Human Reproduction Update 1999, Vol. 5, No. 2 pp. 108–119
European Society of Human Reproduction and Embryology
Mini symposium
New aspects of spermatogenesis
Expression of mitochondrial marker proteins
during spermatogenesis
Andreas Meinhardt, Beate Wilhelm and Jürgen Seitz1
Department of Anatomy and Cell Biology, Philipps-University of Marburg, Robert-Koch-Str. 6, D-35037 Marburg, Germany
Spermatogenesis is a highly complex, hormonally regulated cytodifferentiation process finally leading to the production
of spermatozoa. In addition to other events germ cell differentiation is characterized by a gradual structural modification of many organelles including mitochondria which play a unique role. The morphological and functional development of germ cell mitochondria is a reflection of the permanent change in the testicular microenvironment which occurs
when the germ cells are slowly moving from the base of the seminiferous epithelium to the lumen. Concomitant with the
structural changes, several mitochondrial proteins are known to be expressed and synthesized during distinct phases of
the organelle’s development. This review pays particular attention to these transiently expressed mitochondrial proteins
such as hsp60, Lon protease, sulphydryl oxidase and cytochrome ct. Furthermore, the biological function of this stepwise
gene activation during mitochondrial and germ cell development is discussed.
Key words: chaperones/Lon protease/mitochondria/spermatogenesis/sulphydryl oxidase
TABLE OF CONTENTS
Introduction
Introduction
108
Structure and function of mitochondria
109
Mitochondria in germ cells
109
Heat shock proteins
110
Mitochondrially encoded polypeptides
110
Import and processing of nuclear-encoded mitochondrial
polypeptides
111
Cytoplasmic hsp70 (ct-hsp70) and mitochondrial
hsp70 (mt-hsp70)
111
Mitochondrial hsp60 (mt-hsp60)
112
ATP-dependent Lon protease
113
Sulphydryl oxidase
114
Paracrine factors
115
Conclusions
115
Acknowledgements
117
References
117
According to the endosymbiotic hypothesis, prokaryotes such as
the ‘purple non-sulphur bacteria’ are regarded as the phylogenetic progenitors of mitochondria (Margulis, 1970). This
theory is based mainly on the fact that mitochondria, like
bacteria, contain their own circular DNA and that many
mitochondrial proteins display a very conservative molecular
structure with a high homology to the respective bacterial
proteins. Furthermore, the mitochondria of eukaryotic cells are
generated by cleavage of existing organelles as a direct reaction
to mitotic cell divisions or metabolic alterations. Mitochondria,
therefore, are potentially immortal and continue to exist in the
descending daughter organelles which complete their divided
pool of proteins by an increased expression rate of both
mitochondrially encoded proteins and by the import of nuclear
encoded polypeptides from the cytoplasm. Import and
maturation of these polypeptides is assisted essentially by a
variety of evolutionarily conserved chaperones which show
1To whom correspondence should be addressed at: Institut für Anatomie und Zellbiologie, Robert-Koch-Str. 6, D-35033 Marburg, Germany. Tel:
+49-6421-284001; Fax: +49-6421-285783; E-mail: [email protected]
Mitochondrial differentiation in male germ cells
high homology to the respective bacterial proteins. Also,
mitochondria of the mitotically dividing spermatogonia in the
testis have to be regenerated in order to achieve a full set for
both resulting cells. In the basal compartment of the
seminiferous tubules the spermatogonia have free access to all
of the substances in the intertubular space which originate from
the circulation and the lymph vessels. Although peritubular
cells in some cases can restrict this supply, oxygen, nutrients
and mediators directly influence the metabolism of the stem
cells, providing the biochemical prerequisites for their proliferation (i.e. A spermatogonia). A subpopulation of the spermatogonia, however, only differentiates after further mitotic
divisions via an intermediate form to B spermatogonia and
finally to primary spermatocytes. They later enter meiosis and
are transferred into the apical or luminal compartment. The
border between both compartments comprises the blood–testis
barrier which consists of a tight cell–cell adhesion complex
between neighbouring Sertoli cells (desmosomes and tight
junctions) that cannot be passed by most substances from the
interstitial space, particularly proteins. Consequently, the supply of meiotic and post-meiotic germ cells as well as the control
of their further differentiation is indirectly under Sertoli cell
control.
This dramatic alteration in the surrounding milieu causes a
substantial change in the energy metabolism of the germ cells.
The spermatogonia are able to be nurtured entirely by blood
components, utilizing aerobic pathways for energy production. After passage into the luminal compartment, energyconsuming processes including RNA and protein synthesis
can be fuelled only to a limited extent by glucose turnover.
The germ cells now rely on the breakdown of lactate and
pyruvate provided by the Sertoli cells. This reflects an indirect, yet essential, influence on energy metabolism during
spermatogenesis (Grootegoed et al., 1984) which is accompanied by obvious morphological changes and a sequential expression of different mitochondrial proteins such as hsp60,
Lon protease, sulphydryl oxidase and cytochrome ct.
Structure and function of mitochondria
Mitochondria comprise on average 15–22% of the total cellular volume and deliver 90% of the energy required. Energy is
generated by oxidative phosphorylation resulting in the
formation of ATP, the common currency of energy in all living
cells. Depending on the cell type and the respective functional
status, mitochondria can change their morphology, their location in the cell, fuse to larger units or separate (Bereiter-Hahn
and Vöth, 1994). The total number of these organelles per cell
also varies widely. Some cells contain only a single mitochondrion, whereas male germ cells have 2000–3000, and oocytes
hold >90 000 (Hecht, 1992). Ultrastructural analysis revealed
round-shaped, ellipsoid, cylindrical and filament-like structures of mitochondria. All mitochondria are generally composed of two membranes separated by an intermembrane
109
space. These membranes are joined at numerous ‘contact
sites’, where protein translocation across the membranes in
the matrix occurs. The outer membrane of normal (= orthodox) mitochondria is smooth while the inner membrane is
usually highly convoluted, forming a series of infoldings of
the matrix, known as cristae, which greatly increases their
total surface area. The cristae can form tubular, vesicular,
crest-like or prismatic structures (Bereiter-Hahn and Vöth,
1994). It has also been found that mitochondrial morphology
depends on the fixation technique used to examine the cells
(Bereiter-Hahn, 1990). The tomographic investigations of
Manella et al. showed that the cristae of the orthodox organelle, usually extending for 500–600 nm, are not associated in
parallel bundles as often displayed in many textbooks (Manella et al., 1994). Rather they are found in various orientations
within the matrix. The cristae are open to the intermembrane
space, but have no connections in the matrix, where the breakdown of fatty acids and the intermediates of the glycolysis
occurs. A change in the metabolic rate such as a decreased
oxidative respiration caused by oxygen withdrawal, an increase of intracellular ADP concentration, an inhibition of
glycolysis or the influence of toxins such as rotenon, antimycin or potassium cyanide (KCN) all result in substantial reductions of the intracellular movement as well as alterations of the
ultrastructure. This results in the bright electron-permeable
matrix condensing, becoming electron-dense and staining
darkly. The ‘intracristal spaces’ swell considerably and are
partly connected via ‘tubular extensions’ (Hackenbrok, 1966,
1968; Hackenbrok et al., 1971; Bereiter-Hahn and Vöth,
1983) ultimately changing the mitochondrial morphology
from the orthodox to the condensed type according to the
terminology of Hackenbrok (Hackenbrok 1966, 1968).
Mitochondria in germ cells
Numerous reports have recognized that the mitochondria of
germ cells modify their morphological organization, number
and location during the processes leading to sperm production
(André, 1962; Fawcett, 1970; Machado de Domenech et al.,
1972; De Martino et al., 1974; Kaya and Harrison, 1976;
Hecht, 1995). De Martino et al. (1979) described the morphological, histochemical and biochemical events of mitochondrial differentiation in the course of rat spermatogenesis
(De Martino et al., 1979). First in A-spermatogonia as in most
somatic cells the organelles are orthodox, e.g. ovoid shaped,
containing lamellar cristae and an electron translucent matrix.
The intracristal space begins to dilate in some B-spermatogonia and more prominent during the transition to leptotene
primary spermatocytes. In zygotene and early pachytene spermatocytes the mitochondria elongate and are located in close
proximity to the outer nuclear membrane with their number
steadily increasing. Studies by De Martino et al. show that this
is an indication of an active functional state of mitochondria
during this phase of development (De Martino et al., 1974,
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A.Meinhardt, B.Wilhelm and J.Seitz
1979). Mitochondria form clusters in mid and late pachytene
spermatocytes with an electron-dense material, the nuage,
between them (Fawcett, 1970; Russel and Frank, 1978). Experiments performed by Söderstrom and Parvinen demonstrated novel RNA synthesis in the nuage (Söderstrom and
Parvinen, 1976). Furthermore, Moussa et al. localized essential proteins of the small nuclear ribonucleoprotein particles
(snRNP) in this compartment. This can be interpreted in two
different ways (Moussa et al., 1994). On the one hand the
surrounding mitochondria provide ATP for energy-consuming processes in the nuage such as a modification of the
snRNA–snRNP complex. On the other hand it is possible that
the transcribed mitochondrial pre-RNA strands are exported,
modified by the splicosome in the nuage and subsequently
re-imported into the mitochondria. However, no proven experimental data support either of this hypotheses.
During this phase of meiosis the mitochondria are roundshaped and relatively small with the electron-dense matrix flattened to the outer part of the organelle. The intracristal space is
clearly dilated. The functional status of condensed mitochondria
is unclear. Due to their location beyond the blood–testis barrier,
pachytene spermatocytes receive metabolites which provide less
energy than glucose, such as lactate. Furthermore, this supply is
substituted only indirectly by the Sertoli cells. This causes a
substantial reduction of the respiratory chain activity, resulting in
a decreased oxidative phosphorylation activity and therefore decreased ATP synthesis. However, mitochondria isolated from
pachytene spermatocytes showed an enormous rate of new ATP
synthesis when incubated in normoxic conditions and in the
presence of high concentrations of ADP. This demonstrates their
latent ability to have a normal oxidative phosphorylation turnover
rate (De Martino et al., 1979).
In diplotene and secondary spermatocytes the mitochondria
remain small, round-shaped and condensed. However, mitochondria are not arranged in clusters. In contrast to liver mitochondria no expansion of the intermembrane space is observed.
With the differentiation of germ cells to spermatids a translocation of the organelle beneath the plasma membrane is apparent.
This points to a high energy demand due to an intense exchange
of metabolites with the neighbouring Sertoli cells. However, a
group of elongating and dividing organelles is still visible in the
interior of the cell. From step 8–10 spermatids onwards, approximately, the condensed mitochondria develop crescent-shaped
cristae and the matrix is less condensed. This is defined as the
intermediate organelle form (intermediate spd). Some of them
show a tendency to move in the direction of the developing
flagellum, whereas the remaining mitochondria progressively
group together (De Martino et al., 1974, 1979). These mitochondria leave the developing spermatozoa in the residual bodies and
are probably phagocytosed by the Sertoli cells. In later stages of
development the expanded intracristal spaces of the flagellar mitochondria reduce their size and the cristae distend themselves
into the interior of the organelles. Simultaneously these mitochondria begin to elongate and fuse to a chain-like and twisted
structure around the flagellum. In spermatozoa the cristae develop gradually forming a packed concentric system of membrane
and matrix. In this manner the middle piece mitochondria lose the
morphological appearance typical for the condensed form. At
this stage of sperm maturation the mitochondria reach their maximal elongation and assume a close relationship with the outer
dense fibres of the middle piece (De Martino et al., 1974, 1979).
Finally, the mitochondria are arranged in a helix of 11–13 gyri,
with two mitochondria per gyrus, and deliver ATP to the axoneme for flagellar propulsions.
Heat shock proteins
Heat shock proteins (hsp) have been found in a wide range of
eukaryotic and prokaryotic cells. Heat shock is one of a large
variety of stimuli characterized by a cellular response in the form
of an increased synthesis of a small group of specific proteins, the
heat shock proteins. Physiological data indicate that the production of hsp is essential for cell survival and recovery from stress
(Parsell and Lindquist, 1993). Hsp are amongst the most conserved proteins known (Morimito et al., 1994), and are found in
numerous cell organelles such as the cytoplasm (ct-hsp), the
endoplasmic reticulum (ER), chloroplasts, mitochondria (mthsp) and the nucleus (nuc-hsp), and are classified in hsp families
according to their respective molecular weight: hsp100 (Clp
A,B,C), hsp90 (HtpG), hsp70 (DnaK), hsp40 (DnaJ), hsp60
(GroEL), hsp10 (GroES), hsp23 (GrpE), hsp20 (Ibp A,B), and
hsp8.5 (ubiquitin) family (names in parentheses refer to the term
used for prokaryotes).
Most of the hsp are also expressed by normal (unstressed) cells
where they usually function as molecular chaperones. Chaperones are molecules that support or enable the correct folding and
assembly of newly synthesized proteins, their oligomerization
and their intracellular translocation in mitochondria, peroxisomes
or in the nucleus (Gething and Sambrook, 1992; Georgopoulos
and Welch, 1993; Hartl et al., 1994; Hartl and Martin, 1995).
In the testis the chaperones ct-hsp-90, ct-hsp70, mt-hsp70,
mt-hsp60 and nuc-hsp70 fulfil functions central to the posttranslational maturation and translocation of newly synthesized
polypeptides. They can also act in the heat protection of the germ
cells at temperatures above 35°C by binding unfolded or partially
folded proteins to prevent their aggregation or irreversible thermal denaturation. However, a long-term temperature increase
above 35°C inevitably leads to male infertility (Sarge and Cullen,
1997). Zakeri and Wolgemuth showed that members of the
hsp70-family (hsc70) are also expressed post-meiotically, probably to supply spermatids with a heat protection system (Zakeri
and Wolgemuth, 1987).
Mitochondrially encoded polypeptides
Mitochondrial proteins are synthesized in two different ways.
Firstly, each organelle contains several copies of circular doublestranded DNA in the matrix coding for 37 genes. In human cells
Mitochondrial differentiation in male germ cells
both strands of the mitochondrial DNA (16 569 bp) are transcribed at the same rate, producing two different giant RNA
molecules, each containing a full-length copy of the one DNA
strand. The transcripts made of the strands are extensively processed by nuclease cleavage to yield 13 mRNA for translation,
two ribosomal RNA (12S and 16S) and 22 transfer RNA molecules necessary for mitochondrial protein synthesis. The transcription is constitutive and regulated at the post-transcriptional
level (for review see: Zeviani and Antozzi, 1997). The resulting
translated proteins are components of enzymes or enzyme complexes of the respiratory chain (e.g. polypeptides that are directly
or indirectly involved in energy production) such as subunit I, II
and III of cytochrome c oxidase, subunits six and eight of ATPase, seven subunits of NADH dehydrogenase and apocytochrome b (Attardi, 1986; Hecht, 1992). For nearly 20 years it has
been assumed that mitochondrial DNA (mtDNA), in contrast to
the genes in the nucleus, has an exclusive maternal mode of
inheritance. Even if the midpiece penetrates the ovum, sperm
mitochondrial DNA might be eliminated by a number of
mechanisms (Wallace, 1989). However, studies on hybrids of
two inbred mice strains, whose mtDNA can be distinguished
easily, detected paternally inherited mtDNA at a frequency of
10–4 relative to the maternal contributions (Gyllensten et al.,
1991). This mode of inheritance provides a mechanism for
generating heteroplasmy and may explain mitochondrial disorders exhibiting biparental transmission. Although a similar
mechanism has not yet been shown to occur in humans it is
important to clarify that assisted reproductive technologies
like ICSI do not led to the paternal inheritance of mitochondrial diseases like neuropathies, myopathies or infertility
(Lestienne et al., 1997; Zeviani and Antozzi, 1997). In fact,
spermatozoa with defects in single components of the respiratory chain showed an altered mitochondrial morphology and a
strongly reduced sperm motility (Folgero et al., 1993; Mundy
et al., 1995; Kao et al., 1998). In addition, multiple deletions
of mtDNA were reported in infertile men with asthenozoospermia (Cummins et al., 1994) and oligoasthenospermia
(Lestienne et al., 1997). This may be caused by oxidative
stress (reactive oxygen species) and the lack or non-function
of appropriate DNA repair mechanism resulting in a
5–10-fold higher mutation rate of mtDNA than that of nuclear
DNA. Nevertheless, mtDNA is much more resistant to degradation and thus is used for genetic fingerprinting (i.e. phylogenetic and family tree analyses, and forensic identification).
Import and processing of nuclear-encoded
mitochondrial polypeptides
The vast proportion of mitochondrial proteins are nuclear-encoded. The transcribed mRNA is translated to precursor proteins on cytoplasmic polyribosomes which contain a specific
mitochondrial N-terminal pre-sequence of 10–80 amino acids
forming an α-helix with a relatively high content of basic and
hydroxylated amino acids. The newly synthesized and un-
111
folded polypeptide chain binds to the cytoplasmic chaperone
ct-hsp70 which stabilizes the conformation permitting translocation through the mitochondrial membranes at so-called
‘contact sites’, close associations of the inner and outer membranes. Cytoplasmic proteins of the hsp40 family (DnaJ,
Mdj1, SIS1, Sec63 and SCJ1) are essential for efficient binding of unfolded polypeptides to ct-hsp70 (Cuezwa et al.,
1993). Thereafter, supported by a mitochondrial import stimulation factor (MSF) (Hachiya et al., 1994; Mihara and
Omura, 1996), the pre-sequence binds to the receptor
Tom70/Tom20 on the outer mitochondrial membrane (Komiya et al., 1997) which is part of the protein translocation machinery (for details see Bomer et al., 1997). ATP hydrolysis is
required for the dissociation of the protein chain from cthsp70. In an energy-consuming process depending on the
potential difference (∆Ψ) of the inner membrane, the unfolded polypeptide passes the import channel composed of
Tim17 and Tim23 and binds to the inner mitochondrial import
complex. This is composed of chaperon hsp70 (mt-hsp70), its
membrane anchor Tim44 and the nucleotide exchange factor
mt-GrpE (Horst et al., 1997a). The complex acts as a mechanochemical enzyme that actively pulls precursors across the
inner membrane (Horst et al., 1997b). With most of the polypeptide still buried in the import channel the pre-sequence is
cleaved off by a metalloproteinase (MPP or Mas2p) in the
matrix which in turn is supported by the polypeptide MasP1
(identical to subunit I of cytochrome c oxidase). Additional
signals on the remaining protein chain sort either to the matrix,
the inner membrane (e.g. subunit IV of cytochrome c oxidase)
or direct into the intermembrane space. After ATP hydrolysis
the polypeptide is passed to a folding complex which contains
mt-hsp70, Mdj1 (DnaJ analogue) and Mge1p (mt-GrpE:
Horst et al., 1997b; Dekker and Pfanner, 1997; Azem et al.,
1997). A subset of imported proteins requires additional refolding into the native conformation and/or oligomerization
by the hsp60–hsp10 complex under manifold ATP hydrolysis
(Stuart et al., 1994; Martin, 1997). It should be noted that
some nuclear-encoded mitochondrial proteins can be inserted
into the outer membrane or translocated into the intermembrane space without passage through the import channel.
Cytoplasmic hsp70 (ct-hsp70) and
mitochondrial hsp70 (mt-hsp70)
As described above, both forms of hsp70 play an essential role
during the import of mitochondrial matrix proteins. The cytoplasmic chaperonin receives the newly translated and unfolded
polypeptide chains directly from the ribosome, stabilizes its
conformation in co-operation with hsp40 (DnaJ), and transfers
them to the import channel. Mt-hsp70, an element of the inner
import complex, binds to the precursor sequence after the translocation of the N-terminal segment to avoid premature folding
(Rassow et al., 1994; Horst et al., 1997b). After cleavage of the
pre-sequence by MPP, most of the proteins imported into the
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A.Meinhardt, B.Wilhelm and J.Seitz
matrix are refolded directly by mt-hsp70 in co-operation with
Mdj1 (DnaJ) and mt-GrpE (Rowley et al., 1994; Horst et al.,
1997a). Another subset of imported proteins is converted by
the hsp60–hsp10 complex to their native structure (Stuart et al.,
1994). Furthermore, mt-hsp70 supports the assembly of proteins synthesized in the mitochondria to supramolecular complexes. One example is the ATPase subunit 6 which is kept in a
‘soluble state’ before assembly with the ATPase subunit 9 to
prevent aggregation (Herrmann et al., 1994). Under stress mthsp70 fulfils another important function. Misfolded or denatured proteins exclusively bind to mt-hsp70 and are then
degraded by mitochondrial ATP dependent protease (Lon protease, PIM1 protease; Wagner et al., 1994).
A non-heat-inducible form of ct-hsp70, the protein P70, is
expressed developmentally in rodent testes by the hsp70-2
gene (Allen et al., 1988). In mouse testes, hsp70-2 is first
detectable in pachytene spermatocytes, post-meiotic round
spermatids, the residual body and cytoplasmic droplet from
day 17 post-natally (Rosario et al., 1992). In rat testes, an
analogue of P70 is found first at day 22 post-natally with the
corresponding gene being transcribed in late pachytene spermatocytes, diakinesis and in steps 1–7 of spermiogenesis
(Krawczyk et al., 1988), indicating that this chaperone has an
active role during the differentiation of spermatocytes into
spermatids. This view was clearly supported by the fact that
hsp70-2 knockout mice are infertile and lack spermatids and
spermatozoa (Dix et al., 1996). Further experiments showed
that hsp70-2 interacts with the cyclin B-dependent CDC2
kinase, an enzyme that has a key role in triggering the G2/Mphase transition during mitotic and meiotic cell cycles (Zhu
et al., 1997). In this model hsp70-2 appears to be a molecular
chaperone for CDC2 and is necessary for CDC2/cyclin B1
formation. Disruption of this formation could prevent G2–M
phase transition during meiosis and result in increased levels
of apoptosis (Dix et al., 1996; Zhu et al., 1997).
Our own immunofluorescence experiments using a commercially available monoclonal antibody directed against mthsp70 show localization in mitochondrial of all germ cells up
to pachytene spermatocytes (unpublished data). Therefore,
mt-hsp70 and Lon protease expression overlaps in early primary spermatocytes.
Mitochondrial hsp60 (mt-hsp60)
General remarks
Mt-hsp60 in the mitochondria of eukaryotes or its bacterial
homologue, GroEL, function as a set of high molecular
weight oligomeric structures. They are composed of seven
subunits of 60 kDa with ATPase activity arranged in a single
heptameric toroid structure in eukaryotic cells (Viitanen et al.,
1992). The mt-hsp60 complex functionally co-operates with a
ring-shaped complex of seven 10 kDa subunits (hsp10, anal-
ogous with GroES) which inhibits the ATPase activity of mthsp60 (Ellis and van der Vries, 1991; Hendrick and Hartl,
1993). Mt-hsp60 is essential for the correct folding of the
native structure of imported mitochondrial proteins followed
by a stepwise process of ATP-dependent release.
Rats
Immunostaining of adult rat testis revealed mt-hsp60 localization
in Sertoli and Leydig cells. The seminiferous epithelium showed
a cell type-specific expression of mt-hsp60. In germ cells, mitochondria of spermatogonia and early primary spermatocytes
were mt-hsp60-positive, whereas all other germ cell types were
completely negative. A stage specific-expression of mt-hsp60
was determined from microdissected tubules. High concentrations of mt-hsp60 were found in stages I–V and IX–XIV, and low
levels were detected in the other stages, i.e. VI–VIII. Coinciding
with stages of high mt-hsp60 expression, spermatogonia divide
mitotically, whereas in stages lacking mitosis the mt-hsp60 level
was much weaker (Meinhardt et al., 1995). Of note, the cristae
type of mitochondria (e.g. in Sertoli cells and spermatogonia)
contained mt-hsp60, whereas the condensed type of mitochondria in mid-pachytene spermatocytes and the intermediate (spd)
form in more advanced germ cells was found to be negative. In
the fetal rat gonads, mt-hsp60 was present in the germ cells
organized into sex cords and in the developing Leydig cells of the
testis. In the pubertal testis, Leydig cells were labelled strongly,
whereas spermatogonia and pre-meiotic spermatocytes were labelled moderately. Spermatids remained negative for mt-hsp60
(Paranko et al., 1996). It was concluded that the gene product is
needed primarily during the initial steps of spermatogenesis
where most of the cell divisions occur, while its expression during the differentiation of spermatids and spermatozoa is obviously not necessary. The presence of elevated mt-hsp60
concentrations in stages with proliferating spermatogonia suggest
the existence of a very active mitochondrial protein import and
assembly machinery needed to generate new mitochondria for
the daughter cells. Interestingly, during rodent spermatogenesis,
the testis-specific isoform cytochrome ct has been found to be
expressed at a high level in round spermatids (Goldberg et al.,
1977; Hess et al., 1993; Morales et al., 1993). Cytochrome ct
(testis-specific) replaces cytochrome cs (somatic) in mitochondria of cells that have been shown to be negative for mt-hsp60.
Round spermatids, therefore, lack an essential component of their
refolding machinery, but nevertheless are able to replace this
mitochondrial enzyme. This apparent contradiction is resolved
by the fact that cytochrome c follows a different import pathway,
as do other inner mitochondrial membrane proteins. These proteins are transferred directly from the contact site to the inner
mitochondrial membrane without passing through the matrix.
Imported membrane proteins, in contrast to soluble matrix proteins, obviously do not require mt-hsp60 chaperonin action (for
review see Glick et al, 1992). However, their folding is mediated
by mt-hsp70 (see above).
Mitochondrial differentiation in male germ cells
Human
To evaluate the potential role of mt-hsp60 as a marker of spermatogenic efficiency, testicular biopsies from adult men with
disturbed fertility were investigated. In normal unaffected tubules, mt-hsp60 was localized to spermatogonia, early primary
spermatocytes and Sertoli cells. In spermatogonia, a difference
in cytoplasmic and mitochondrial labelling could be discriminated. In general, the number of mt-hsp60-positive spermatogonia decreased with the loss of spermatogenic function as
with maturation arrest of spermatogenesis at the level of primary spermatocytes (Werner et al., 1997). In addition, this
decrease correlated with the diminution of cytoplasmic mthsp60 immunolabelling. Werner et al. suggested that a low
level of mt-hsp60 in mitochondria of spermatogonia may lead
to a different pattern of protection, which could result in low
spermatogenic (Werner et al., 1997). It is important to note that
mt-hsp60 is known more as a chaperone and less as a heat-inducible protein. Its detection in the cytoplasm is unusual. Under
normal circumstances mt-hsp60 concentration in the cytoplasm is below the level of detection, because it is translocated
immediately into the organelle after synthesis on cytoplasmic
ribosomes. Therefore, the increased cytoplasmic level could
result from an inefficient or disturbed protein import in mitochondria of germ cells.
Non-human primates
After a decrease of mt-hsp60 in germ cells of men had been
shown to be associated with disabled spermatogenesis, the
hormonal regulation of mt-hsp60 was examined in a primate
animal model. Mt-hsp60 production in the testes of the cynomolgus monkey Macaca fascicularis and animals that had
been treated with the GnRH antagonist Cetrorelix for 25 days
were studied. In addition, testes of the untreated adult rhesus
monkey Macaca mulatta and immature animals either exposed to human chorionic gonadotrophin (HCG), human follicle stimulating hormone (FSH) or HCG and FSH in
combination were analysed (Meinhardt et al., 1998a). In both
sets of adult monkeys, specific mt-hsp60 staining was observed in Sertoli cells, Leydig cells, spermatogonia and early
primary spermatocytes, which was consistent with the observations in human and rat (Meinhardt et al., 1995; Werner et
al., 1997). In the testis of immature rhesus monkeys, mthsp60 was visible in gonocytes, spermatogonia and Sertoli
cells, whereas interstitial cells were negative. The mt-hsp60
pattern was unaffected by GnRH antagonist treatment and
human FSH alone. However, application of HCG alone or in
combination with FSH caused a substantial and marked upregulation of the chaperonin in Leydig cells (Meinhardt et al.,
1998a). It was therefore concluded that HCG is an important
regulator of Leydig cell mt-hsp60 expression during development, whereas FSH in immature animals and GnRH in adult
monkeys is of less relevance.
113
ATP-dependent Lon protease
Mitochondria contain different enzymes with proteolytic activity. These can be divided into those that use ATP hydrolysis
for allosteric activation and non-ATP-dependent proteases
such as the above-mentioned MPP, which have the exclusive
function of cutting off the pre-sequence of newly imported
mitochondrial matrix proteins. ATP-dependent proteases
have been found in all three mitochondrial compartments: (i)
matrix enzymes: PIM1 (Van Dyck et al., 1994); Lon proteases
of rat liver (Desautels and Goldberg, 1982a,b), in the cortex of
bovine adrenal (Watabe and Kimura, 1985) and in human cell
cultures (Wang et al., 1993, 1994); (ii) proteases associated
with the inner membrane of yeast mitochondria as Afg3p
(Guzelin et al., 1996), IMP1 (Schneider et al., 1994), the
YTA10–12 complex (Arlt et al., 1996) and (iii) corresponding
enzymes in the intermembrane space (Sitte et al., 1995).
The function of the ATP-dependent proteases in higher animals is not yet understood. However, recently published articles
about bacteria and yeast mutants lacking the genes for the respective proteases revealed some essential functions. As there is a
high sequence homology between the prokaryotic and eukaryotic
proteases, similar functions in eukaryotes can be assumed.
The bacterial Lon protease as well as PIM1 binds to and
stabilizes TG-rich promotor regions of mitochondrial DNA and
becomes proteolytically almost inactive (Sonezaki et al., 1995;
Fu et al., 1997). Yeast mutants defective of PIM1 protease expression lose mitochondrial DNA integrity thereby rendering a
deficient respiratory chain (Teichmann et al., 1996).
Some of the proteases are working in close co-operation
with the molecular chaperones mt-hsp70 and Mdj1p (Atj1,
DnaJ) by degrading misfolded or ageing matrix proteins
(Wagner et al., 1994; Chloupkova and Luciakova, 1995). The
Mdj1-mediated binding of misfolded polypeptide chains to
hsp70 prevents their aggregation and in consequence they are
presented to the respective proteases. Proteolytic degradation
is increased after a heat shock resulting in dissociation of the
protease from the DNA (Sonezaki et al., 1995). However, also
the ‘normal’ mitochondrial matrix proteins are degraded by
ATP-dependent proteases after reaching their average life
span of 3.5–5 days.
The metalloproteinases Afg3p (Guzelin et al., 1996; Rep et
al., 1996) and YTA10–12 (Arlt et al., 1996) as well as the
metallopeptidases m- and i-AAA protease (Langer and Neupert, 1996) are involved in the degradation of malformed subunits of the respiratory chain complexes III and IV as well as
of the ATP synthase (complex V) located in the inner membrane. Therefore, an intact and co-ordinated oxidative phosphorylation and ATP synthesis is guaranteed.
The human homologue of the bacterial Lon protease was
cloned recently and localized by immunofluorescence in mitochondria of cultured human adrenal cells (Wang et al., 1993,
1994). The native enzyme consists of a hexamer complex of
600 kDa that after reduction in sodium dodecyl sulphate–
114
A.Meinhardt, B.Wilhelm and J.Seitz
polyacrylamide gel electrophoreseis separates into six homologue subunits of 100 kDa. Proteinase activity can be blocked
by serine and cysteine proteinase inhibitors. In rat testes a
polyclonal antibody raised against an evolutionarily conserved peptide sequence of hLon reacted specifically with
mitochondria in early meiotic cells (leptotene and zygotene
spermatocytes), but not with other germ cell types (Möbius et
al., 1995; Seitz et al., 1995). Western blot and reverse transcriptase–polymerase chain reaction analysis confirmed the
identity of Lon protease in rat testes (Meinhardt et al., 1997).
Lon protease, therefore, is a new and specific marker for an
intermediate type (spc) of mitochondria, which are just starting to condense into the form characteristic of mid-meiotic
cells. However, it remains unclear why Lon is expressed only
during such a short time frame of mitochondrial differentiation. Interestingly, the last phases of mt-hsp60 and Lon protease expression overlap and it is tempting to postulate that
Lon may be involved in mt-hsp60 breakdown. On the other
hand, removal of Lon protease itself requires either an autolytic process or the timely involvement of other mitochondrial
proteases, like the recently discovered PIM1 protease (Van
Dyck et al., 1994). Our own Western blot analysis showed a
cross-reaction of an antibody directed against PIM1 protease
with Lon protease isolated from the mitochondrial matrix.
The structural and functional homology of PIM 1 protease and
Lon protease is so evident that PIM1-deficient cells transfected with Lon protease display an almost complete protection of the mitochondrial DNA integrity and ongoing capacity
to degrade malfolded proteins (Teichmann et al., 1996). Furthermore, the mitochondrial proteases PIM1 and Lon show a
high homology to the protease in the cytoplasmic and nuclear
proteasome which, in co-operation with (cyt) and (nuc) hsp70
and dependent on ubiquitin, degrades of malfolded proteins
(Dahlmann et al., 1995).
et al., 1990), of hamster, juvenile (Bergmann et al., 1991) and
adult (Kumari et al., 1990) as well as in the axolotl Ambystoma mexicanum, a lower vertebrate species possessing testes
with a cystic organization (Oehmen et al., 1991), have shown
that SOx is localized in a stage-specific manner during development of spermatogenesis, involution and recrudescence
(Bergmann et al., 1990) and during normal spermatogenesis.
Sulphydryl oxidase
Human
Sulphydryl oxidase (SOx, or thioloxidase, EC 1.8.3.2.) is an
enzyme that catalyses the oxidation of sulphydryl compounds
such as glutathione, cysteine and thioglycerol, utilizing molecular oxygen as an electron acceptor. The newly formed
disulphide bonds are thought to result in conformational
changes of membrane proteins such as in spermatozoa
(Haugaard, 1968) that are required for certain membraneassociated processes. An antibody raised against isolated rat
seminal vesicle sulphydryl oxidase (Seitz et al., 1988) localized the antigen to secretory granules of the seminal vesicles.
Using the same immunohistochemical and immunoelectron
microscopical approach, a cross-reactive species of the antigen was found in the matrix of mitochondria in several tissues such as striated ducts of salivary glands, distal tubules in
the kidney and certain cell types in the testes of rat, hamster
and man (Seitz et al., 1988). More detailed analyses in the
testes of rat, juvenile (Seitz et al., 1990) and adult (Kumari
In the mature human testis moderate SOx amounts are found in
Leydig cells, whereas no SOx has been reported for Sertoli
cells and peritubular cells (Aumüller et al., 1991). The Adark
spermatogonia contain SOx in their mitochondria, whereas
Apale spermatogonia only in stage V of spermatogenesis are
significantly positive. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes have only moderate
SOx amounts, which are highest in pachytene spermatocytes of
stages I–IV, decreases in stage V, and are low during diakinesis
and in secondary spermatocytes. Late spermatids generally
showed higher SOx levels than early spermatids. The midpieces of human spermatozoa are free of SOx-positive mitochondria, whereas in residual bodies small amounts of SOx are
visible (Aumüller et al., 1991). Compared to rat and hamster
testis, SOx distribution in the human testis is less clearly stage
dependent and not confined to certain germ cell types. The
striking difference in the human seminiferous epithelium is the
Rat and hamster
In the seminiferous epithelium of rat and hamster testes SOx
appears in a stage-dependent manner. It is first seen in mitochondria of pachytene spermatocytes at stage I in both animal species
(Kumari et al., 1990). The fate of such mitochondria is speciesspecific. In rat, the immunoreactive mitochondria aggregate during the maturation phase and are retained in the residual bodies.
Hence, spermatozoa free of SOx are released into the lumen. On
the contrary, in the hamster the immunoreactive mitochondria
arrange themselves around the midpiece of spermatozoa while
the residual bodies lack SOx. During photoperiodically induced
testicular involution and recrudescence in the Djungarian
hamster, mitochondrial SOx does not change in pachytene spermatocytes and spermatids as long as these cells are present within
the seminiferous epithelium. Its disappearance coincides with the
degeneration of spermatocytes in phases IV and V of involution
and reappears during recrudescence, when the first spermatogenic wave has reached the pachytene stage (Bergmann et al.,
1990). Post-natally SOx is present in pre-spermatogonia and the
first population of spermatogonia type A within the seminiferous
epithelium of the hamster (Bergmann et al., 1991). The pattern of
SOx distribution during spermatogenesis is identical to that of
adult hamsters. Sertoli cell SOx immunoreactivity persists only
until the onset of meiosis at day 11, whereas Leydig cells show
weak SOx staining during all developmental stages (Bergmann et
al., 1991).
Mitochondrial differentiation in male germ cells
occurrence of SOx predominantly in Adark spermatogonia that
are regarded as the stem cell population (Paniagua et al., 1987).
Bergmann et al. investigated SOx in the seminiferous epithelium of human biopsy material in order to evaluate the possible
value of this mitochondrial marker in the diagnosis of male
(Bergmann et al., 1992). In biopsies of oligozoospermic men
showing hypospermatogenesis a significant increase of SOxlabelled spermatogonia was associated with a significant decrease of sperm concentrations in the ejaculate (Bergmann et
al., 1992). SOx in Sertoli cells is only found in single degenerating cells and in tubules with Sertoli cell-only syndrome
(SCO), but not in these cells of immature seminiferous cords.
This indicates that Sertoli cell SOx is rather a sign of physiological alterations in degenerating cells than dependent on the
stage of differentiation and suggests SOx as a marker for spermatogenic efficiency.
Paracrine factors
Very little is known about the factors that regulate or trigger
the various steps of mitochondrial differentiation during spermatogenesis. Recent studies indicate the involvement of factors secreted by Sertoli cells which act in a paracrine fashion
on primary spermatocytes (Seitz et al., 1995). In-vitro the
normal condensed shape of mitochondria was maintained
during co-culture with Sertoli cells. The same result is
achieved in germ cell-only cultures incubated in conditioned
medium of testosterone-stimulated Sertoli cells (SCCM). The
absence of SCCM or the degradation of the proteinaceous
components of SCCM resulted in a ‘dedifferentiation’ back to
the mitochondrial isotype between orthodox and condensed
(Seitz et al., 1995). The nature of this proteinaceous substance, originally termed paracrine mitochondrial maturation
factor (PMMF), is still unclear, but it may represent one or
more of the known products of the Sertoli cell.
Numerous reports in the past showed that both Sertoli cells
and germ cells secrete various growth factors which act in a
local or paracrine manner (Schlatt et al., 1997). The majority
of these proteins bind to heparin. The PMMF activity can be
isolated from SCCM by heparin-affinity chromatography,
suggesting that PMMF contains one or several growth factors.
In-vitro experiments using isolated leptotene, zygotene and
pachytene spermatocytes incubated with physiological concentrations of recombinant nerve growth factor (NGF), fibroblast growth factor (FGF)-1 and FGF-2 as well as epidermal
growth factor (EGF) demonstrated that all of these polypeptides induce, to a different degree, the formation of condensed
mitochondria in early meiotic germ cells and stabilize the
condensed type in pachytene spermatocytes. This effect could
be reversed by the addition of the respective neutralizing antibody. Furthermore, the addition of these antibodies to SCCM
almost completely inhibited its activity to stabilize the condensed form of mitochondria in pachytene spermatocytes.
This is in contradiction to some reports in the literature which
115
propose an expression and secretion of the above-mentioned
growth factors by germ cells and not by the Sertoli cells (Ackland et al., 1992). Our own Western blot analysis, however,
indicates the presence of polypeptides in the secretion of testosterone-stimulated Sertoli cells that are immunologically
related to the growth factors mentioned above. Another possible explanation is the presence of proteinaceous substances in
PMMF stimulating primary spermatocytes to secrete growth
factors that finally act in an autocrine manner on mitochondrial differentiation. Taken together, these data strongly imply
that PMMF consists of several biologically active factors.
Recently, activin was found to be one of them. The activins
are homo- and heterodimeric proteins consisting of a combination of the βA and βB subunits of inhibin. Activin A (βAβA),
activin B (βBβB) and activin AB (βAβB), each have the capacity to stimulate FSH secretion by the pituitary gland. In addition
to their widespread biological effects in many tissues, the local
production of activins in the testis may result in paracrine effects. In the testis, activin A is a product of the Sertoli cells (de
Winter et al., 1993) and activin type IIB receptors are found on
many germ cell types (de Winter et al., 1992; Kaipia et al.,
1993). Further, Krummen et al. showed binding of 125I-labelled activin to spermatocytes and round spermatids (Krummen et al., 1994). The action of activin involves several
transmembrane serine-threonine kinase receptors (for review
Mathews, 1994), as well as being modulated by the activin
binding and neutralizing protein follistatin (Nakamura et al.,
1990; Sugino et al., 1993; Schneyer et al., 1994).
Activin A showed a similar ability to SCCM to maintain a
high percentage of spermatocyte mitochondria in the condensed state, while inhibin has no effect (Meinhardt et al.,
1996). The addition of an antiserum specific for activin A
resulted in a neutralization of the effect caused by activin A or
SCCM. Therefore, activin A is the first Sertoli cell product that
has been identified to influence differentiation of male meiotic
germ cells. Knowledge of the physiology of acitivin is intricately linked to an understanding of the actions of follistatin.
Follistatins are a product of a single gene of ~6 kb consisting of
six exons (Shimasaki et al. 1988). Alternative splicing results in
two different forms of follistain (FS344 and FS317). Both variants are proteolytically processed, with FS344 yielding FS315
and FS300 while FS317 yields FS288. Variable glycosylation
of each of these products results in at least six forms of this
protein (Robertson et al., 1987; Ueno et al., 1987). The capacity of each form of follistatin to bind activin is similar, but
FS288 binds strongly to heparan sulphate proteoglycans whilst
FS315 shows no similar capacity (Sugino et al., 1993). In rat
testis follistatin mRNA has been located in many germ cells.
Small amounts of follistatin mRNA were found in pre-leptotene and leptotene primary spermatocytes, but the expression
declined in the late leptotene and early zygotene stages (stages
XI and XII). The expression increased slightly in early pachytene stages (stages XIV–V) and reached a maximum in the late
pachytene and diplotene stages (stages VIII–XIV). Follistatin
116
A.Meinhardt, B.Wilhelm and J.Seitz
Figure 1. Mitochondrial differentiation during male germ cell development. Boxes indicate expression of mitochondrial marker proteins in the
different germ cell types. hsp60 = heat shock protein 60; SOx = sulphydryl oxidase; mt-hsp70 = mitochondrial heat shock protein 70; cytochrome ct = testicular isoform of cytochrome c; intermediate spc = intermediate form of mitochondria in spermatocytes; intermediate spd = intermediate form of mitochondria in spermatids.
remained relatively high in spermatids from step 1 to step 10,
declined thereafter and was not detected from step 16 to step 19
(Meinhardt et al., 1998b). Follistatin was also found in Sertoli
cells and endothelial cells, but no mRNA could be detected in
Leydig cells (Meinhardt et al., 1998b).
Conclusions
Taken together, these data resulted in the following conclusions and functional hypothetical model of mitochondrial
differentiation during spermatogenesis.
Due to the direct access of glucose and oxygen originating
from testicular blood vessels, the metabolism of spermatogonial mitochondria is comparable to that of other tissues and
organs. The final differentiation of germ cells to the haploid
spermatozoa, however, implies a dramatic functional change
of the organelles. This becomes apparent by the striking morphological change from the orthodox type via a condensed
form to the intermediate (spc) type (Figure 1). Mature spermatozoa require only a limited number of mitochondria for their
motility, which are located in the proximal flagellum. The
majority of germ cell mitochondria are segregated and then
released within the residual body. One of the driving forces in
the alteration of mitochondrial structure at the onset of meiosis is certainly influenced by the switch to pyruvate and
lactate instead of glucose to generate ATP. However, there is
clear evidence that proteinaceous factors also cause the
change in mitochondrial morphology and the transient stabilization of the individual forms during development. These
mediators are secreted by hormone-stimulated Sertoli cells
and act on germ cells in a paracrine fashion. One such factor,
activin, has been shown to stabilize the condensed type of
mitochondria in pachytene spermatocytes in vitro. The finding of activin binding protein follistatin in cells other than step
1–11 spermatids suggests activin neutralization by follistatin
could account for the observed change in mitochondrial morphology to the intermediate type (spd) in post-meiotic germ
cells by activin neutralization. It cannot, however, be excluded that other factors also are involved
The studies of de Martino et al. showed that in spite of the
substantial change of mitochondria during meiosis from the
orthodox to the condensed type the structure and functional
integrity of the inner membrane is constantly maintained (de
Martino et al., 1979). In-vitro isolated condensed mitochondria
kept under normoxic conditions and in the presence of ADP
demonstrated an increased ability for oxidative phosphorylation, i.e. the production of ATP. However, the question arises if
the mitochondria located beyond the blood–testis barrier have
the same high metabolic rate. Indeed our own indirect evidence
indicates a strongly decreased ability of sperm mitochondria
for ATP synthesis. ATP is not only required for ‘normal’ cellular function and metabolism, but also for the import and refolding of mitochondrial proteins. Most if not all the steps of the
import sequence are energy-demanding, such as the release of
Mitochondrial differentiation in male germ cells
newly synthesized proteins from ct-hsp70 and the subsequent
translocation through the import channel. Furthermore, the
binding to and release from mt-hsp70 as well as refolding and
assembly of imported polypeptides by the mt-hsp60/hsp10
complex require ATP hydrolysis. Malfolded matrix proteins
and those of the inner mitochondrial membrane bind in an
energy-dependent fashion to mt-hsp70. Potentially, this step
could enable the ATP-dependent proteolysis by the Lon/PIM1
protease. For this purpose the protease specifically is released
from its inactivating binding to the DNA. However, in mitochondria of leptotene and zygotene spermatocytes a change in
the energy-rich steady state is observed that is either caused by
local factors and/or by the translocation of cells beyond the
blood–testis barrier. The activated ATP-dependent protease
seems to be responsible not only for the degradation of normal
matrix proteins, such as mt-hsp60, but also for components of
the ATP-synthesizing system in the inner mitochondrial membrane. The involvement of mt-hsp70 in this degradation process has not yet been resolved, but appears to be likely. Because
the ATP-dependent Lon protease is not detectable beyond the
mid-pachytene stage, either a suicidal breakdown or a degradation by other proteases is possible. During intact oxidative
phosphorylation sufficient amounts of NADH/NADPH and
reduced glutathione (GSH) are synthesized. GSH keeps many
essential proteins in a reduced state (free cysteine residues) and
prevents oxidative damage potentially caused by reactive oxygen species. We postulate that the concentrations of these reductive substances are reduced from meiosis onwards. This
correlates with the appearence of the mitochondrial matrix protein SOx. Our in-vitro experiments using purified SOx show an
almost complete inhibition of the enzyme activity by physiological concentrations of ATP, NADH/NADPH and GSH, respectively. An in-vitro decrease of these three factors to <20%
of the physiological concentration, however, resulted in a dramatic increase of SOx enzyme activity. Assuming that these
respective metabolites decrease from mid to late pachytene
spermatocytes onwards (which still has to be verified experimentally) the SOx also could be activated in vivo to cross-link
matrix proteins via disulphide bridges. This could contribute to
the condensation of the organelles or prepare a subset of mitochondria for degradation in the residual body. Nevertheless,
even haploid germ cells lacking the essential import component mt-hsp60 have been shown to replace cytochrome cs by
the testis-specific isoform cytochrome ct. However, this inner
mitochondrial membrane protein follows a different import
pathway that does not require passage through the matrix and
refolding by mt-hsp60 or mt-hsp70.
The obviously very complex and sophisticated interactions
in the seminiferous epithelium and between seminiferous tubules and the interstitium in the mammalian testis that finally
lead to germ cell differentiation are just beginning to be understood. Many more polypeptides and proteins may play specific
roles in the initiation and regulation of particular steps that have
not yet been revealed.
117
Acknowledgements
The authors are very thankful to Drs Con Mallidis, Andrew Laslett and to Prof. Monika Löffler for critically reading the manuscript and to Sebastian Seitz for design of the figure.
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Received on July 9, 1998; accepted on January 13, 1999