marine sponges (Lilien, 1969; Morris and Moscona, 1970; Moscona, 1962, 1968; Phillips and Steinberg, 1969; Steinberg, 1963). It is within this conceptual framework that the present study has been initiated employing juvenile chicken testicular cells in uitro. In this report, I describe the behavior of dissociated testicular cells in suspension and monolayer cultures. The ability of cells to reaggregate in oitro, the “sorting out” of different cell types in monolayer cultures, and the influence of sera on these phenomena will be described. At least one differentiated cell type has been defined by histochemical methods. The usefulness of this culture system in the study of 322
REAGGREGATION
cell interactions cussed.
AND
and related
MATERIALS
REORGANIZATION
phenomena
AND
OF
in the testis
CELLS
will
323 be dis-
METHODS
Chemicals. Dehydroisoandrosterone (DHA), 17a-hydroxypregnenolone, A5-pregnen-3/3-ol-20-one (pregnenolone), and nitro blue tetrazolium were obtained from Sigma Chemical Company. Trypsin (l-250) was purchased from Nutritional Biochemical Corporation. Tryptose phosphate broth (TPB) was obtained from Difco, Inc. Other chemicals were purchased from Fisher Chemical Company. Culture media. Horse serum was obtained from Hyland Laboratories, Los Angeles, California. Fetal calf and calf serum were obtained from Grand Island Biological Company, Grand Island, New York. The two synthetic media used were Ham’s F12 (Ham, 1965) modified to contain 2~ amino acids (Lee et al., 1968) and M-199 (Grand Island Biological Company). These two synthetic nutrient mixtures were supplemented with various sera (SC, serum containing). Some experiments were done with serum-free (SF) media (Tables 1 and 2). It was found later that F12 with 10% TPB and 1% horse serum gave identical results for monolayer cultures. This mixture was, therefore, routinely used unless otherwise stated. All culture media were also supplemented with penicillin, 50 units/ml; streptomycin sulfate, 50 pg/ml; amphotericin B, 2.5 pg/ml, and NaHCO,, 1.5 g/l. “Conditioned” medium was the medium in which monolayer cultures were cultivated for a week to 10 days. This medium was refiltered to ensure sterility. Cell cultures. Testes containing no mature sperm were obtained from juvenile chickens (lo-13 weeks posthatching) of White Leghorn varieties. In several experiments, Barred Plymouth Rock varieties were used and gave identical results. Testes were decapsulated, minced to small fragments, and washed with Puck’s saline-G. In order to obtain dissociated cells, tissue fragments from a testis of l-l.5 cm in length were first incubated in 10 ml of trypsin solution (0.125% in saline G) at 37°C with mild agitation. After 15 minutes, the suspension was drawn up-and-down several times in a pipette and 5 ml more of trypsin solution was added. This procedure was repeated once or twice depending on the yield of single cells. Cells which remained in this trypsin solution for as long as 1 hour did not appear to be damaged as judged by their ability to multiply. At the end of this period, 5
324
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ml of medium containing horse serum was added to stop the action of trypsin. Large tissue fragments were sedimented out by standing for a few minutes. The supernatant fraction, containing mostly single cells and loose cell clumps, were centrifuged in a clinical centrifuge. The cell pellet was resuspended in medium and the suspension was filtered through Nitex-102 nylon mesh (Tobler, Ernst and Traber, Inc., New York). This cell suspension, which consisted of more than 99% single cells, was washed twice with the appropriate culture medium and counted using a hemocytometer. Dissociated testicular cells obtained in this manner were cultivated at 37°C in an atmosphere of 5% CO, in air and 98% humidity. Each culture dish (Falcon, 60 mm) contained 2 x lo6 cells in 5 ml of medium. The medium was not changed until the cells had become attached to the bottom of the culture dish. Studies on cell reaggregation, mediated by mechanical agitation, were carried out at 37°C in a reciprocal shaker-bath (Thermo-shake, Forma Scientific Company) set at 80 strokes (4.0 cm per stroke) per minute in an atmosphere of 5% CO, in air. The cells were placed in the 30 mm Falcon tissue culture dishes at 3 X lo6 per ml, and 3 ml of cell suspension were used. At intervals, several random fields were photographed using an inverted microscope at low magnification. The sizes of the aggregates were measured from these photographs. Histochemical methods. Monolayer cultures were washed three times with saline G and frozen on crushed dry-ice. The frozen cultures can be stored at -70°C for at least 1 month before being used. The histochemical test which was used to determine the presence of A”-3/3hydroxysteroid dehydrogenase was that published by Levy et al. (1959). The quantities of substrates were, however, doubled. The optimal incubation duration was found to be 100 minutes at 37°C. Shorter incubation than 90 minutes gave weak or negative reactions, and longer incubation than 110 minutes gave nonspecific reactions. These nonspecific reactions may have been those already pointed out by Pearse (1960) and called “nothing-dehydrogenase.” After incubation, the cultures were washed thoroughly with phosphate-buffered saline and fixed in 10% formalin with 1% CaCI,. Cover glasseswere mounted with glycerin jelly. RESULTS
The modified Ham’s F12 nutrient mixture supplemented with horse serum was chosen initially because it has been used successfully to support differentiation of embryonic chick muscle (Lee et al., 1968)
REAGGREGATION
AND
REORGANIZATION
OF
CELLS
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FIG. 1. Behavior of dissociated testicular cells in stationary cultures. (la) Eight hours after seeding in F12 with 15’;; horse serum. Phase contrast, x 132. (lb) Reorganization in monolayer. Five days after seeding in the same medium. F, fibroblastic cells; S, steroidogenic cells. Phase contrast, X 132.
and cartilage (Coon and Cahn, 1966) cells in clonal cultures. Eight hours after seeding in stationary cultures, dissociated testicular cells of juvenile chickens form small aggregates spontaneously as shown in Fig. la. The aggregates remain unattached and gradually increase in size for about 24 hours. Within 4 days after seeding, fibroblastic cells are observed to attached to the bottom of the culture dish while most of the aggregates still remain floating. A few of the aggregates do attach to the culture dish. This attachment is however very loose, and aggregates can be detached by gently shaking the dish. On the fifth day after seeding, the cultures have become monolayers. The monolayer shows an interesting and distinct pattern (Fig. lb) similar to those observed previously (Lee, 1969). At least two morphologically distinct cell types are present, fibroblastic (F) and epithelial-like (later shown to be steroidogenic, referred to as S cells). The latter are organized into discrete colonies surrounded by fibroblastic cells (Fig. lb). Furthermore, a refractile substance begins to appear in the intercellular spacesbetween the S cells. These phenomena, spontaneous reaggregation and reorganization, are exceedingly reproducible. So far
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IN VARIOUS
1
Spontaneous Beaggregation 8 hours after seeding
Medium
F12 + 15% HS” F12 + 15% FCS F12 + 15% CS Ml99 + 15% HS Ml99 + 1% FCS 4% cs Ml99 + 10% TPB F12 + 10% TPB F12 + 10% TPB 1% HS M-199 F12 Saline G a Abbreviations: tryptose phosphate
HS, horse broth.
CELLS FROM
MEDIA OF DISSOCIATED TESTICULAR Z-MONTH-OLD CHICKENS
serum;
CS,
calf
serum;
Reorganization days after seeding
+ + -
+ + -
+ +
+ +
-
+ + +
FCS,
fetal
calf
serum;
5
TPB,
these results have been obtained in more than 40 experiments when chicken tests containing no mature spermatozoa have been used. To further examine the media suitable for the cultivation of juvenile chicken testicular cells, two synthetic media with different supplements were tested. The behavior of the dissociated testicular cells, which is illustrated in Fig. 1, is summarized in Table 1 for the various culture media. As shown in Table 1, M-199 or F-12 supplemented with either horse serum or fetal calf serum, can support aggregation as well as reorganization of the cells. Reaggregation is not found with either calf serum or SF media. Reorganization, however, is observed in media which has not been supplemented with sera. The experiments also show that it is necessary to supplement serum-free media with tryptose phosphate broth in order to obtain reorganization. Synthetic media without supplements and saline G are unable to support either the reaggregation or the reorganization of the cells. Figure 2 illustrates a colony of S cells cultivated for 8 days in M-199 supplemented only with 10% TPB. Note that the intercellular space is filled with refractile material. This material is also observed in cultures maintained in serum-containing (SC) media. The only difference between cells cultivated with SC and SF media is that the cells can
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reach confluency in the former whereas they begin to die in the latter roughly on the 9th to the 10th day after seeding. It has also been found that the behavior of the testicular cells, namely reorganization and the ability to reach confluency, is the same when cultivated in modified Ham’s F12 which is supplemented with only 1% horse serum and 10% TPB. We are now using this mixture as a routine medium for monolayer cultures. However, no spontaneous reaggregation was seen 8 hours after seeding (Table 1) in this medium. In order to study more efficiently the phenomena of aggregation in vitro, we employ the reciprocal-shaking-mediated procedure. The results of these experiments are presented in Table 2. As shown in Table
FIG. 2. Intercellular material between steroidogenic 199 with 10% TPB. Phase contrast, x 165. DIAMETERS
cells in serum-free
TABLE 2 (p) OF AGGREGATES IN DIFFERENT 15 minutes
medium,
M-
MEDIAO 45 minutes
Medium
F12, F12, Fl2, F12, Saline
15% HS 5% HS, conditioned serum-free serum-free, conditioned G
a Reciprocal
shaker,
80 strokes/minute,
Range
Average
124-214 94-111 118-159 69-156
169 102 139 111 -
37°C
5% CO,
Range 170-258 107-157 91-105 98-120 75-101 in air.
Average 214 132 98 108 88
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FIG. 3. Localized formazan deposits due to AS-3/SHSDH reactions in the testicular cell cultures. (3a) Phase contrast photograph of the cells after the reaction using DHA as the substrate. X 315 (3b) Same field as 3a but taken with ordinary optics. Formazan deposits (arrows) are clearly shown in the reorganized steroidogenic cells but not in the fibroblastic cells. (3c and 3d) These are similar to 3a and 3b, respectively, except that pregnenolone is the substrate. x 315.
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2, within 15 minutes the size of the aggregates in SC medium is much greater than in other media. In fact, in SF medium, the size of the aggregates after 45 minutes is smaller than at 15 minutes. The average diameter of the aggregates in SC medium at 45 minutes is obviously much greater than in others. Such experiments demonstrated that the enhancement of reaggregation of testicular cells depends on some factor(s) present in the serum. Also, “conditioned” media do not enhance reaggregation. Although the dissociated testicular cells from juvenile chickens have the adhesive property of differentiating embryonic cells (Moscona, 1962; Steinberg, 1963), it was not clear whether or not they were functionally differentiated. That question was the next to be examined. The enzyme A5-3P-hydroxysteroid dehydrogenase (HSDH) oxidizes A5-3P-hydroxysteroids to A”-3-ketosteroids, a metabolic step involved in the synthesis of practically all steroids with hormonal activity (Baille et al., 1966). It has been successfully used to localize the steroidogenic cells in frozen sections of an adult and differentiating gonads of the chick (Narbaitz and Kolodny, 1964; Narbaitz and De Robertis, 1968; Woods and Domm, 1966). Using this enzymatic marker in conjunction with the formazan reaction, we have demonstrated that the epithelial-like cells, surrounded by fibroblastic cells, are steroidogenic. Figure 3a is a phase contrast photograph of the frozen culture after the enzymatic reactions with DHA used as the substrate. The formazan
FIG. 4. Control ture. No formazan
sister cultures of Fig. 3. No substrates deposits in any cell types. X 315.
added
to the incubation
mix-
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reaction granules are obscured by the cellular components. Figure 3b is the same field, but taken, however, with bright-field optics showing clearly the tetrazolium depositions only in the steroidogenic colonies, but not in the surrounding fibroblastic cells. Figures 3c and 3d are sister cultures of fig. 3a and 3b but with pregnenolone used as the substrate. Similar results were obtained when 17-a-hydroxypregnenolone was used as the substrate. Controls (Figs. 4a and 4b) for the cytochemical test are those in which no substrate was added to the incubation mixture. No tetrazolium deposit is observed in the control cultures. The cellular details are not clear because the cultures are not fixed with formalin until after the enzymatic reactions. DISCUSSION
Differentiating cells in suspensions from vertebrate embryos have been repeatedly demonstrated to have the ability to reaggregate and “sort out” according to cell type in uitro (Moscona, 1962; Steinberg, 1963). Studies on cell differentiation in monolayer cultures require the supplement of macromolecules, such as sera, embryo extracts, or collagen, in the culture medium (Coon and Cahn, 1966; Hauschka and Konigsberg, 1966). Cell interactions in the genesis and maintenance of organ-specific characteristics have been examined in monolayer cultures (Hilfer, 1968; Hilfer et al., 1968). It is within the conceptual framework of cell adhesion and the requirement for attaining a stable cellular organization that the present communication will be discussed with respect to juvenile chicken testis. Results presented here and in a previous study (Lee, 1969) indicate that the addition of horse or fetal calf sera, but not calf serum, supports cell reaggregation in cultures spontaneously, i.e., without mechanical agitation. Macromolecules present in the sera may not play any specific role in specific cell adhesion or in the phenomena of reorganization among testicular cells. This statement is supported by the fact that steroidogenic cells, cells having A5-3@hydroxysteroid dehydrogenase, form discrete colonies with fibroblastic cells around them in the absence of sera as seen in the monolayer cultures. To test the possibility that biogenesis of macromolecules, which are subsequently released to the environment, is necessary for reaggregation, “conditioned” medium was used. Results show that “conditioned” media, with or without sera, do not enhance cell reaggregation. The inability of “conditioned” medium containing serum to enhance reaggregation indicates that no specific factors are released by the testicular cells for reaggregation. The role of sera in this aspect is most likely nonspecific
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because cells in SF media did reaggregate although not to the same extent. The maintenance of a relatively stable organization of homotypic cells, however, may require cell surface-associated substances. The refractile substance observed only in the intercellular space of the steroidogenic cells, and studies by Khan and Overton (1969), tend to support this contention. Furthermore, the steroidogenic cells are those that form discrete colonies, not the fibroblastic cells. Whether or not these steroidogenic cells are in fact steroidogenic while in suspension as they undergo reaggregation has not yet been examined. Neuroblastoma cells do not show the structural characteristics of the differentiated neuron until their attachment to the substratum has been established (Schubert et al., 1969). Although the initial phase of specific cell adhesion may not require specific cell-surface factor, the attainment of morphogenetic stability may require cell-surface associated factors characteristic of different differentiating cell types. Another aspect of the present study concerns heterotypic cell interactions during the development and maturation of testicular tissue. Earlier reports (Steinberger and Steinberger, 1966; Steinberger, et al., 1967) indicated that rat testicular cells in vitro become fibroblastic. The present study clearly establishes the presence of at least two cell types with different morphology. One of these, the nonfibroblastic type, is steroidogenic even though the cells have been in culture for 10 days. Thus, the culture system does support differentiation of the steroidogenic cells. Although no cell counts were taken at intervals after seeding, the fact that the culture as a whole can reach confluency and still remain an organized pattern demonstrates the ability of the testicular cells to divide in the present culture conditions. One can ascertain, using the present system, whether or not the steroidogenic cells require the presence of other cell types to be functional and the growth potential of one cell type as compared to others. Recently, Lam et al. (1970) demonstrated that different cell types from rat testis can be separated by their difference in sedimentation velocities. These specific properties plus the biochemical markers, such as the ability to synthesize specific hormones, thus provide an excellent system to investigate heterotypic and homotypic cell interactions in uitro. A specific interaction has been demonstrated between secretory and capsule cells of the embryonic chick thyroid using monolayer cultures as one of the steps in the experimental procedures (Hilfer, 1968; Hilfer et al., 1968). The in vitro system employing juvenile chicken testis presented here may well prove to be equally useful to study cell-cell interactions
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in differentiation and morphogenesis as other systems (DeHaan, 1968; Miura and Wilt, 1970; Morris and Moscona, 1970; Steinberg, 1963). Interactions between the nongerminal and germinal populations of cells in relation to initiation of spermatogenesis could then be studied. The development of testicular cell culture represents an additional system to investigate the effects of oncogenic virus on differentiation at the cellular level (Ebert and Kaighn, 1966). SUMMARY
A culture system has been developed which maintains the characteristics of the androgen-producing cells together with fibroblastic cells dissociated from juvenile chicken testes. The behavior of these cells, including their ability to reaggregate in vitro and reorganize into distinct patterns in monolayer cultures, indicates that the maintenance of a stable arrangement of homotypic cells is achieved by intercellular materials holding the cells in discrete groupings. I am grateful to Dr. James D. Ebert, Carnegie Institution of Washington and Dr. Malcolm Steinberg, Princeton University, for their constructive criticism of the study. The techical assistance of Miss Suzanne Hinr is acknowledged. This investigation is aided by a grant (E-553) from The American Cancer Society, Inc. REFERENCES BAILLE, A. H., FERGUSON, M. M., and HART, D. M. (1966). “Development in Steroid Histochemistry.” Academic Press, New York. BISHOP, D. W., (1968). Testicular enzymes as fingerprints in the study of spermatogenesis. In “Reproduction and Sexual Behavior.” (M. Diamond, ed.), pp. 261-286. Indiana Univ. Press, Bloomington, Indiana. CONNELL, G. M., CONNELL, C. J., and EIK-NES, K. B. (1966). Testosterone synthesis by the two-day-old chick testis in uitro. Gen. Camp. Endocrinol. 7, 158-165. COON, H. G., and CHAN, R. D. (1966). Differentiation in uitro: Effects of Sephadex fractions of chick embryo extract. Science 153, 1116-1119. DEHAAN, R. H. (1968). Form and function in the embryonic heart. In “The Emergence of Order in Developing System” (Michael Locke, ed.), pp. 208-250. Academic Press, New York. EBERT, J. D., and KAIGHN, M. E. (1966). The key to change: Factors regulating differentiation. In “Major Problems in Developmental Biology” (Michael Locke, ed.), pp. 29-84. Academic Press, New York. HAM, R. G. (1965). Clonal growth of mammalian cells in a chemically defined synthetic medium. hoc. Nat. Acad. Sci. U. S. 53, 288-293. HAUSCHKA, S., and KONIGSBERG, I. R. (1966). The influence of collagen on the development of muscle clones. Proc. Nat. Acad. Sci. U. S. 55, 119-126. HILFER, S. R. (1968). Cellular interactions in the genesis and maintenance of thyroid characteristics. In “Epithelial’Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 177-199. William and Wilkins, Baltimore, Maryland.
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HILFER, S. R., LSZARD, L. B., and HILFER, E. K. (1968). Follicle formation in the embryonic chick thyroid. Z. Zellforsch. Mikrosk. Amt. 92, 256-269. KHAN, T., and OVERTON, T. (1969). Staining of intercellular material in reaggregating chick liver and cartilage cells. J. Exp. Zool. 171, 161-173. LAM, D. M. K., FURRER, R., and BRUCE, W. R. (1970). The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc. Nat. Acad. Sci. U. S. 65, 192-199. LEE, H. H. (1969). Aggregation and histogenesis in vitro of dissociated chick testicular cells. J. Cell Viol. 43, 76a. LEE, H. H., KAIGHN, M. E., and EBERT, J. D. (1968). Induction of thymidine-3H incorporation in multinucleated myotubes by Rous sarcoma virus. ht. J. Cancer 3, 126136. LEVY, H., DEANE, H. W., and RUBIN, B. L. (1959). Visualization of steroid 3B-01 dehydrogenase activity in tissues of intact and hypophysectomized rats. Endocrinology 65, 932-943. LILIEN, J. E. (1969). Toward a molecular explanation for specific cell adhesion. Curr. Top. Deuelop. Bio2. 4, 169-195. MIURA, Y., and WILT, F. H. (1970). The formations of blood islands in dissociated-reaggregated chick embryo yolk sac cells. Exp. Cell Res. 59, 217-226. MORRIS, J. E., and MOSCONA, A. A. (1970). Induction of glutamine synthetase in embryonic retina: Its dependence on cell interactions. Science 167, 1736-1738. MOSCONA, A. A. (1962). Analysis of cell recombination in experimental synthesis of tissues in uitro. J. Cell. Camp. Physiol. 60, Suppl. 1, 65-80. MOSCONA, A. A. (1968). Cell aggregation: Properties of specific cell-ligands and their role in the formation of multicellular system. Deoelop. Biol. 18, 250-277. NARBAITZ, R., and KOLODNY, L. (1964). A5-3P-hydroxysteroid dehydrogenase in differentiating chick gonads. Z. Zellforsch, Mikrosk, Amt. 63, 612-617. NARBAITZ, R., and DEROBERTIS, E. M., JR. (1968). Postnatal evolution of steroidogenic cells in chick ovary. Histochemie 15, 187-193. P!UFZjE, A. G. E. (1960). “Histochemistry.” Little, Brown, Boston. PHILLIPS, H. M., and STEINBERG, M. S. (1969). Equlibrium measurements of embryonic chick cell adhesiveness, I. Shape equilibrium in centrifugal fields. hoc. Nat. Acad. Sci. U. S. 64, 121-127. SHIN, S. (1967). Studies on interstitial cells in tissue culture: Steroid biosynthesis in monolayers of mouse testicular interstitial cells. Endocrinology 81, 440-448. SHIN, S., YASUMURA, Y., and SATO, G. H. (1968). Studies on interstitial cells in tissue culture. II. Steroid biosynthesis by a clonal line of rat testicular interstitial cells. Endocrinology 82, 614-616. SCHUBERT, D., HUMPHREYS, S., BARONI, C., and COHN, M. (1969). In vitro differentiation of a mouse neuroblastoma. Proc. Nat. Acad. Sci. U. S. 64, 316-323. STEINBERG, M. S., (1963). Reconstmction of tissues by dissociated cells. Science 141, 401-408. STEINBERGER, A., and STEINBERGER, E. (1966). In vitro culture of rat testicular cells. Exp. Cell Res. 44, 443-452. STEINBERGER, E., STEINBERGER, A., VILAR, O., SALAMON, I. I., and SUD, B. N. (1967). Microscopy, cytochemistry and steroid biosynthetic activity of Leydig cells in culture. Testis Ciba Found. Colloq. Endocrinol. 16, 56-78. Little, Brown, Boston. STEINBERGER, A., and STEINBERGER, E. (1967). Factors affecting spermatogenesis in organ cultures of a variety of mammalian testes. J. Reprod. Fertil., Suppl. 2, 117-124.
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G. M. H., and SETCHELL, B. P. (1969). Physiology of the testis, epididymis and scrotum. In “Advances in Reproduction Physiology” (Anne McLaren, ed.) Vol. 4, pp. l-63. Academic Press, New York. WOODS, J. E., and DOMM, L. V. (1966). A histochemical identification of the androgenproducing cells in the gonads of the domestic fowl and albino rat. Gen. Comp. Endocrinol. 7, 559-570. YOUNG, W. C. (Ed.) (1961). “Sex and Internal Secretions,” Vol. 1. Williams and Wilkins, Baltimore, Maryland. WAITES,