/ . Einhryol. exp. Morph. Vol. 44, pp. 297-302, 1978
Printed in Great Britain (p Company of Biologists Limited 1978
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SHORT PAPER
Morphogenesis by dissociated immature rat
testicular cells in primary culture
By JOHN C.DAVIS 1
From the Department of Population Dynamics,
School of Hygiene and Public Health,
The John Hopkins University, Baltimore, U.S.A.
SUMMARY
Dissociated cells from immature (15-day) rat testes maintained in primary culture were
shown to undergo morphogenesis. Dissociated cells (1-4 x 106) were cultured in 2 ml medium
NCTC-135 containing 10% rat serum at 32-5 °C under an air:CO2 atmosphere (95%:5%).
During the initial culture period (1-3 days) the dissociated cells attached to the culture dish
and formed a monolayer. After 4-5 days of culture, aggregates of cells formed at discrete
foci within the monolayer; these structures averaged 50/tm in diameter and numbered
3-4 x 103/dish. Within 5-6 days of culture some of these aggregates (2-3 x 102) released their
contact with the substratum. Histological examination of released aggregates indicated a more
regular, tissue-like organization with increasing time in culture. Aggregates at earlier time
periods (4-5 days) exhibited concentric rings of cell while by day 6 some aggregates exhibited
a vesicular appearance with a single layer of columnar epithelium surrounding a fluid-filled
lumen. The type of structure formed varied with plating density; at densities less than
2 x 10°/dish the structures were spheroid while at greater densities tubes were formed. Timelapse observations indicated that spheres formed by movements of cells into an aggregating
center while tubes formed by a rolling from the edge of the monolayer. The cells comprising
the vesicle epithelium appeared to be predominantly one cell type as determined by light
microscopy. These results therefore indicate that dissociated testicular cells have the ability to
reform tissue-like associations in culture.
INTRODUCTION
Dissociated cells from the immature rat testis form colonies in response to
follicle-stimulating hormone in primary culture (Davis & Schuetz, 1975a).
Although the testis contains a number of germinal and somatic cell types, these
colonies contained primarily one cell type. Recent evidence has suggested that
these colonies are formed by cell-specific aggregation and not cell division and
are Sertoli cells (Davis & Schuetz, 1977). In the present study, further differentiation of dissociated rat testicular cells in primary culture was observed,
1
Authors'1 address: Department of Physiology and Cell Biology, 108 Snow Hill, The
University of Kansas, Lawrence, Kansas 66045, U.S.A.
298
j . c. D A V I S
namely the reorganization of monolayers of rat testicular cells to form tissuelike structural units resembling those seen in situ.
In vitro cell-specific aggregations have been noted with cells from a number of
non-testicular tissues of several species (Galtsoff, 1925 Moscona, 1957 Steinberg, 1963). Such aggregations have, in some cases, differentiated into tissue-like
structures in primary culture (Okada, 1965; McGrath, Nandi & Young, 1973).
These structures have been typically spheroid or 'dome-shaped'. Depending on
initial cell concentrations, dissociated rat testicular cells form spheroids or tubes.
These observations indicate that dissociated rat testicular cells have the capacity
for morphogenesis in vitro.
METHODS
I. Cell dissociation
Testes from five 15-day-old Sprague-Dawley rats (Flow Laboratories) were
removed and decapsulated. The teased tubules were incubated in 15 ml of
0-1 % trypsin (Difco 1:250) in Ca, Mg-free phosphate-buffered saline (pH 7-35)
in a 50 ml trypsinizing flask. The tissue-free supernatant was collected every
10 min, diluted with 8 % fetal calf serum (1:1), and filtered through 28/* Nitex
cloth (TET-Kressilk) (Davis & Schuetz, 19756). After 50 min of dissociation,
the pooled cells were collected by centrifugation at 1000 g for 10 min.
II. Culture conditions
Cells were cultured in 2 ml NCTC-135 containing 10% rat serum (Difco),
at 32-5° under air:CO 2 (95 %:5 %) with penicillin 100 i.u./ml and streptomycin
100/*g/ml in 35 mm dishes. In most cases, cells were plated at 1-6 x 106 cells/
dish. The cell suspension contained 1-16±005 cells/clump (n = 4, mean±
S.E.M., 300 cells counted); viability (trypan blue dye exclusion) ranged from
93 to 96 %. In all cases new media was added on day 4; care was taken not to
pipet aggregates during media changes. Cell number was determined on a
Biophysics Cytograf; only particles with sizes greater than red blood cells were
counted.
Detached aggregates were collected and fixed in 4 % formalin (12 h), embedded in 5 % agar, and the agar block embedded in paraffin and sectioned at
5 jum. Sections were stained for 4 min in hemotoxylin. In one experiment movement of cells within the monolayer was followed by time-lapse cinephotomicroscopy.
RESULTS
Description of aggregation
Single-cell suspensions of rat testis were plated at 1 x 106 cells/dish in NCTC135 medium containing 10 % rat serum. After 24 h of culture, cells were attached
to the culture dish; however, no cells were seen in closely packed groups,
colonies, with culture in 10 % rat serum as has been noted in media containing
Testis cell movement and reaggregation in culture
299
Fig. 1. Phase-contrast photomicrograph of cells in the monolayer of a 4-day
culture. Cells were plated at 1 x 106/dish. Cells can be seen in multiple layers at the
center of the figure (arrow). Bar = 10 /*m.
Fig. 2. Phase-contrast photomicrograph of cells in an aggregate in a 5-day culture.
Cells were plated at 1 x 10°/dish. Bar = 10 /xm.
Fig. 3. Section of a detached vesicle at 4 days of culture. At this point little organization is seen within the structure. Bar = 10 /<m.
Fig. 4. Section of detached vesicle at 5 days of culture. An epithelial arrangement
with a clear lumen is seen at this time. Bar = 10/tm.
Fig. 5. Dish (35 mm) containing a concentric ring of tubes (arrows) at 6 days of
culture.
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J. C. DAVIS
10 % fetal calf serum (Davis & Schuetz, \975a). By day 3 of culture, aggregates
of cells were seen attached to the dish substratum (Fig. 1). Unlike the 'colonies'
noted in the previous study (Davis & Schuetz, 1975a), which contained closelypacked groups of cells attached to the monolayer, the aggregated cells formed
mounds or multiple layers with some cells losing contact with the substratum.
The cells were loosely arranged within the aggregate. At 4 days of culture,
aggregates had enlarged and were spheroid in shape (Fig. 2). Cell orientations
within all aggregates examined appeared to be random. The number of attached
aggregates varied between 3-4 x 103/dish (n = 6).
After 4-5 days in culture, most aggregates detached from the dish bottom.
Histological examination of the released aggregates showed some internal
organization (Fig. 3) with concentric layers of cells around a central lumen.
By 5 days (Fig. 4), an epithelial-like layer of cells surrounding a central lumen,
similar to that noted in the germ-cell-depleted testis in situ (Means & Huckins,
1974), was seen. The cells forming the epithelium appear to be of one cell type.
Other released vesicles showed an epithelial-like layer surrounded by concentric
rings of cells; in others, the concentric layer was seen inside the epithelial layer.
Some aggregates resembling those seen at day 4 (Fig. 3) were still present at
day 5.
At cell concentrations below 2 x 106/dish, vesicles were formed; at greater cell
concentrations, tubules were formed (Fig. 5). The time course of tubule formation was identical to that of vesicles. At concentrations greater than 4 x 106/dish,
a concentric ring of tubes was seen at the outside of the dish with vesicles in the
middle (Fig. 5). Size of the tubes varied from 500 fim to 1-2 cm in length. The
number of tubes varied from 1 to 15 per dish, number was inversely proportional to size, and the time course of tube formation was identical to aggregate
formation. Whereas the aggregates were formed by movement of cells into one
center, tubes were formed by rolling of the edge of the monolayer. The rolling
edge was always initiated at the point where the confluent monolayer was in
contact with the dish edge. The absence of tube formation at plating densities
below 2 x 106/dish is probably due to the lack of monolayer confluence.
To rule out the possibility that the detached vesicles formed from epithelial
cells of the tubuli recti, the tubulus portion of testis from 15-day-old rats was
removed and discarded prior to cell dissociation. Under these conditions,
aggregation still occurred, indicating that the tubulus cannot be the sole source
of reaggregating cells.
DISCUSSION
It is now well established that embryonic cells from several tissues can form
tissue-specific associations in vitro (Moscona, 1957; Steinberg, 1963). Abraham
(1960) has noted specific growth patterns in monolayers of embryonic chick
testis cells. Tissue-like reassociations of adult mammary cells have also been
noted (McGrath et al. 1973). The in vitro reassociations reported in the present
Testis cell movement and reaggregation in culture
301
paper exhibit several characteristics identical to testicular cells in situ, including
(1) formation of a somatic (Sertoli) cell epithelium, (2) lumen formation, and (3)
formation of tubular structure. The testis cells were obtained after birth, but at
an age when the cells involved are still differentiating (Flickinger, 1967).
The ability of a mixture of cells to form an epithelium comprising predominately one cell type clearly implies the ability for cell recognition, either by
cell-surface or extrinsic signals (Moscona, 1960). The geometry of the resulting
structures may be explained by the hypothesis of Steinberg (1963), according
to which cell aggregates form spheres in vitro because this maximizes the cellcell and minimizes the cell-media interactions. Tubes, however, are initiated at
the cell-dish interface, the only position at which a cell in the monolayer lacks
an interaction with the surface of another cell. Since the cells in the axis parallel
to that interface are already in direct contact with one another, this puts a constraint on movement in that direction, so that the resulting structure of least
free energy is a cylinder or tube.
The testis of the 15-day-old rat contains germinal and non-germinal cells.
However, only non-germinal (somatic) cells attach to the culture dishes (Steinberger, 1965). The cells comprising the epithelial layer of the vesicles appear
to be Sertoli cells (Davis & Schuetz, 1977). Close scrutiny of the Sertoli cell
in situ offers some insight into the forces involved in the movement. The Sertoli
layer in the seminiferous tubules in vivo is polarized, the basal portion lying in a
plasma-like environment and the apex in the unique ionic environment of the
tubule (Tuck, Setchell, Waites & Young 1970); this polarization may be involved,
in the movements noted.
1 would like to thank Drs Allen Schuetz, Zenus Sykes, and Young Kim for their helpful
discussions; Dr Allen Cohen's help with the time-lapse photomicroscopy is greatly appreciated. This research supported by a contract HD3-2794 from the National Institutes of Child
Health and Human Development. Funds for some equipment were provided by a grant from
the Rockefeller Foundation (grants awarded to A. W. Schuetz).
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{Received 9 March 1977, revised 18 July 1977)