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Mitochondrial pleiomorphism in sustentacular cells of Xenopus laevis.

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Mitochondrial Pleiomorphism in Sustentacular
Cells of Xenopus laevis
MARVIN R. KALT
Department of Anatomy, University of Connecticut Health Center
Farmington, Connecticut 06032
ABSTRACT
The mitochondrial population of sustentacular cells in the testis
of the amphibian, Xenopus laevis, has been examined by electron microscopy.
Three distinct types of mitochondria have been observed. The first and most
common mitochondrial type is a “typical” organelle with a rod-like profile containing tubular to plate-like cristae. The second mitochondrial form is charac-
terized externally by irregular bulbous protrusions and internally by increased
numbers of tubular cristae. The third mitochondrial type, which is usually ovoid
in profile, has the most unusual internal membrane configuration, consisting of
pleated folds pierced by regular rows of fenestrations. Mitochondrial type one is
found in all sustentacular stages, mitochondrial type two first appears in sustentacular cells surrounding early spermatids, and mitochondrial type three is
observed only in sustentacular cells surrounding spermatozoa. These observations indicate that discrete subclasses of mitochondria are present in sustentacular cells of Xenopus.
Male germ cells in the anuran testis are
surrounded through their development by
somatic gonadal elements known as sustentacular cells (Brokelmann, ’64; Burgos
and Vitale-Calpe, ’67a,b; Lofts ’68, ’72).
Sustentacular cells may be divided into two
subclasses based upon morphology, which
is in turn related to the germ cell stage
that they envelop. Squamous or cuboidal
sustentacular cells surrounding gonial and
meiotic prophase germ cell stages are referred to as follicle cells, while columnar
sustentacular cells surrounding postmeiotic
germ cells and mature spermatozoa are
referred to as Sertoli cells, after their
mammalian counterparts. These two sustentacular cell forms actually represent different developmental stages of one cell
type, the columnar Sertoli cell being derived from the flattened follicle cell during
the growth and maturation of the seminiferous epithelium. Together, germ and sustentacular cells make up the spermatocyst,
the basic unit of the anuran seminiferous
tubule.
The general cytological and histochemical features of sustentacular cells have
been described in a number of anurans
(Brokelmann, ’64; Burgos and Vitale-Calpe,
ANAT. REC., 182: 53-60.
’67a,b; Lofts, ’68, ’72), including Xenopus
(Wiebe, ’70; Reed and Stanley, ’ 7 2 ) , and
will not be considered here. The present
study has instead concentrated on the ultrastructure of the mitochondrial population in sustentacular cells during the evolution from follicle to Sertoli cell stage. The
results of this investigation indicate that
while follicle cells contain only a single
class of mitochondrion, Sertoli cells possess
multiple forms of this organelle, some of
which contain highly unusual ordered arrangements of the internal mitochondrial
membrane.
MATERIALS AND METHODS
Adult male Xenopus laevis supplied by
the South African Snake Farm, Cape Hoek,
South Africa, were used in this investigation. Sample specimens were taken from
animals maintained in the laboratory for
periods of between one week and one year,
so that an entire breeding cycle was examined. Animals were terminally anesthetized with ether, the testes dissected out,
and the surrounding connective tissue capsule partially removed with forceps. In
most cases, the testis was then placed imReceived July 5, ’74. Accepted Nov. 5, ’74.
53
54
MARVIN R. KALT
mediately in 2.5% glutaraldehyde and
2% formaldehyde (half-strength Karnovsky’s fixative) (Karnovsky, ’65) in 0.1 M
phosphate buffer, pH 7.2, and was fixed
for 8-16 hours at room temperature.
Tissues were then washed in phosphate
buffer and subsequently were postfixed in
2% osmium tetroxide in 0.1 M phosphate
buffer, pH 7.2, for an additional six hours
at room temperature. In some cases, specimens were placed directly in osmium without prior fixation in aldehydes. After
osmication, all samples were rinsed in distilled water, dehydrated in graded acetones,
and embedded in an Epon-epoxy mixture
(Kalt and Tandler, ’71). Thin sections were
cut with diamond knives on a Sorvall MT
2B or LKB Ultratome I11 ultramicrotome.
Sections were collected on copper grids
covered with carbon coated Padgett’s film
(Padgett, ’63), stained with uranyl acetate
(Locke and Krishnan, ’71) and lead citrate
(Venable and Coggeshall, ’65), and were
examined in a Philips 300 electron microscope operated at an accelerating voltage
of 80 kV.
OBSERVATIONS
Xenopus laevis in the wild normally
breeds in the Fall, but animals examined
at any time in the year contain the full
range of germ and sustentacular cell
stages. The sustentacular cell population,
therefore, remains qualitatively constant,
so that all animals contain all of the mitochondrial forms described below.
Follicle stage sustentacular cells in
Xenopus contain a mitochondrial complement consisting of ovoid to rod-shaped
organelles with either plate-like or tubular
protrusions of the inner mitochondrial
membrane. In aldehyde-osmium fixed specimens, the cristae are moderate in number
and extend into an electron dense inner
mitochondrial matrix. Specimens fixed in
osmium alone have the same membrane
configuration, but much of the mitochondrial matrix appears lost (fig. 1).
As sustentacular cells begin the transition from follicle to Sertoli stage, new mitochondrial configurations begin to appear.
One of the first changes noted in some
mitochondria is an increase in the number of cristae. This increase is coupled with
a change in the overall shape of the mito
chondrion from a rod-like configuration to
a more convoluted profile, often resulting
in bulbous or doughnut-shaped protrusions
at one end of the organelle (fig. 2). As
earlier, aldehyde-osmium fixed specimens
have an electron dense mitochondrial matrix (fig. 2 ) , while specimens fixed in
osmium alone appear to have lost much of
this material.
Mature Sertoli stage sustentacular cells
surrounding spermatozoa contain a third
type of mitochondrion in addition to the
two types found in earlier stage sustentacular cells. The external profile of this third
mitochondrial type, while variable, is not
unique, ranging from ovoid to rod-like. As
before, the only obvious difference in preservation between single and double fixed
specimens is in the density of the mitochondrial matrix, which again appears
washed out with osmium alone. As compared to more “typical” mitochondria
within the cell, both types of fixation reveal
extremely prominent intramitochondrial
dense granules (compare fig. 2 to figs. 3,
5). The most unusual characteristic of
this third mitochondrial type, however, is
the organization of the cristae projecting
from the internal mitochondrial membrane. In both aldehyde-osmium and osmium fixed specimens, the cristae occur
in pleated sheet-like configurations. Each
crista, in turn, is pierced by regular alternating rows of fenestrations in the folds of
the pleats (figs. 3, 6, 8 ) . These fenestrations are continuous with the inner compartment spaces between cristae. En. face
views of fenestrations demonstrate a high
degree of order in their arrangement (figs.
3, 6, 8 ) . Tangential sections further reveal
the pattern which makes up the infoldings
of the inner membrane, and demonstrate
membrane profiles which indicate that apposed rows are set at an angle to each
other (figs. 5, 7). The probable structure
of these cristae is diagrammatically represented in figure 9.
In addition to the above mitochondrial
forms, mature Sertoli cells also contain
irregularly shaped mitochondria with two
or more distinct internal membrane configurations; one segment showing the
pleated tubule pattern, others showing the
SUSTENTACULAR CELLS OF X E N O P U S
more common plate-like or tubular configuration (fig. 4). Together, these transitional forms and the pure “pleated” form
account €or approximately ten to fifteen
percent of the mitochondria seen in sections of Sertoli cells and show a random
distribution within the cytoplasm of individual cells.
DISCUSSION
The present study demonstrates that sustentacular cells of Xenopus Zaevis normally
possess multiple forms of mitochondria.
These different mitochondrial configurations show neither preferential location
within the cell, nor qualitative seasonal
variation, being present in all animals examined. There does exist, however, a difference in the distribution of organelles between follicle and Sertoli stages, since it
is only in the latter that the more unusual
morphological variations are observed.
Mitochondria with a complex tubular
structure similar to that described here
have been reported in the cytoplasm of an
amoeba (Pappas and Brandt, ’59). While
the similarities between these two cases
appear to be fortuitous, it is of interest to
note that, in general, mitochondria with
tubular cristae are characteristic of steroid
secreting cells (Christensen and Gillim,
’69). Furthermore, mitochondria with paracrystalline “braids” of tubular cristae have
been reported in steroid secreting cells derived from the Sertoli cells of the urodele
testis (Picheral, ’68, ’70). While histochemical tests for some steroid pathway
enzymes in Sertoli cells of Xenopus have
been negative (Wiebe, ’7O), there are many
more enzymes which have not yet been
examined. Therefore, i t is still possible that
these cells may be active in some aspect of
steroid metabolism. Since evidence exists
in other anurans that Sertoli cells can play
a role in steroid metabolism (Christensen
and Gillim, ’69; Lofts, ’68, ’ 7 0 ) , further
examination of this question in Xenopus is
warranted, In any case, the pleiomorphic
nature of mitochondria in the Sertoli cell
Xenopus has been established, and the restricted presence of such an unusual inner
mitochondrial membrane structure suggests that these organelles may play a
unique role in the physiology of Sertoli
cells.
55
ACKNOWLEDGMENTS
This study was supported by grants from
the University of Connecticut Research
Foundation, National Science Foundation
grant GB-41836, and National Institute of
Child Health and Human Development
grant HD-08268.
LITERATURE CITED
Brokelmann, J. 1964 Uber die Stiiz-und Zwischenzellen des Froschhodens Wahrend des
Spermatogenetischen Zyklus. 2. Zellforsch.
Mikrosk. Anat., 64: 429-461.
Burgos, M. H., and R. Vitale-Calpe 1967a The
fine structure of the Sertoli cell-spermatozoan
relationship in the toad. J. Ultrastruct. Res.,
19: 221-237.
1967b The mechanism of spermiation
in the toad. Am. J. Anat., 120: 227-252.
Christensen, A. K., and S . Gillim 1969 The
correlation of fine structure and function in
steroid-secreting cells with emphasis on those
of the gonads. In: The Gonads. K. W. McKerns,
ed., Appleton-Century-Crofts. New York, pp.
415-488.
Kalt, M. R., and B. Tandler 1971 A study of
fixation of early amphibian embryos for electron microscopy, J. Ultrastruct. Res., 36: 633645.
Karnovsky, M. J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolality for use
in electron microscopy. J. Cell Biol., 27: 137A.
Locke, M., and N. Krishnan 1971 Hot alcoholic
phosphotungstic acid and uranyl acetate as
routine stains for thick and thin sections.
J. Cell Biol., 50: 550-556.
Lofts, B. 1968 Patterns of testicular activity.
In: Perspectives in Endocrinology: Hormones
in the lives of lower vertebrates. E. J. W.
Barrington and C. B. JWgensen, eds., Academic
Press, New York, pp. 239-304.
1972 The Sertoli cell. Gen. Comp. Endocrinol. Suppl., 3: 636-648.
Padgett, F. 1963 Some observations on new
fixation and staining procedures of biological
material and on a new supporting film. Sci.
Instruments, 8: 27-33.
Pappas, G. D., and P. W. Brandt 1959 Mitochondria. I. Fine structure of the complex patterns in the mitochondria of Pelomyxa carolinensis Wilson (Chaos chaos L.). J. Biophys.
Biochem. Cytol., 6: 85-89.
Picheral, B. 1968 Les tissues Claborateurs dhormones steroides chez les amphibiens urodeles.
I. Ultrastructure des cellules du tissu glandulaire du testicule de Pleurodeles zualtlii Michah.
J. Microscopie, 7 : 115-134.
1970 Les tissues elaborateurs d’hormones steroids chez les amphibiens urodeles.
IV. Etude en microscopie Clectronique et photonique du tissu glandulaire du testicule et de
la glande interrenale apres hypophysectomie,
56
MARVIN R. KALT
chez Pleurodeles waltlii Michah. 2. Zellforsch.
n5krosk. Anat., 107: 68-86.
Reed, S. C., and H. P. Stanley 1972 Fine
structure of spermatogenesis i n the South
African clawed toad Xenopus laevis Daudin.
J. Ultrastruct. Res., 41: 277-295.
Venable, J. H., and R. Coggeshall 1965 A
simplified lead citrate stain for use in electron
microscopy. J. Cell Biol., 25; 407-408.
Wiebe, J. P. 1970 The mechanism of action of
gonadotrophic hormones in amphibians: The
stimulation of A5-3p hydroxy steroid dehydrogenase activity in testis of Xenopus laevis Daudin. J. Endocrinol., 47: 4391150.
Figures 1 and 7 are micrographs of specimens fixed in osmium alone.
All other figures are micrographs of aldehyde-osmium double fixed
specimens.
PLATE 1
EXPLANATION OF FIGURES
Typical rod shaped mitochondrion from a follicle stage sustentacular
cell showing both platelike and tubular cristae. Note the rather
sparse mitochondrial matrix and the moderate size of the intramitochondrial dense granules (arrows). x 36,000.
Modified mitochondrion with a doughnut shaped protrusion from a n
early Sertoli stage sustentacular cell. A cytoplasmic area ( C ) containing a membrane whorl is visible i n the “hole” of the doughnut.
Note the increased number of cristae and increased density of the
mitochondrial matrix compared to figure 1. x 41,000.
Ovoid mitochondrion with fenestrated cristae from Sertoli stage sustentacular cell. The cristae are obliquely sectioned so that several
rows are seen at an angle. The fenestrations (marked 1 ) are delimited
by the surface of the inner mitochondrial membrane and are filled
with the dense matrix material of the inner mitochondrial compartment. The lighter “holes” (marked 2) are cross sections of tubular
elements of the cristae, and are delimited by the internal side of the
inner mitochondrial membrane. The space enclosed, therefore, is that
between the inner and outer mitochondrial membrane, or the intracristal space. Compare this configuration to that of a more “typical”
mitochondrion in the same section (M). x 90,000.
SUSTENTACULAR CELLS OF XENOPUS
Marvin R. Kalt
PLATE 1
57
PLATE 2
EXPLANATION OF FIGURES
4
Irregularly shaped mitochondrion from a mature Sertoli cell. Note the
three distinct patterns of cristae within this single organelle (A, B, C ) ,
and the external investment with profiles of smooth endoplasmic
reticulum (SER). E, external (intracristal) compartment; I, internal
(matrix) compartment. X 66,000.
5
Mitochondrion with fenestrated cristae from a Sertoli stage sustenacular cell. The plane of section is such that angular tubular inner
membrane profiles can be seen to better advantage. Numbers correspond to those in figure 3.
83,000.
x
58
6
En face view of crista from a Sertoli cell mitochondrion showing the
regular arrangement of fenestrations (1) and the continued presence
of tubule cross sections ( 2 ) at the point where the plane of section
changes. X 75,000.
7
Section of a mitochondrion s i m i l a to that shown in figure 6, except
that the plane of section is at right angles to figure 6. The separate,
corrugated nature of the cristae is apparent. I, internal compartment;
E, external compartment. x 46,000.
8
Another tangential view of a Sertoli cell mitochondrion with the
fenestrated pattern. x 46,000.
SUSTENTACULAR CELLS OF XENOPUS
PLATE 2
Marvin R. Kalt
59
PLATE 3
SUSTENTACULAR CELLS OF X E N O P U S
Marvin R. Kalt
EXPLANATION O F FIGURE
9 Artist’s conception of the surface of a pleated inner membrane showing a regular array of alternating fenestrations. The fenestrations,
which are circular en face, appear heart-shaped due to the perspective.
60
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