BIOLOGY OF REPRODUCTION 52, 798-807 (1995)
Expression of Mitochondrial Heat Shock Protein 60 in Distinct Cell Types and Defined
Stages of Rat Seminiferous Epithelium'
ANDREAS MEINHARDT, 3 4' MARTI PARVINEN, 6 MICHAEL BACHER,5 GERHARD AUMULLER, 4
HARRI HAKOVIRTA, 6 AHMED YAGI, 6 and JORGEN SEITZ2 "4
Department of Anatomy and Cell Biology4 and Department of Experimental Immunology 5
Philipps University of Marburg, D-35037 Marburg, Germany
Department of Anatomy, 6 University of Turku, FIN-20520 Turku, Finland
ABSTRACT
Changes in the level of the gene transcript of heat shock protein (hsp)60, a mitochondrial chaperonin, during the cycle of
rat seminiferous epithelium and its cellular localization were studied. The seminiferous epithelium showed a cell type-specific
expression of hsp60. Immunostaining of adult rat testis revealed localization in Sertoli and Leydig cells. In germ cells, mitochondria of spermatogonia and early primary spermatocytes were immunoreactive for hsp6O. Mitochondria of all other germ cell
types were completely negative for hsp60. Stage-specific expression of hsp60 was determined from pooled segments of stagespecific microdissected tubules by a combination of Western blotting and polymerase chain reaction (PCR). High concentrations
of hsp60 were found in stages I-V and IX-XIV, and low levels were detected in the other stages, i.e., VI-VIII. In stages with high
hsp60 expression, spermatogonia divide mitotically, whereas in stages lacking mitosis, the hsp60 level was much weaker. In
seminiferous epithelium, two different types of mitochondria are present. Therefore, immunoelectron microscopy was used to
differentiate these two morphologically distinct types of mitochondria. The crista type of mitochondria (e.g., in Sertoli cells and
spermatogonia) reacted with the antibody against hsp60, whereas hsp60 was negative in so-called "condensed"-type mitochondria
found in midpachytene spermatocytes and more advanced germ cells. It could be shown for the first time that expression of the
hsp60 gene is regulated during the cycle of the seminiferous epithelium. The results indicate that the gene product is primarily
needed during the initial steps of spermatogenesis in which most of the cell divisions occur, while its expression during the
differentiation of spermatids and sperm is obviously not necessary. The presence of hsp60 in stages with mitotic activity suggests
a very active mitochondrial protein import and protein assembly machinery that generates further mitochondria for the dividing
cells.
INTRODUCTION
A heat shock is one of a large variety of stimuli characterized by cellular response in the form of increased synthesis of a small group of specific proteins known as heat
shock proteins [1]. Heat shock proteins (hsp) have been
found in a wide range of eukaryotic and prokaryotic cells.
Physiological data indicate that the production of hsp during stress is essential for cell survival and recovery from
stress [2]. Hsp are highly conserved during evolution [3].
Mammalian hsp exhibit extensive sequence similarities that
are consistent with those observed in related proteins from
yeast, prokaryotes, and plant chloroplasts (40-50% identical residues plus an additional 25-30% conservative replacement). This indicates that these proteins constitute one
of the most highly conserved groups of proteins known [3].
Hsp60 in eukaryotes and its bacterial homologue, GroEL,
function as a set of high-molecular-mass oligomeric strucAccepted December 2, 1994.
Received April 4, 1994.
'Support for this research was provided by grants from the Deutsche Forschungsgemeinschaft (DFG Se 370/4-2) and from the Academy of Finland (project no.
1071023).
'Correspondence: Dr. Jilrgen Seitz, Philipps Universitit Marburg, Institut fur Anatomie und Zellbiologie, Robert-Koch-Str. 6, 35037 Marburg, Germany. FAX: 49-6421/
28-5783.
'current address: Institute of Reproduction and Development, Monash University, Monash Medical Centre, Clayton, Victoria 3168, Australia.
tures. They are composed of two stacked seven-subunit rings
with ATPase activity [4]. The GroEL protein in E. coli functionally cooperates with groES, a ring-shaped complex of
seven 10-kDa subunits that inhibits the ATPase activity of
GroEL [5]. A homologue complex is localized in the mitochondrial matrix and plays an important role in mitochondrial protein import and protein assembly [6]. As a
member of the so-called "chaperonin" family, hsp60 is essential for the correct folding in the native structure of imported mitochondrial proteins [7, 8]. Folding may occur by
a step-wise process of ATP-dependent release from the oligomeric hsp60 or GroEL structure [9].
Spermatogenesis provides a good model for the investigation of regulation and sequential activation of genes
during differentiation. Well-defined synchronous stages with
the cell associations circling the tubule are most commonly
defined by morphology of the developing acrosomes and
the nuclei of the young spermatids [10]. Along the seminiferous epithelium, these stages follow one another in regular fashion, giving rise to the wave of seminiferous epithelium in most mammals. Spermatogenesis starts with the
mitotic amplification of germinal stem cells (spermatogonia), with some spermatogonia first undergoing six mitoses
and then moving on to proceed to meiosis. Subsequent to
the final mitotic step, spermatogonia differentiate into primary spermatocytes, undergoing further growth and meiotic
division followed by transformation of the resulting hap798
hsp60 GENE EXPRESSION IN TESTIS
loid spermatids into mature spermatozoa during a process
termed spermiogenesis. Germ cells, however, do not develop independently. Their differentiation is influenced by
specialized somatic cells (Sertoli cells, Leydig cells) through
paracrine and/or cell-cell interactions [11, 12].
Mitochondria in germ cells undergo rapid morphological changes during spermatogenesis. Mitochondria in prepubertal germ cells or adult spermatogonia do not differ
morphologically from those of somatic cells. During meiosis,
however, "condensed"-type mitochondria appear in pachytene spermatocytes. They are vacuolized and have a less
differentiated structure with poor or no cristae [13]. It has
been proposed that morphological changes of mitochondria coincide with those of function [14].
The present study addresses the distribution of mitochondrial hsp60 and its role in the various differentiation
and developmental steps of spermatogenesis. We have shown
a correlation between high levels of hsp60 expression and
mitotic stages in spermatogenesis.
MATERIALS AND METHODS
Immunocytochemistry
Adult rats were obtained from Charles River (Kislegg,
Germany). Animals were anesthetized with ether and killed
by cervical dislocation. Testes were removed and fixed for
approximately 24 h in Bouin's fluid, dehydrated, and
embedded in paraffin. Sections were cut at 5-Im thickness,
deparaffinized, and subsequently passed through decreasing concentrations of alcohol into water. They were then
immersed in PBS. Pretreatment with 3% H2 0 2 in PBS was
performed for 30 min to inactivate endogenous peroxidases. Tissue sections were incubated in blocking solution
(2% skimmed milk powder in PBS) in order to minimize
nonspecific antibody binding. Sections were rinsed in PBS
and incubated overnight at 40C with the primary polyclonal
antibody against hsp60 (1:800 in PBS); this was followed by
three washes in PBS containing 0.05% Tween and then incubation with swine anti-rabbit IgG (1:200, Dako, Copenhagen, Denmark) for 30 min at room temperature. Sections
were washed thoroughly with PBS/Tween and then incubated with the soluble peroxidase-anti-peroxidase (PAP)
complex (1:200 in PBS; Dako). Through use of 3,3'-diaminobenzidine (DAB) as chromogen (0.035% DAB and 0.015%
H2 02 in 50 mM sodium acetate buffer, pH 6.0), the enzyme
reaction was terminated after 5 min at room temperature.
After rinsing, sections were counterstained with hematoxylin, dehydrated, and then mounted in resin and coverslipped. Nonimmune serum and PBS were used as negative
control instead of as a primary antibody.
Rabbit anti-hsp60 antibody used in this study was gratefully received from Dr. F.U. Hartl (Laboratory of Cellular
Biochemistry and Biophysics, Rockefeller Research Laboratory, Sloan-Kettering Institute, NY).
799
Immunoelectron Microscopy
Small testis tissue samples were fixed in 0.05% glutaraldehyde and 4% paraformaldehyde in 0.05 M phosphate
buffer (pH 7.2) for 4 h. Tissue samples were dehydrated in
70% ethanol. The samples were incubated in 2:1, then in
1:1, and finally in at least 1:2 ethanol (100%):LR white resin
(v/v) for 30 min each at room temperature. Thereafter,
samples were infiltrated with pure LR white resin three times
for 1 h. Polymerization occurred at 4°C for 5 days under
UV illumination via a Grau HBO 100 (Plano, Marburg, Germany) lamp.
Ultrathin sections of 50-100-nm thickness were collected on formvar-coated nickel grids and blocked with 0.5%
egg albumin, 2% milk powder in PBS for 20 min. After
treatment with polyclonal rabbit anti-hsp60 antibody (1:10,
1 h at room temperature), sections were washed in 1% BSA,
0.05% Triton X-100, and 0.05% Tween 20 in PBS (pH 7.4)
and decorated with anti-IgG-coated gold particles (15 nm,
1:40; Biocell, Cardiff, UK) for 1 h. The sections were contrasted with uranyl acetate for 10 min and examined in a
Zeiss EM-10 electron microscope (Carl Zeiss, Oberkochen,
Germany).
Cell Fractionation
Adult rat testes were obtained as described above. Testes
were rinsed in ice-cold homogenization buffer (250 mM
sucrose, 5 mM 3-[N-morpholino]propanesulfonic acid
(MOPS), and 1 mM EDTA/0.01% ethanol; pH 7.4). All succeeding steps were carried out at 4C. The decapsulated
testes were cut into small pieces and homogenized carefully in 5 ml homogenization buffer in a Potter Elvehjem
(Braun Biotech, Melsungen, Germany) homogenizer (3
strokes). The homogenate was centrifuged (500 x g) for
10 min. The supernatant was collected, and the pellet was
resuspended in 2 ml buffer and homogenized again. Supernatants from both homogenizations were pooled and
centrifuged for 10 min at 1000 x g, removing intact cells,
cell debris, and nuclei. The supernatant was collected and
the mitochondrial fraction was precipitated by centrifugation at 12 000 x g for 20 min. The pellet containing mitochondria was washed twice with homogenization buffer.
The lysosomes were disrupted by incubation for 30 min by
gentle stirring in ice-cold hypotonic buffer (20 mM sodium
phosphate, 10 mM EDTA; pH 7.4). Mitochondria were pelleted again (12 000 x g for 20 min) and resuspended in
20 mM phosphate buffer (pH 7.4). For subfractionation, mitochondria were sonicated on ice at four 20-sec intervals
(Braun) interrupted for 1 min to prevent warming of the
sample. Samples were transferred to Eppendorf cups and
centrifuged (14 000 x g for 20 min). Supernatant containing the mitochondrial matrix and a pellet (mitochondrial
membranes) were separated for further studies. Protein
concentration was determined according to Bradford [15].
Both fractions were also prepared for SDS-gel electrophoresis.
800
MEINHARDT ET AL.
Both the enhanced chemoluminescence system (ECL system; Amersham, Braunschweig, Germany) and the DAB
method were used as detection systems. Developed x-ray
films (ECL system) were used to permit quantification of
different protein concentrations in the microdissected stages
with the System Scan Pack (Biometra, Gottingen, Germany)
quantification system.
RNA Preparation
FIG. 1. Western blots of mitochondrial subfractions of rat testis. Samples (10 ILg)of mitochondrial matrix proteins (lane A) and mitochondrial
membrane proteins (lane B) were run on 10% and 7.5%-20% SDS-PAGE,
respectively. Protein bands were transferred to nitrocellulose membranes.
Related bands were detected with polyclonal rabbit antibodies against hsp60
(lane A) and COx-IV (lane B). A specific reaction was achieved at 60 kDa
for hsp60 (lane A) and at 17 kDa for COx-IV (lane B). The peroxidase-DAB
reaction was used for visualization. Molecular mass markers are indicated
in kDa.
Transillumination-AssistedMicrodissection of the
Seminiferous Tubules
Testes of 3-5-mo-old Sprague-Dawley rats were used.
Testes were decapsulated and placed in PBS solution on
ice. Different stages were determined under transillumination light according to light absorption criteria as previously described [16]. Segments containing at least one
complete wave of the seminiferous epithelium without obvious modulations were selected and cut sequentially at 2mm intervals [17, 18]. Pools at stages I, II-III, IV-V, VI, VlIab,
VIIcd, VIII, IX-XI, XII, and XIII-XIV were collected (total length
used for Western blot was 8 cm; for RNA analysis total length
was 9 cm). The wet weight of 1 cm of the tubule was approximately 1 mg.
SDS-PAGE and Western Immunoblotting
SDS-PAGE was performed as described by Laemmli [19]
using 4% stacking and 10% resolving slab gels. To summarize, the samples were divided into equal amounts and
loaded on two gels: one for analysis of hsp60, the other for
cytochrome-c-oxidase subunit IV (COx-IV) as reference (the
antibody was generously supplied by Prof. B. Kadenbach,
Marburg, Germany). Samples were diluted 2:1 in reducing
standard sample buffer [19] containing 5 mM dithiothreitol
before being loaded on the gel. Proteins were transferred
onto nitrocellulose membranes using a Bio-Rad Labs. (Richmond, CA) transblot cell [20]. Nitrocellulose membranes were
incubated with the polyclonal rabbit antibodies against hsp60
(1:800) or COx-IV (1:300), followed by treatment with swine
anti-rabbit immunoglobulin (1:100) and PAP complex (1:200).
Frozen microdissected tubule segments were obtained
from transillumination-assisted microdissection (see above).
They were disrupted in 4 M guanidium isothiocyanate, 0.025
M sodium citrate (pH 7.0), and 0.1 M beta-mercaptoethanol
[21] and extracted in phenol-chloroform. RNA was precipitated with isopropanol (1:2 [v/v]) and resuspended in sterile water. RNA concentration was determined spectrophotometrically at 260 nm [22].
Polymerase Chain Reaction (PCR)
PCR was started with 1 g of the total RNA from each
tissue aliquot studied. First strand cDNA synthesis was performed as described by Lee and Caskey [23]. After denaturation of the mRNA-cDNA duplex (10 min at 95°C, then
quenching on ice), 2 Rzl of the cDNA reaction mixture was
used as a template for PCR according to Innis et al. [24]. A
46
.5-1l PCR reaction mixture containing 2 l cDNA, 5 l
PCR buffer (10-strength), and 39.5 l1 sterile water was processed on a Hybaid Omni Gene (MWG Biotech, Ebersberg,
Germany) programmable block. After an initial denaturation step for 20 min at 95°C, the following substances were
added to the reaction mixture to a final volume of 50 l:
1 l1each of sense and antisense primers (50 pmol/l), 1
,l dNTPs (10 mM each nucleotide), and 0.2 al Taq polymerase (1 U; Pharmacia, Freiburg, Germany). Twenty cycles
were performed, each consisting of denaturation at 95°C
for 1 min, primer annealing at 68°C for 1 min (hsp60) or
600C (GAPDH), and primer extension at 72°C for 1.5 min.
After 20 cycles, the reaction was continued at 72°C for 5
min. PCR products were analyzed on 1.5% agarose gels in
single-strength TBE (90 mM Tris-HCl, 90 mM sodium tetraborate, and 2 mM EDTA) and stained with ethidium bromide as described by Sambrook et al. [22].
Rat GAPDH primers [25] were purchased from MWGBiotech (Ebersberg, Germany): 5' sense-5'-CGTCTTCACCACCATGGAGA; 5' antisense-5'-CGGCCATCACGCCACAGITT; 307-bp fragment.
Rat hsp60 primers [3] were synthesized by IMT (Marburg, Germany): 5' sense-5'-GCGGATGCTCGAGCCTTAAT; 5' antisense-5'-ATGACCAAGGGCTTCCGGTG; 702bp fragment.
Slot-Blot Hybridization and DensitometricAnalysis
To permit quantification of gene expression, 10- 1 l aliquots from the log phase of PCR were collected after in-
801
hsp60 GENE EXPRESSION IN TESTIS
FIG. 2. Western blot analysis of seminiferous tubule segments microdissected from defined stages of the epithelial cycle. Total cellular protein (each lane contains 10 Lg protein) was resolved on 10% SDS-PAGE, blotted onto
a nitrocellulose membrane, and probed with the antibodies against hsp60 (A) and COx-IV (B). Molecular masses are
indicated in kDa. Immunodetection was performed on x-ray films using ECL to permit signal quantification.
tervals of 10, 15, and 20 cycles. DNAs were transferred [26]
onto nylon membranes (Hybond N; Amersham Buchler,
Braunschweig, Germany) using a Many-fold II filtration unit
(Schleicher & Schiill, Dassel, Germany). Membranes and
DNA were UV-crosslinked using a Fluo-Link crosslinker (1.29
J/cm 2 ; MWG-Biotech). DNA probes were labeled with digoxigenin according to the manufacturer's guidelines (digoxigenin labeling kit; Boehringer-Mannheim, Mannheim,
Germany). Membranes were prehybridized in sealed plastic bags for at least 1 h at 42°C in hybridization solution
before incubation with the probes in fresh hybridization
solution at 42°C for 18 h as described by Bacher et al. [271.
The membranes were exposed to x-ray films at room temperature for 2 h. DNA concentrations were quantified using
the commercially available System Scan Pack (Biometra).
Protein (see above) and DNA autoradiograms were analyzed for the relative intensity of signals by densitometry.
Autoradiograms were put through a densitometer (System
Scan Pak, Biometra) three times each with the axis of absorbance normalized. Intensity of DNA- and protein-derived signals of hsp60, COx-IV, and GAPDH were obtained
by integrating the areas of the absorbance peaks combined
with measurements of their relative densities. Background
subtraction was performed according to the manufacturer's
guidelines.
All experiments were repeated five to six times. Data are
presented as the mean + SEM.
RESULTS
Western Blotting
Specificity of the two polyclonal antibodies used in the
present work was verified by Western blotting of isolated
rat testis mitochondrial matrix (anti-hsp60-Ab; for antibody
specificity see Ostermann et al. [91) and mitochondrial
membrane proteins, respectively (anti-COx-IV-Ab). The antibody against hsp60 gave a single band at 60 kDa (Fig. 1A),
and the anti COx-IV antibody yielded a band at 17 kDa (Fig.
1B). Differences in the hsp60 protein level were studied
through the use of pooled isolated stages of 10 pools of
microdissected segments of seminiferous epithelium. Immunoblotting using the antibody against hsp60 showed a
single band of hsp60 at all stages of the cycle of seminiferous epithelium (Fig. 2A). The lowest levels of hsp60 were
found in stages VI-VIII. Highest levels in hsp60 were found
in stages I, II-III, IV-V, and I-XIV (Figs. 2A and 6A). Highest
levels of hsp60 correlate with stages at which A and intermediate spermatogonia divide mitotically (stages I, IV, IX,
XII, and XIV). Identical pools of microdissected tubule segments were studied with the antibody against COx-IV (Figs.
2B and 6B). This ubiquitous mitochondrial protein showed
a prominent presence throughout all stages examined without any obvious variations.
802
MEINHARDT ET AL.
Immunohistochemical Localization of hsp60 in
Seminiferous Epithelium
Localization of hsp60 was studied immunohistochemically in paraffin sections of adult rat testis (Fig. 3). In germ
cells, hsp60 was found in the basal portion of seminiferous
epithelium. Spermatogonia were always immunoreactive,
most of them exhibiting an intense spotted intracellular labeling pattern (Fig. 3). In primary spermatocytes, immunoreactivity became apparent as early as stage VII in preleptotene spermatocytes. Zygotene spermatocytes were
labeled from stage XII to XIII, whereas in pachytene spermatocytes the immunoreactivity decreased and only very
early stages (up to stage IV-V) were stained (Fig. 3). The
intracellular staining pattern was granular, reflecting the aggregation of mitochondria in small clusters. Other spermatogenic cells including mid- and late pachytene primary
spermatocytes, secondary spermatocytes, spermatids, residual bodies, and sperm were completely negative for hsp60
at all stages (Fig. 3). In Sertoli cells, the immunoreaction
was found to be most intense in the apical cell processes,
the adjacent germ cells (e.g., spermatids) being negative. A
consistent result was the strong positive immunoreactivity
of Leydig cells. Peritubular cells were negative for hsp60.
Immunogold Electron Microscopy
Ultrathin sections were stained through use of the immunogold method, which revealed a clear-cut difference
between morphologically distinct types of mitochondria
present in testis. Mitochondria of the usual crista type present in Sertoli cells, spermatogonia, and early primary spermatocytes were hsp60-positive (Fig. 4). Figure 4A illustrates
the decoration of cristae or "orthodox"-type mitochondria
in Sertoli cells with the gold particles. During the time when
mitochondria modify their morphological organization
[28, 29] to the "condensed" type, these organelles remained
unstained or were labeled at background level (Fig. 4B).
This process is initiated in mid-pachytene spermatocytes,
where the inner space with its dense matrix is flattened
against the outer membrane by a considerable expansion
of one or more intercristal spaces (Fig. 4B).
Expression of HSP60 mRNA during Spermatogenesis
FIG. 3. Immunohistochemical analysis of hsp6O protein in paraffin
sections of rat testis. Sections (A, B) were counterstained with hematoxylin.
Panel A shows two tubules at stage IV-V. Long arrows indicate pachytene
spermatocytes stained with the hsp60O antibody. B) Pachytene spermatocytes (long arrow) do not stain with the anti-hsp60 antibody in stage VII
tubules; short arrows point to immunopositive preleptotene spermatocytes. Negative control with nonimmune serum was not counterstained
(panel C). Magnifications are x32 (A, B) and x16 (C).
The level of hsp60 mRNA expression was studied with
PCR before quantitation of data by slot-blot analysis. Because the total RNA amount prepared from microdissected
pools of the spermatogenic wave was too low for a direct
analysis of hsp60 by Northern blotting, a previous amplification by PCR was performed. Slot blotting was used for
quantification. The PCR products were analysed by agarose
gel electrophoresis. Hsp60O mRNA was expressed at all stages.
A strong signal was found in stages I, II-V, IX-XI, XII, and
XIII-XIV (Figs. 5A and 6C). The other stages investigated
exhibited a weak signal; these included stages VI and VIIab,
VIIcd, and VIII. The hsp60 mRNA expression level was the
803
hsp60 GENE EXPRESSION IN TESTIS
FIG. 4. Immunoelectron microscopic analysis of hsp60O protein in adult testis. A) Labeled mitochondria in a Sertoli
cell. B) The matrix of condensed mitochondria (arrows) shows a decrease in volume as consequently the volume of
the intercristal space increases, often occupying as much as 60% of the total mitochondrial volume. Bars, 500 nm.
same as revealed for-hsp60 protein. This means that there
was correlation with the stages at which mitosis of the different types of A and intermediate spermatogonia occurred
(stage I, dividing A4 spermatogonia; stage IV, dividing intermediate spermatogonia; stage IX, Al spermatogonia in
mitosis; stage XII, A2 spermatogonia in cell division; stage
XIV, A3 spermatogonia in mitosis). The control reaction was
run using the constitutively expressed GAPDH mRNA; an
almost equal level of expression was found in the sper-
matogenic cycle, proving that approximately equal amounts
of mRNA had been applied (Fig. 5 and Fig. 6, C and D).
DISCUSSION
In the present study, we investigated the localization,
mRNA expression, and protein level of hsp60, a mitochondrial chaperonin, in rat testis. We found that the localization
of this heat shock protein is restricted to distinct cell types
FIG. 5. Agarose gel of PCR amplification products of rat hsp6O cDNA (A) and GAPDH cDNA (B) in segments of
defined stages of seminiferous epithelium. First strand cDNA synthesis was followed by 20 amplification cycles. The
cDNAs of the defined stages were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized
in UV light. The hsp60 PCR product was a 702-bp cDNA fragment (A), and the GAPDH cDNA fragment was 307 bp
long (B). Lane C (negative control): PCR without template DNA. M: 100-bp ladder.
804
MEINHARDT ET AL.
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FIG. 6. Relative levels of hsp60 protein (A) and hsp60 mRNA (C)obtained from autoradiograms of Western blots
and slot-blot hybridization (slot-blot not shown; see Materials and Methods). In A, the levelof hsp60 protein was
compared to the constitutively present protein COx-IV. In C,the amount of hsp60 mRNA was related to GAPDH mRNA
as a loading control (D). Highest concentrations of hsp60 were found in stages I-V and IX-XIV. Data are expressed as
mean - SE of five to six experiments.
hsp60 GENE EXPRESSION IN TESTIS
(Leydig, Sertoli, and early spermatogenic cells). This is consistent with the observation that hsp60 is not expressed in
germ cells with "condensed"-type mitochondria. High levels of hsp60 expression were found, however, in stages of
the cycle of seminiferous epithelium, where mitosis of
spermatogonia, mainly the different forms of A spermatogonia, occurs (stages I, IV, IX, XII, XIV).
Spermatogenesis in the seminiferous tubules can be divided into three main phases: (i) spermatogonial multiplication, (ii) meiosis, and (iii) spermiogenesis. Spermatogonia differentiate and proliferate through six mitotic divisions
and one meiotic DNA synthesis, all of which are located in
defined stages of the cycle of the seminiferous epithelium
[30]. The stages can be recognized by their transillumination pattern and become available by microdissection for
biochemical and molecular biological analysis [16,17].
Western blotting as well as PCR analysis followed by
quantification showed that hsp60 expression obviously differs depending on the stages of the cycle of seminiferous
epithelium. High levels were found in stages IX-XI, XII, XIIIXIV, I, and II-V (Figs. 2, 5, and 6). In control experiments,
the ubiquitous mitochondrial protein COx-IV was analysed
within the same pool of samples. These showed an equal
level of COx-IV antigen at all stages (Fig. 2B). Similar resuits were obtained with the constitutively expressed GAPDH
gene in PCR analysis. A nearly identical level of GAPDH mRNA
expression indicated the presence of comparative concentrations of total RNA in the investigated samples (Fig. 5 and
Fig. 6, C and D). High levels of hsp60 expression correlated
with those stages in which A and intermediate spermatogonia divide mitotically (stages IX, XII, XIV, I, and IV). Type
A spermatogonia have been postulated to provide the stem
cell population [30]. Their high level of proliferation is
thought to provide a significant contribution to the stagespecific expression of hsp60 mRNA. Mitochondria import
new proteins and are divided by fission to provide new
organelles for the daughter cells. As a prerequisite, hsp60
has to be expressed and imported into mitochondria to ensure correct refolding and assembly of the proteins temporary denatured during import [8]. At stages IV and VI,
other types of spermatogonia undergo mitosis (stage IV: intermediate spermatogonia develop into B spermatogonia;
stage VI: B spermatogonia form preleptotene primary spermatocytes); however, no significant increase in hsp60 level
could be detected at stage VI. It is, therefore, evident that
B spermatogonia as well as preleptotene spermatocytes,
which are present at stages VI-VIII, can correctly assemble
imported mitochondrial proteins but do not transcribe the
hsp60 gene at the high levels typical for stages at which A
and intermediate spermatogonia proliferate. Shakoori et al.
[31] also found maximal hsp60 levels during active proliferation in osteoblasts and promyelotic leukemia cells and
a subsequent decline post-proliferatively. Lu and Seligy [32],
however, observed completely opposite effects on the level
of hsp60 mRNA expression when inducing differentiation
805
in two diverse cell lines; this suggests significant differences
in the involvement of mitochondria in the differentiation
of these cell lineages. This might provide an explanation
for the high hsp60 protein concentrations of stages II-III,
which are comparable to those of stages I and IX-XIV (Fig.
2A), although no mitosis occurs in stages II-III.
Numerous studies have revealed that mitochondria
undergo dramatic morphological changes during spermatogenesis [14, 29,33]. The mitochondria in A spermatogonia are ovoidal in shape and have lamellar cristae characteristic of the "crista" or "orthodox" appearance. In
leptotene spermatocytes, the space delimited by the' two
membranes of lamellar cristae increases in size. In pachytene spermatocytes, the matrix is flattened against the outer
membrane, displaying the "condensed" type. In spermatids, the "condensed" mitochondria gradually develop more
numerous and convoluted cristae. Parts of the mitochondria show the tendency to move towards the flagellum to
later become incorporated in the sperm midpiece. The remaining mitochondria leave mature spermatids or spermatozoa within the residual bodies [14]. Our results give
rise to the hypothesis that the hsp60-depleted "condensed"
mitochondria have lost the ability to import and refold freshly
synthesized proteins. Because the intact hsp60 complex is
required for proper refolding and assembly of imported
mitochondrial proteins [9,34], it is concluded that mitochondria from mid-pachytene spermatocytes and more advanced spermatogenic cells do not depend on an efficient
import machinery. But why should these cells get rid of a
central part of the factors that they require for import and
assembly of mitochondrial proteins? The following facts may
explain this unusual disappearance of hsp60 in germ cells.
Because of the blood-testis barrier, transport of essential
nutritional components between Sertoli and germ cells is
critical for germ cell survival and metabolism [35]. Sertoli
cells provide, e.g., coenzymes, nucleotides, amino acids,
lactate, and pyruvate as energy substrates to the spermatogenic cells sequestered in a serum-free microenvironment [36]. Mitochondria in cells of the adluminal compartment are not the unique source of newly synthesized ATP,
because anaerobic glycolysis plays an important role in the
metabolic pathways of pachytene spermatocytes, spermatids, and sperm [37]. The testis-specific type of lactate dehydrogenase appears earlier, but it increases in abundance
after meiosis [38]. This enzyme plays a crucial role in providing energy from anaerobic glycolysis occurring in the
cytoplasm. Subsequently, as observed in the current study,
hsp60 is present only in cells having more or less direct
access to components of the circulation. Later in spermatogenesis, germ cells depending on metabolites provided from
Sertoli cells (lactate, pyruvate) change in their mitochondrial ultrastructure into the so-called "condensed" type and
are negative for hsp60 (Fig. 4).
Furthermore, male germ cells do not contribute to the
mitochondrial pool in the zygote. In developing sperma-
806
MEINHARDT ET AL.
tozoa, one portion of mitochondria is degraded within residual bodies. Mitochondria of the mature sperm midpiece
remain excluded from entering the egg. In relation to a
very limited postmeiotic haploid gene expression, where
no mitochondrial protein is known to be expressed from
spermatids on [39], it is likely that developing germ cells
degrade parts of the mitochondrial protein import machinery, as the latter are obviously not destined to become part
of a new generation.
Hsp70 is another example of differential gene expression during spermatogenesis. The testis-specific isoform of
hsp70 (hst70) was localized in the cytoplasm of late primary spermatocytes and early spermiogenesis. High levels
were found in stages XII to early VII, and an involvement
in the differentiation of spermatocytes into spermatids was
speculated upon [40]. Hsp60O is also precisely regulated in
the cycle of the seminiferous epithelium and is no longer
detectable from early pachytene spermatocytes onward.
Several proteases located in the mitochondrial matrix or
their bacterial homologues are known to degrade a number of proteins [41]. These proteases are an important component of developmental pathways [42]. Mitochondrial proteins display heterogeneous rates of degradation (half-lives
of 1 to 40 h) that vary according to the physiological state
of the cell [43]. It is likely that the alterations in structure
and composition of mitochondria in response to regulatory
signals or changes in nutritional conditions may depend on
the differential degradation of preexisting proteins and the
synthesis of new ones.
ACKNOWLEDGMENTS
We thank Dr. K.P. Radtke for critical reading of the manuscript and Jim McFarlane
for editorial help. The excellent technical assistance of Mrs. T. Seitz, Mrs. I.
Dammshauser, Mr. G. Jennemann (University of Marburg, Germany), and Mr. L.H.
Wikgren (University of Turku, Finland) is gratefully acknowledged.
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