BIOLOGY OF REPRODUCTION 51, 50-62 (1994)
Ultrastructural Distribution of Calcium in the Rat Testis'
N. RAVINDRANATH, V. PAPADOPOULOS, W. VORNBERGER, D. ZITZMANN, and M. DYM2
Department of Cell Biology, Georgetown University Medical Center, Washington, District of Columbia 20007
ABSTRACT
Despite the important role of calcium in the growth and differentiation of a variety of cell types, its exact location and function
in the somatic and germ cells of the testis remain to be determined. In the present study, we examined the subcellular distribution
of calcium in the immature and adult rat testis. Calcium was localized at the electron microscopic level by ion-capture cytochemistry using combined oxalate and pyroantimonate procedures. Calcium-containing precipitates localized primarily within
the nuclei, mitochondria, and cytosol of somatic and germ cells. Differences in the size and quantity of the calcium precipitates
were observed among the various cellular compartments. In the somatic cells (Sertoli, Leydig, and myoid), the nuclei exhibited
large round-shaped calcium-containing precipitates, whereas the mitochondria in these cell types contained numerous smaller
precipitates. The cytoplasmic vesicles possessed single precipitates. These vesicles could be calciosomes, which have been described in other non-muscle cell types. Among germ cells, round spermatids exhibited a large number of vesicular, calsiosomelike structures in the cytoplasm containing single precipitates. The elongating spermatids from adult testis showed calcium localization within the nuclear matrix unassociated with the nuclear envelope, or in a peripheral alignment of precipitates along
the nuclear envelope. Calciosome-like structures were also seen in round spermatids. Spermatogonia and spermatocytes exhibited
calcium in nuclei, mitochondria, and cytoplasmic vesicles. These results demonstrate a differential distribution of calcium within
the various cell types of the testis. The presence of calcium in the nucleus may suggest a role in cell growth and differentiation;
calsiosome-like structures may represent the active exchangeable pool of calcium, and the differential type of distribution of
calcium in elongating spermatids suggests a role for calcium in spermatid differentiation.
INTRODUCTION
antimonate-based method [6] has been used extensively for
electron microscopic visualization of intracellular storage
sites of calcium. Alternatively, an oxalate-glutaraldehyde
method has been used to detect intracellular calcium deposits [7]. But both of these methods have inherent drawbacks and lack of reproducibility. A combination of these
two methods has been developed and used successfully with
reproducible results in different tissues [8-10]. The rationale of this combined procedure is first to selectively precipitate loosely bound calcium with oxalate and then to wash
out other cations before subsequent conversion of the precipitate into insoluble electron-dense antimonate. Using this
method, we investigated the distribution of calcium in immature and adult rat testes. The data obtained may be indicative of the specialized role of calcium in testicular cell
types.
Calcium plays a predominant role in the regulation of
many functional processes of eukaryotic cells. Although very
little is known about its involvement in the functions of
Sertoli, Leydig, and myoid cells, recent studies have suggested a role for calcium in Sertoli cell estradiol biosynthesis [1] and transglutaminase activation [2], in Leydig cell
steroidogenesis [3], and in endothelin action on myoid cells
[4]. It has also been demonstrated that calcium is essential
for the maintenance of cell shape and the regulation of protein secretion by Sertoli cells [5]. However, the literature
available on the role of calcium ions in the different germ
cell types that are present in the testis is very scanty. In
addition, the exact location of the intracellular storage sites
of calcium in different cell types of the testis is not known.
Furthermore, very little information is available on calcium
homeostasis in these cell types. Intracellular storage organelles along with the plasma membrane play a major role
in the maintenance of homeostasis by regulating the quantity of free cytosolic calcium. Changes in free cytosolic calcium levels can be measured by using the calcium-binding
florescent probes, fura-2 and indo-1. However, this method
can only detect the ionic concentration of calcium in the
cytosol and cannot resolve the localization of calcium within
subcellular sites and intracellular organelles. Thus far, an
MATERIALS AND METHODS
Adult male rats (90-120 days old) and immature male
rats (10-35 days old) were anesthetized and fixed by perfusion through the heart, the abdominal aorta, or the testicular artery for 15-30 min. Perfusion fixation was chosen
over immersion fixation in order to complex and stabilize
the intracellular calcium as quickly as possible, thereby limiting the expected ion translocations during immersion of
tissue blocks. The primary fixative consisted of 2% glutaraldehyde, 2% formaldehyde, 90 mM potassium oxalate, and
1.4% sucrose, pH 7.4. After the perfusion, the testes were
removed, cut into 1-mm blocks, and immersed in the primary fixative at 4°C for an additional 12-24 h. The tissues
were postfixed in 1% osmium tetroxide containing 2% po-
Accepted March 16, 1994.
Received January 6, 1994.
'This work was supported by an NIH grant (HD24633) to M.D. and by an NIH
Research Career Development Award (HD01031) to V.P.
2
Correspondence: Dr. Martin Dym, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road, N.W., Washington, DC 20007. FAX: (202)
687-1823.
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LOCALIZATION OF CALCIUM RESERVOIRS IN THE RAT TESTIS
51
FIG. 1. Electron micrograph of a section of a seminiferous tubule from an immature rat showing calcium-containing precipitates in the nucleus, mitochondria (M), and vesicles (V) of a Sertoli cell. Note the absence of large precipitates in the nucleolus (N) and heterochromatin (H). There are also calcium
deposits in the space between the outer and inner leaflets of the nuclear envelope (arrowheads). x 10000.
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RAVINDRANATH ET AL.
FIG. 2. A) Electron micrograph from an immature rat testis showing calcium precipitates within the mitochondria in a Sertoli cell. x20 000. B) Highmagnification electron micrograph of an individual mitochondrion in a Sertoli cell. The calcium-containing precipitates (arrowheads) are mainly associated
with the cristae formed by inner mitochondrial membrane. x40 000.
LOCALIZATION OF CALCIUM RESERVOIRS IN THE RAT TESTIS
53
FIG. 3. Electron micrograph depicting calcium localization in the nucleus and in vesicular structures (V) in the cytoplasm of a Sertoli cell from an
adult rat testis. Arrowheads point to calcium deposits in the nuclear envelope. Two germ cells (G) are visible in the upper right and upper left of the
micrograph. x33 000.
tassium pyroantimonate for 2-3 h at 4C. After a rinse for
15 min in cold distilled water brought to pH 10 with potassium hydroxide, the tissues were dehydrated in ascending grades of ice-cold ethanol (50%, 70%, and 95%) and at
room temperature in absolute ethanol for 2 h. The tissues
were infiltrated in 50% and 100% Epon after 30 min of
treatment with propylene oxide and later embedded in 100%
Epon:2% DMP-30 mixture. To assess the specificity of the
reaction, unstained thin sections were treated with 10 mM
EGTA. Ultrathin sections. were examined either unstained
or after brief counterstaining with uranyl acetate and lead
citrate. For the other controls, tissues were fixed with 5%
glutaraldehyde in collidine buffer, or the primary fixative
lacked oxalate, or the pyroantimonate was omitted in the
postfixation step. AJEOL 1200EX electron microscope (Tokyo, Japan) was used to view the sections at 60 V of current.
The results presented are derived from five independent
studies performed in groups of six immature and six adult
rats with corresponding controls.
RESULTS
Perfusion fixation of the testes with the primary fixative
containing 2% glutaraldehyde, 2% formaldehyde, 90 mM
potassium oxalate, and 1.4% sucrose did not provide the
firmness to the tissue observed in tissues fixed with 5%
glutaraldehyde in collidine buffer. But the general morphology of the tissue was good and was comparable to 5%
glutaraldehyde-fixed tissues. No major fixation artifacts were
observed.
In tissue sections derived from young and adult rats, Sertoli cells exhibited numerous calcium-containing precipitates within their nuclei. At 8000-10 000X magnification,
these precipitates appeared as large round electron-dense
bodies distributed throughout the nuclear matrix. The nucleoli and heterochromatin were devoid of any large precipitates. Tiny precipitates were observed in the nucleolus
of some cells but not in the heterochromatin. A number of
precipitates were found within the bilaminar structure of
the nuclear envelope (Fig. 1). Numerous mitochondrial
precipitates were present as fine singular deposits distributed within the matrix (Fig. 2A). At higher magnification
(40 000x), they appeared to localize within the cristae of
the mitochondria (Fig. 2B). The precipitates did not appear
to be associated with the outer mitochondrial membrane.
Apart from mitochondria, small vesicular structures in the
cytoplasm exhibited single round precipitates (Fig. 3). Very
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RAVINDRANATH ET AL.
FIG. 4. Electron micrograph of a tight junction between adjoining Sertoli cells in a section of testis from an immature rat. No calcium precipitates are
located in the immediate region of the adjoining Sertoli cell plasma membranes (white arrowheads). The associated endoplasmic reticulum (ER) shows
calcium-containing precipitates. M, mitochondrion. x38 000.
few precipitates unassociated with any organelle were observed. Generally, the adjoining plasma membranes at the
tight junctional complexes between Sertoli cells had very
few precipitates; however, the associated endoplasmic reticulum showed calcium-containing precipitates (Fig. 4). At
the Sertoli and germ cell interface, no precipitates were
observed.
Leydig cells also exhibited a large number of round precipitates in the nucleus. The heterochromatin and nucleolus were devoid of any precipitates. Mitochondrial precipitates were small and numerous. The plasma membrane did
not show any associated precipitates, and the cytoplasmic
lipid droplets were devoid of any calcium deposits (Fig.
5A). At higher magnification (30 000 x ), several precipitates
were found to be associated with the endoplasmic reticulum. Few precipitates were localized within vesicles (Fig.
5B).
The peritubular myoid cells manifested fewer calcium
deposits within their nuclei in comparison to Sertoli and
Leydig cells. These precipitates were associated with euchromatin only. The nuclear envelope showed some calcium precipitates. The vesicular structures infolded from
the plasma membrane did not exhibit any precipitates. Vesicles within the cytoplasm presented tiny deposits. Mito-
chondria and endoplasmic reticulum showed membranebound deposits (Fig. 6).
The spermatogonial cells also contained a large number
of electron-dense calcium deposits within their nuclei. Unlike the situation for the Leydig and myoid cells, some precipitates were found near the nucleoli and associated heterochromatin. The mitochondria possessed mostly small
precipitates. The Golgi apparatus presented few large precipitates within its membranous structure. Some of the Golgi
vesicles also contained tiny calcium deposits. Endoplasmic
reticulum did not show any precipitates (Fig. 7).
In pachytene spermatocytes, the nuclei exhibited numerous large precipitates with both the nucleoli and heterochromatin devoid of calcium deposits. Occasionally,
precipitates were observed on the synaptonemal com-
FIG. 5. A) Electron micrograph of a Leydig cell from an immature rat
testis showing calcium precipitates in the nucleus and mitochondria (M).
Note the lack of precipitates in the nucleolus (N), lipid (L), and heterochromatin (H). x10 000. B) High-magnification electron micrograph of a Leydig
cell depicting calcium deposits in the nucleus, but not in the nucleolus (N),
and in the cristae of mitochondria (M). The arrowhead points to a calcium
precipitate localized within the bilaminar structure of the nuclear envelope.
Round precipitates distributed within the ER are also shown. x35 800.
LOCALIZATION OF CALCIUM RESERVOIRS IN THE RAT TESTIS
55
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RAVINDRANATH ET AL.
FIG. 6. A peritubular myoid cell from an adult rat testis exhibiting a few calcium deposits within its nucleus, but not in the heterochromatin (H), is
depicted in this electron micrograph. Note the calcium precipitates within the inner and outer leaflets of the nuclear envelope (arrowheads). There is an
absence of precipitates in the pinocytotic vesicles (V) near the plasma membrane. x30 000.
plexes. Mitochondria showed fewer precipitates, and precipitates unassociated with any organelle were also seen in
the cytoplasm. The nuclear envelope, endoplasmic reticulum, and plasma membrane did not show any precipitates
(Fig. 8).
The round spermatids exhibited large-sized precipitates
in the nucleus. However, the nucleolus and heterochromatin were devoid of any precipitates. The acrosomal cap
exhibited one or two precipitates. There were few precipitates in the mitochondria (Fig. 9A). A large number of vesicles containing a single precipitate appeared in the cytoplasm. Very few precipitates unassociated with any organelle
were seen in the cytoplasm (Fig. 9B).
The elongating spermatids in the adult testis presented
two different kinds of calcium localization. One group of
spermatids exhibited a few large nuclear deposits unassociated with the nuclear membrane. The acrosomal cap was
totally devoid of any deposits. Very few calcium-containing
precipitates were observed in the cytoplasm (Fig. 10A). In
another group of spermatids, deposits were localized along
the inner leaflet of the nuclear envelope. One or two precipitates appeared within the central matrix portion of the
nucleus (Fig. 10B).
In control reactions, the exposure of sections to EGTA
resulted in complete removal of precipitates in tissues fixed
in oxalate-pyroantimonate (Fig. 11). No precipitates were
observed when pyroantimonate was deleted from the postfixative solution. In the absence of potassium oxalate in the
primary fixative, pyroantimonate in the postfixative solution
binds to all cations in the cell and therefore precipitated
as a granular deposit all over the tissue sections.
DISCUSSION
Calcium regulates cell function by acting as a primary
modulator of the cellular environment and as a second
messenger in signal transduction pathways. Eukaryotic cells
contain millimolar concentrations of calcium. However, the
ionic form of calcium in the cytosol is very limited (nanomolar concentrations), suggesting that most of the calcium is not free but bound to calcium-binding molecules
and sequestered in intracellular organelles. A number of
attempts have been made to identify these molecules and
organelles within the cell in a variety of tissues. In the testis,
there is no information on the subcellular localization of
calcium. In the present study, we have localized calcium
using the oxalate-pyroantimonate technique [8-10]. The rationale of this cytochemical technique is the initial precipitation of calcium in ice-cold oxalate-glutaraldehyde fixative
to limit translocation of precipitates, followed by conver-
LOCALIZATION OF CALCIUM RESERVOIRS IN THE RAT TESTIS
57
FIG. 7. Electron micrograph of a type B spermatogonia from an immature rat testis showing calcium deposits in the nucleus, Golgi, and mitochondria
(M). Some precipitates are found near the nucleolus (Nu) and associated heterochromatin (H). Careful inspection of the Golgi apparatus reveals tiny calcium
precipitates, in addition to several larger ones, among the saccules and vesicles. x10 000.
sion into electron-dense antimonate deposits by subsequent postfixation with osmium-pyroantimonate. Treatment of these sections with EGTA chelates calcium from
calcium oxalate-pyroantimonate complex, rendering antimonate deposits soluble in water [11]. This cytochemical
technique has been further validated by x-ray microanalysis
[12-14] and proton probe microanalysis [15]. Finally, the
results presented here are from several perfusion fixation
experiments undertaken both in immature and adult rats.
The presence of a large number of calcium-containing
precipitates within the nuclei of all the cell types in the
testis suggests that calcium may play an important role in
the process of cellular growth and differentiation. The requirement for intracellular calcium in the nuclei in the early
stages of DNA synthesis during both nuclear envelope
breakdown and cytokinesis is well established [16]. In the
testes, Sertoli and Leydig cells are under hormonal regulation by FSH and LH, respectively. Although both of these
hormones act via the cAMP second messenger pathway, a
role for calcium has been invoked in their hormone action.
Treatment of Sertoli cells with FSH results in a decrease in
calcium-regulated phosphodiesterase enzyme, which hydrolyzes cAMP, and an increase in intracellular nonexchangeable calcium [17]. Recently, Grasso et al. [1] and
Gorczynska and Handelsman [18] have demonstrated FSHinduced calcium uptake by Sertoli cells. The mitochondrial
precipitates observed may represent the nonexchangeable
pools of calcium. However, it is not known whether the
mitochondrial pools of calcium participate in any specific
cellular function other than regulating mitochondrial matrix enzymes [19]. Similarly, in Leydig cells, calcium may
enter the nonexchangeable pool to facilitate cAMP action
or may be involved in specific hormone action as suggested
by Sullivan and Cooke [3]. The presence of single precipitates within vesicular structures in both Sertoli and Leydig
cells suggests that they may represent "calciosomes" described in other non-muscle cells [20]. Absence of precipitates at the tight junctions between Sertoli cells and in the
submembranous regions of the plasma membrane is possibly due to the fact that calcium bound to the phospho-
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RAVINDRANATH ET AL.
FIG. 8. Electron micrograph of a pachytene spermatocyte from an adult rat testis exhibiting numerous nuclear calcium precipitates. x10 000. Other
precipitates can be seen in the mitochondria (M) and also unassociated with any organelle in the cytoplasm (arrows). The heterochromatin (H) and occasionally the synaptonemal complexes (S) also exhibit calcium precipitates. x12 000.
lipids of membranes does not form stable complexes with
pyroantimonate [12]. Sertoli cells, being secretory in nature, could be expected to possess calcium pools at the
plasma membrane region for exocytosis of secretory products. FSH and calcium have been implicated in the process
of exocytosis of proteins [5]. Annexins, a class of calciumbinding proteins, have been localized in these sites in other
cell types in the body. They form the link between microtubules, calcium, and membrane phospholipids [21].
The peritubular myoid cells (smooth muscle cell types)
are thought to provide structural integrity for the seminiferous tubules, and they interact in a paracrine manner with
Sertoli cells. They are also involved in tubule contraction
for the progression of spermatozoa along the lumen of
seminiferous tubules [22]. The presence of endoplasmic reticulum-bound deposits suggests that this exchangeable pool
of calcium may be involved in the contractility of myoid
cells. In support of this, the vasoconstrictor endothelin-1
binds to myoid cells and increases cytosolic free calcium
concentrations via the inositol phosphate pathway [4].
Among the germ cell populations, spermatogonia, unlike
the somatic cells, exhibited calcium deposits in nucleoli and
heterochromatin in addition to euchromatin. The significance of calcium in the nucleoli is not known. Mid-pachytene nucleoli actively engaged in ribosomal RNA synthesis
have been reported to display calcium-containing precipitates, particularly in the dense fibrillar component of nucleoli [14]. However, in our study, only spermatogonia exhibited deposits in the nucleoli. Pachytene spermatocyte
nucleoli were devoid of the precipitates.
The most interesting observation came from the screening of a large number of spermatids in sections from adult
FIG. 9. A) Electron micrograph of a round spermatid, at step 8 of development, from an adult rat testis showing large nuclear calcium deposits.
The Golgi apparatus (G), mitochondria (M), and vesicular structures (V) in
the cytoplasm exhibit calcium deposits. x12 000. B) Higher-magnification
electron micrograph showing single precipitates in the numerous vesicles
of the cytoplasm (V) and in the mitochondria (M). Precipitates unassociated
with any organelle were also seen in the cytoplasm. x15 000.
LOCALIZATION OF CALCIUM RESERVOIRS IN THE RAT TESTIS
59
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RAVINDRANATH ET AL.
LOCALIZATION OF CALCIUM RESERVOIRS IN THE RAT TESTIS
61
FIG. 11. Electron micrograph of a section of a Sertoli cell from an adult rat testis after treatment with EGTA (10 mM). The holes (arrowheads) within
the nucleus, mitochondria (M), and other parts of the cytoplasm represent the calcium precipitates chelated by EGTA. x20 000.
rat testis. The round spermatids showed large precipitates
within their nuclei. The acrosomal cap exhibited one or
two precipitates. A large number of vesicles containing a
single precipitate appeared in the cytoplasm. These could
be conveniently called "calciosomes," since calciosomespecific calreticulin protein has been identified in spermatids [23]. In elongating spermatids, two different types of
arrangement of calcium precipitates were seen: a diffuse
type of distribution within the nucleus and a peripheral
alignment of deposits along the inside of the nuclear envelope. The significance of this observation is not yet known.
Even though the calcium-binding proteins, annexins and
calreticulin, have been immunolocalized in the acrosome
[23, 24], our studies failed to localize calcium in the acrosome of elongating spermatids.
In conclusion, our results unequivocally demonstrate the
presence of sequestered calcium associated with the nu-
FIG. 10. A) Electron micrograph of a section of adult testis showing
elongating spermatids. This group of spermatids shows large nuclear calcium precipitates. x10000. B) Electron micrograph of a section of adult
testis showing another group of elongating spermatids with calcium deposits localized in'the nuclear envelope (arrowheads). x10,000.
cleus, mitochondria, and cytoplasm of different cell types
in the testis. The "calciosome"-like structures seen in Sertoli cells, Leydig cells, and round spermatids indicate that
an active exchangeable pool of calcium is present in these
cell types. The differential distribution of calcium precipitates in elongating spermatids may suggest different stages
of their development and function. Further characterization
of these calcium reservoirs within the cell will be necessary
to define the role played by calcium in different cellular
events.
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