Download The role of heat shock proteins in reproduction

Human Reproduction Update 2000, Vol. 6 No. 2 pp. 149–159
© European Society of Human Reproduction and Embryology
The role of heat shock proteins in reproduction
A.Neuer1,2,*, S.D.Spandorfer1, P.Giraldo1,3, S.Dieterle2, Z.Rosenwaks1 and S.S.Witkin1
1
Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, New York, USA, 2Division of
Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Witten/Herdecke, Germany, and
3
Department of Gynecology and Obstetrics, University of Campinas, Sao Paulo, Brazil
Received on June 9, 1999; accepted on October 22, 1999
Heat shock proteins (HSP) were first identified in cells after exposure to elevated temperature. Subsequently HSP
have been identified as a critical component of a very complex and highly conserved cellular defence mechanism to
preserve cell survival under adverse environmental conditions. HSP are preferentially expressed in response to an
array of insults, including hyperthermia, free oxygen radicals, heavy metals, ethanol, amino acid analogues,
inflammation and infection. HSP interact with intracellular polypeptides and prevent their denaturation or incorrect
assembly. In addition HSP are also involved in several processes essential for cellular function under physiological
conditions. HSP production is enhanced during in-vitro embryo culture and they are among the first proteins
produced during mammalian embryo growth. The spontaneous expression of HSP as an essential part of embryo
development is well documented and the presence or absence of HSP influences various aspects of reproduction in
many species. Finally, HSP are immunodominant antigens of numerous microbial pathogens, e.g. Chlamydia
trachomatis, which have been recognized as the main cause of tubal infertility. Many couples with fertility problems
have had a previous genital tract infection, have become sensitized to microbial HSP, and a prolonged and
asymptomatic infection may trigger immunity to microbial HSP epitopes that are also expressed in man. Antibodies
to both bacterial and human HSP are present at high titres in sera and hydrosalpinx fluid of many patients
undergoing in-vitro fertilization (IVF). In a mouse in-vitro embryo culture model, these antibodies impaired the
mouse embryo development at unique developmental stages. Recent studies indicate an association between a
previous infection, immunity to HSP and reproductive failure.
Key words: Chlamydia trachomatis/embryo development/heat shock proteins/infection and immunity/IVF
TABLE OF CONTENTS
Introduction
General properties of heat shock proteins
Reproductive functions of heat shock proteins
Pathology of heat shock proteins: implications
for reproductive outcome
HSP60 and IVF outcome
Acknowledgements
References
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Introduction
Heat shock proteins (HSP) are highly conserved cellular stress
proteins present in every organism from bacteria to man. The first
description of a cellular heat stress response was made >37 years
ago. It was first observed in 1962 that the salivary gland
chromosomes of the fruit fly, Drosophila melanogaster, exhibited
a characteristic puffing pattern after exposure to heat (Ritossa,
1962). The first gene products of this chromosomal puffing were
identified 12 years later and the term ‘heat shock proteins’ was
created (Tissiere et al., 1974). Since this time many other stimuli
which induce a heat shock response have been identified. The
chromosomal location of the genes coding for these proteins have
been identified, the genes have been sequenced, the conformation
of the resulting proteins have been described, and the mechanism
of gene activation by nuclear heat shock transcription factors
characterized (Westwood et al., 1991). Since the heat shock
response is a vital cellular survival mechanism, it is
understandable that HSP have gained considerable interest in
almost every medical field including reproductive medicine,
immunology and infectious diseases (Mizzen, 1998).
Drugs modulating HSP expression (thus protecting integrity
and homeostasis of cells and tissues) are undergoing clinical trials
(Biro et al., 1996; Vigh et al., 1997). In the following paragraph a
number of crucial characteristics that define this family of proteins
are summarized.
* To whom correspondence should be addressed at: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University
of Witten/Herdecke, Olpe 19, 44135 Dortmund, Germany. Tel: (49) 231/5575 450; Fax: (49) 231/ 5575 4599; [email protected]
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General properties of heat shock proteins
All organisms studied ranging from prokaryotic bacteria to
mammals, including man, respond to an increase in temperature
by switching off the synthesis of most proteins and commencing
large-scale synthesis of a few HSP. Even thermophilic organisms,
whose optimal growth temperature lies between 50–90°C,
respond to a sudden temperature rise with a rapidly increased
expression of HSP.
The amino acid composition of HSP has not changed very much
during evolution. HSP of highly divergent organisms are very
similar to one another (their structure has been conserved).
Some members of the HSP families are strictly inducible by
stress, whereas others are constitutively expressed at normal
temperature and are only slightly induced by heat shock. The term
HSP refers to inducible protein products while HSC describes
constitutively expressed HSP.
HSP serve two major functions: firstly, under physiological
conditions, they act as molecular chaperones (intracellular
housekeeping proteins) which are involved in mediating the
folding and transport of other intracellular proteins and in some
cases their assembly into oligomeric structures. HSP act as
chaperones by participating in the assembly of proteins without
being part of the final protein structure (Ellis, 1987). Moreover
they fulfil crucial roles in intracellular transport, the maintenance
of proteins in an inactive form and the prevention of protein
degradation. Secondly, they are induced in response to cellular
stresses which include changes in temperature, the presence of
free oxygen radicals, viral and bacterial infections, heavy metals,
ethanol, and ischaemia (Lindquist, 1986; Welch, 1992). The
stress-elicited activation of heat shock genes is called the heat
shock response. This heat shock response is frequently found in
clinical situations, e.g. ischaemia, infection and circulatory and
haemorrhagic shock. Cellular stress disturbs the tertiary structure
of proteins and has adverse effects on cellular metabolism.
However, pretreatment of cells with a mild stress, just sufficient to
induce the expression of HSP, results in protection to subsequent
insults. This phenomenon has been called ‘stress tolerance’ and is
probably caused by the resolubilization of proteins that were
denatured during the initial stress. In addition, it has been
suggested that cellular structures like microfilaments and
centrosomes but also cellular functions like transcription and
translation are more stabilized during a second stressful event in
stress tolerant cells. Due to their ubiquitous and essential role in
the production, quality control and disposal of other proteins it is
not surprising that HSP are among the most highly conserved gene
products in nature.
HSP are classified into different families according to their
molecular weight measured in kDa rather than by their function.
Most scientific knowledge has been accumulated on four families
of HSP. These are the ‘small’ 27, 60, 70 and 90 kDa HSP.
Recently the expression of another high molecular weight HSP
during embryo development has been investigated (Hatayama et
al., 1997). In the context of reproduction, HSP60 and HSP70 are
most important. The HSP60 family consists of proteins that are
highly expressed in a constitutive manner and are moderately
stress inducible. The main localization of HSP60 is in the
Figure 1. Heat shock protein (HSP) expression in human first trimester
decidua. The epithelium of endometrial glands (arrow) and some stromal cells
show an intense immunoreactivity for HSP60. Fresh frozen decidual tissue
sections were immunostained by the avidin–biotin–peroxidase complex. 3,3′
diaminobenzidene was used as a chromogen. Original magnification ×200.
mitochondria (Jindal et al., 1989). However, other loci including
cell surface exposition of HSP60 has been documented (Soltys
and Gupta, 1996). The HSP70 family comprises several proteins
that are localized in distinct cellular compartments. The
constitutively synthesized HSC70 is found in the cytosol and
nuclei of cells and is only moderately stress inducible. The HSP70
family represents the most conserved group of proteins within the
HSP superfamily (Hunt and Morimoto, 1985).
For several reasons, the above described characteristic features
of HSP increase their potential to become a target antigen in the
pathogenesis of autoimmune diseases (Kaufmann, 1990). Firstly,
HSP are phylogenetically conserved. In practical terms, there is a
>50% sequence homology between prokaryotic HSP and HSP of
mammalian cells (Lamb et al., 1989; Jones et al., 1993). Secondly,
HSP are immunodominant antigens for many common microbes,
which means that these infectious agents are mainly recognized by
the immune system through recognition of their HSP epitopes.
This is important for reproduction and assisted reproductive
medicine, because many infertile couples have been sensitized
during the course of a previous microbial infection. Finally, HSP
are overexpressed at sites of acute and chronic inflammation (Van
Eden, 1999). Thus, in a susceptible individual, exposure to an
infectious agent could result in an immune response to the
infecting agent’s HSP and/or could also cross-react with self
directed, organ-specific proteins, resulting in an autoimmune
disease.
Reproductive functions of heat shock proteins
Heat shock protein expression in reproductive tissue
The presence of HSP has been demonstrated in different tissues
relevant to human reproduction. Tabibzadeh and colleagues
described the full complement of human HSP in the endometrium
of healthy women (Tabibzadeh et al., 1996). Similarly, HSP
expression can be detected in the decidua during the first trimester
of pregnancy (Neuer et al., 1996, Figures 1 and 2). In an ongoing
The role of heat shock proteins in reproduction
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Figure 4. Heat shock protein (HSP) 27 expression in Fallopian tubes. Same
patient as Figure 3. Original magnification ×400.
Figure 2. Heat shock protein (HSP) expression during first trimester decidua.
Decidual stromal cells immunoreactive for HSP70. Original magnification
×400.
Figure 3. Heat shock protein (HSP) 27 expression in Fallopian tubes. Strong
immunoreactivity in tubal epithelial cells of a pregnant patient with ectopic
pregnancy. Original magnification ×200.
study we have been able to demonstrate the presence and
differential expression of HSP in Fallopian tube tissue of women
with and without ectopic pregnancy (Figures 3 and 4).
Maximum values of HSP27, HSP60 and HSC70 in the
endometrium are expressed after ovulation and in the early
secretory phase, which is the critical period of ‘endometrial
receptivity’ for an implanting embryo. However, since oestrogen
and progesterone receptors are associated with HSP, these HSP
are constantly involved in the modulation of steroid function in the
endometrium (Renoir et al., 1990; Bagchi et al., 1991).
Recently the prevention of cytotoxic damage by cytokines has
been proposed as another function of HSP in the endometrium
(Tabibzadeh and Broome, 1999). In the endometrium leukocytes
can produce high levels of reactive oxygen species and cytokines.
Both products can modulate the expression of HSP (Jaquire-Sarlin
et al., 1994). Since leukocytes and cytokines, e.g. tumour necrosis
factor-α (TNF-α), accumulate progressively during the secretory
phase, it is possible that HSP protect endometrial cells from the
adverse side-effects of this leukocyte accumulation and cytokine
release (Tabibzadeh and Broome, 1999). Cells transfected with
HSP70, for example, are protected from cytotoxic damage by
TNF-α (Jaattela 1993). In addition it has been suggested that
HSP70 can prevent DNA strand breaks, protect mitochondrial
structure and function and thus inhibit apoptosis (Jaquire-Sarlin
et al., 1994).
Finally HSP are present in the human placenta (Divers et al.,
1995; Ziegert et al., 1999). Immunohistochemical results revealed
that HSP were more evident on the apical surface of the
syncytiotrophoblasts than on stromal or muscle cells. Placental
HSP expression did not differ between preterm and term
pregnancies, indicating that their production was part of the
physiological pregnancy process. However, immune complexes
between immunoglobulin (Ig)G antibodies and HSP60 or HSP70
were detected only in the placentae of women who delivered
preterm (Ziegert et al., 1999). This correlation suggests that
autoimmunity to HSP might be involved in immune-mediated
preterm labour.
HSP expression and spermatogenesis
During spermatogenesis, three distinct phases can be discerned:
mitotic proliferation of spermatogonia; meiotic development of
spermatocytes; and post-meiotic development of spermatids and
maturation of the spermatozoon (Eddy et al., 1991). Since all
these developmental stages represent situations where dramatic
transformations and cellular differentiation take place, it is not
surprising that spermatogenesis is accompanied by the expression
of different HSP (Dix, 1997; Meinhardt et al., 1999). During
mouse and rat spermatogenesis, the constitutive form of HSP70
(HSC70) accumulates (Allen et al., 1988a,b). Also mRNA coding
for proteins related to HSP86 was found in rat and human testis
(Lee, 1990). In infertile men it has been demonstrated that the
number of HSP60-expressing spermatogonia paralleled the loss of
spermatogenic function (Werner et al., 1997). These observations
suggest that a low level of HSP60 expression in spermatogonia
might lead to a decreased level of protection, which in turn could
be involved in low spermatogenic efficiency. In a recent study,
Dix et al. showed in a mouse model that the disruption of the
HSP70-2 gene by gene targeting results in failed meiosis, germ
cell apoptosis and male infertility (Dix et al., 1996).
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Spermatocytes of mice in which the HSP70-2 gene had been
knocked out became arrested during meiosis. Morphological
examination revealed that these animals had testes only one third
the size of control mice. This failure of meiosis was associated
with an increase in spermatocyte apoptosis (Mori et al., 1997).
Thus, with minor exceptions (Mori et al., 1999), HSP70-2
participation during spermatogenesis is required for successful
completion of meiosis in mouse spermatocytes.
Induction of heat shock proteins by human semen
The mRNA for one of the heat shock proteins, HSP70, has also
been shown to be induced by cell-free seminal fluid as well as by
isolated motile spermatozoa (Jeremias et al., 1997, 1998, 1999). In
peripheral blood mononuclear cells and human cervical epithelial
tumour cells (HeLa) in vitro, and in endocervical cells in vivo,
exposure to semen resulted in transcription of the HSP70 gene.
The mechanism of semen-induced HSP70 gene activation and the
biological consequences of this activity remain a matter of
speculation. Human semen is a rich source of prostaglandins,
proteases, polyamines and other products which conceivably
could induce a stress response in cells after physical contact. This
response and HSP70 expression may activate lymphocytes that
were previously sensitized to cross-reacting regions common to
microbial HSP70s. By this mechanism, the immune system might
initiate a rapid response to micro-organisms in semen, even to
those organisms never previously encountered. HSP70 gene
activation, by promoting suppression of pro-inflammatory
immune responses (Cahill et al., 1996), may also inhibit an
immune response to spermatozoa in the female reproductive tract.
This, however, might contribute to the sexual transmission of
disease pathogens, as suggested by Kelly et al. (1997) and
Jeremias et al. (1997, 1998).
HSP expression and oogenesis
The female germ line like the mammalian male germ line is
sensitive to hyperthermic as well as to other environmental stress
factors. Similar to spermatogenesis, HSP expression is an integral
process during oogenesis in a number of species. These include
distant species like insects (Ambrosio and Schedl, 1984), fish and
amphibians (Heikkila et al., 1985, 1997), but also mammals
(Heikkila et al., 1986). The conservation of HSP expression in
evolutionary diverse organisms supports the assumption of a
fundamental role of HSP during germ cell development. HSP are
found, for example, in ovarian nurse cells of Drosophila where
they are subsequently transported to the oocyte (Zimmerman
et al., 1983). In mammalian oocytes a ‘window’ for heat induction
of HSP exists, which is regulated by the specific stage of oocyte
development. In mouse oocytes, the heat shock response is
maximized during the growth period of the oocyte and declines
with acquisition of the full oocyte size. Finally it is shut off with
the terminal oocyte and follicle differentiation (Curci et al., 1987,
1991). Thus, the ability of mouse oocytes to mount an inducible
heat shock response is highest during early follicular growth and
disappears prior to ovulation. Growing oocytes spontaneously
express high levels of the constitutive 70 kDa HSP (HSC70). Thus
HSC70 is found at high levels in the pre-ovulatory oocyte (Curci
et al., 1991). Later, its synthesis ceases shortly after germinal
vesicle breakdown and it is undetectable in the ovulated oocyte at
the time of fertilization. After meiosis, HSC70 synthesis has
vanished completely. This is interesting to note, because it is
known that mammalian oocytes are very heat sensitive. Since
fully developed oocytes are unable to express the heat inducible
HSP70 form, this could explain why mammalian oocytes exhibit
an atypical and degenerate morphology after an exposure to
hyperthermic stress (Baumgartner and Chrisman, 1981). The
observed abnormalities included multinuclear eggs and an
increase in size of the first polar body. In vitro, elevated
temperature reduces the number of oocytes proceeding to
metaphase II and decreases the rate of fertilization (Lenz et al.,
1983). The above-described block of heat shock gene induction
during oocyte differentiation seems to represent a general feature
in oogenesis, even though it may follow different time schedules
in different species. It is interesting to speculate about the role of
HSP during the ovulation process. Since ovulation is
characterized by the cardinal features of an inflammatory reaction
(Espey, 1994), it is possible that HSP play a role in the ovulation
process and the maintenance of the postovulatory metabolic
activity and survival of the oocyte. The presence of HSP60 in
human follicular fluid of patients undergoing in-vitro fertilization
(IVF) has recently been demonstrated (Neuer et al., 1997). In the
rat, HSP70 induction mediates luteal regression (Khanna et al.,
1995). However at present no further detailed knowledge on the
function of HSP during this time of the reproductive cycle exists.
Heat shock proteins and embryo development
The successful completion of the fertilization process and the
initiation of the first cleavage steps mark the beginning of embryo
development. Before questioning the role of HSP during embryo
development it is important to realize that almost all of the present
knowledge on the function and role of HSP in reproduction relies
on information obtained from animal studies or from human
somatic cell lines which can be induced to differentiate. Although
data from animal models may suggest similar mechanisms in man,
the exact role of HSP for human embryo development remains
speculative. Due to technical difficulties and ethical restrictions
most of the existing studies focus on HSP and early mammalian
embryo development from a zygote up to the expanded
trophoblast stage. Less experimental knowledge exists on the role
of HSP for advanced embryo and organ development. Since the
mouse is often used as a model in the study of mammalian
development and since some of our own data are derived from a
mouse embryo model, we will mainly focus on findings
concerning the role of HSP in mouse embryo development.
Although the HSP70 family has received special attention in this
context, members of the 60 and 90 kDa HSP are also synthesized
by the murine preimplantation embryos (Bensaude et al., 1983).
Figure 5 shows an example of HSP60 expression in a murine
2-cell embryo.
As for all mammals, the embryological development of the
mouse can be subdivided in two main phases. The preimplantation
period, which can be easily assessed in vitro and, secondly, the
post-implantation period. On the role of HSP during early
implantation and attachment to endometrial surfaces no
information is presently available. The preimplantation period
comprises the time span after ovulation and fertilization in the
oviduct before complete implantation in the maternal uterus. In
the mouse there is a 4–5 day preimplantation period. During this
The role of heat shock proteins in reproduction
Figure 5. Heat shock protein (HSP)60 expression in a murine 2-cell embryo.
A scattered fine immunoreactivity is observed in the cytoplasm. The
connective area between the two blastomeres is particularly positive.
time embryos develop from the zygote to the blastocyst stage and
migrate freely from the oviduct to the uterus.
Distinctive features of HSP expression are directly linked to
major events occurring during the preimplantation phase: (i)
spontaneous, constitutive HSC70 expression begins with the onset
of zygotic genome activity and at the early 2-cell stage. During the
same period inducible HSP70 expression is still absent (Bensaude
et al., 1983; Morange et al., 1984); (ii) the constitutive form,
HSC70, is the predominant HSP expressed up to the blastocyst
stage (Morange et al., 1984); (iii) in mouse embryos, the induction
of HSP synthesis by heat shock begins at the blastocyst stage
(Wittig et al., 1983); and (iv) since blastocyst formation marks the
differentiation of two types of embryonic cells forming the inner
cell mass and the outer cell mass, progressive acquisition of HSP
inducibility is associated with continuing embryonic
differentiation. Thus inducible HSP70 expression and the
formation of heat shock protein expression appear to be
developmentally regulated.
It seems to be a common feature of mammalian embryos that
very early stages of development are characterized by a lack of
induced HSP synthesis. This inability generally reflects the
absence of embryonic gene transcription. As soon as transcription
resumes, most heat shock genes become stress inducible.
Experiments using nuclei transfers have demonstrated that ageing
of the egg cytoplasm directs the onset of heat shock gene
transcription (Barnes et al., 1987; Howlett et al., 1987). At the
8-cell stage, the mouse embryo does not yet synthesize inducible
HSP70 even after heat shock, but it does synthesize very high
levels of the cognate HSC70. However, when an 8-cell stage
nucleus is transferred into a 1-cell embryo devoid of its pronuclei
the reconstructed 1-cell embryo does not synthesize any HSC70 in
the first hours that follow the manipulation. However, after
allowing time for cell division, the reconstructed embryos
synthesize both the inducible HSP70 and the cognate HSC70 at
the correct time relative to the development of the recipient
cytoplast. Thus, distinct features of HSP expression are directly
linked to major events occurring during the preimplantation phase
(Dix et al., 1998). However, after implantation the expression of
HSP is less co-ordinated and no uniformity exits in the HSP
expression pattern of different tissues (Loones et al., 1997).
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Recently several studies focused on the molecular mechanisms
regulating the expression of HSP, in particular on the properties of
the two best studied heat shock transcription factors HSF1 and
HSF2 (Christians et al., 1997a,b). Heat shock transcription factors
(HSF) bind to the promoters of heat shock genes on conserved
heat shock sequence elements (HSE). HSF1 is unable to bind to
HSE in the absence of stress, while HSF2 is active under normal
temperatures. HSF2 is believed to be the major factor for
constitutive HSE binding activity. Studies in the mouse suggested
that HSF2 might be involved in the control of heat shock gene
expression during embryogenesis (Mezger et al., 1994a,b). Even
earlier HSF1 is already present at the 1-cell stage. The relative
abundance of HSF1 is correlated with the high amount of HSP70
gene expression at the 2-cell stage described previously
(Christians et al., 1997b). Recently, additional HSFs, including a
new human HSF have been discovered (for review, Morange
et al., 1998). This multiplicity of HSF reflects the involvement of
heat shock genes and their gene products in various essential
cellular processes.
Pathology of heat shock proteins: implications for
reproductive outcome
In addition to the above-described physiological properties of
HSP in reproductive events, several pathological conditions have
also been associated with HSP. The origin of the pathogenicity of
HSP for reproduction is based on several mechanisms. Firstly,
HSP can induce a persistent inflammatory response. Secondly,
HSP molecules serve as antigenic targets for the immune system.
Finally, the extensive amino acid sequence homology between
human and microbial HSP could result in autoimmune mediated
reproductive failure.
Properties of bacterial HSP
Members of the HSP60 and HSP70 families have been recognized
as immunodominant antigens of many microbial pathogens. These
include bacteria, e.g. Chlamydia trachomatis, one of the most
frequently found sexually transmitted microbial pathogens in
patients of reproductive age (Cerrone et al., 1991; Zhong and
Brunham, 1992). Like eukaryotic cells, microbial pathogens
express a constitutive level of HSP required for the maintenance
of essential house keeping functions. During an infection the
microbial stress protein synthesis is up-regulated and the
increased amount of microbial HSP at sites of an infection can
contribute to immunodominance of the microbial HSP. In recent
years it has become clear that HSP play a major role in the acute
inflammatory response and in the persistence of inflammatory
reactions (Moseley, 1998). HSP released from infectious
organisms or infected host cells can induce cytokine release and
provoke an immune response (Tabona et al., 1998). After invasion
into a host tissue, the pathogen is subjected to environmental
conditions, such as elevated temperature, nutrient deprivation
changes in pH and exposure to oxygen radicals, which induce a
stress response. Thus, during an infection enhanced microbial
HSP synthesis may be part of the protective response of the
pathogen to host defences and can contribute to microbial
virulence (Mizzen, 1998). This is important for reproduction since
many couples seeking infertility treatment have had a previous
exposure to microbial pathogens. C.trachomatis infections of the
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Table I. Properties of the 60 kDa family of heat shock proteins
Detectable in all eukaryotic and prokaryotic organisms
Essential chaperone proteins involved in transport, folding and
assembly of protein subunits
Production is elevated in response to environmental stress factors in
order to minimize protein denaturation
Amino acid sequence is highly conserved throughout evolution. The
human and bacterial proteins share a sequence homology of ∼50%
Immune responses to conserved regions of heat shock proteins have
been implicated in autoimmune phenomena.
HSP60 and IVF outcome
IVF bypasses the requirement for patent Fallopian tubes and thus
has become the treatment of choice for women with occluded or
damaged Fallopian tubes. However it is only recently beginning to
be appreciated that the cause of tubal blockage and sensitization to
chlamydial and human HSP may also negatively influence postfertilization events (Spandorfer et al., 1999a; Moomjy et al.,
1999). Based on the above-described model (Table II) we tried to
elucidate the role of HSP in early embryo development and the
consequences of immune sensitization to HSP60 for reproductive
outcome of IVF patients.
Immunity to chlamydial HSP60 and pregnancy outcome
female genital tract for example are the major cause of infertility
due to occluded Fallopian tubes. Several studies have revealed
that sensitization to HSP60 of C.trachomatis and subsequent
expression of the highly homologous human HSP60 can lead to
unsuspected infertility problems.
60 kDa chlamydial and human HSP
HSP60 is one of the best-characterized molecular chaperones of
both eukaryotic and prokaryotic organisms. The major properties
of HSP60 are delineated in Table I. Typically, during the course of
an acute infection, immunity is restricted to HSP60 epitopes that
are specific to the invading micro-organism, e.g. C.trachomatis.
Most patients with infertility problems due to tubal occlusion
have experienced a chronic persistent chlamydial infection
(Witkin et al., 1997). In contrast to an acute infection, cells
chronically infected with C.trachomatis synthesize only low
levels of structural components but continue to produce
chlamydial HSP60 at high levels (Beatty et al., 1993). Thus, some
women with asymptomatic and untreated C.trachomatis
infections and tubal infertility have experienced a long-term
exposure to chlamydial HSP60. Since bacterial and human HSP
share ∼50% amino acid sequence homology (Shinnik 1991), it has
been proposed that a prolonged exposure of the immune system to
chlamydial HSP60 and a concomitant exposure to both the
chlamydial and human HSP60 may lead to autoantibody
formation (Witkin et al., 1997). A possible model for impairment
of early-stage pregnancy after immune sensitization to conserved
regions of the C.trachomatis HSP60 has been outlined previously
(Witkin et al., 1996) and is summarized in Table II.
In one of our initial studies, the prevalence of IgA antibodies to
chlamydial HSP60 in the cervix of 216 women undergoing IVF
treatment was determined (Witkin et al., 1994). None of the
investigated women ever had a recognized chlamydial infection.
However, antichlamydial HSP60 IgA was identified in 41 (20.7%)
patients. This is remarkable, because the presence of this antibody
is considered to reflect an acute immune response to chlamydial
HSP60 and was, in addition, associated with unsuccessful IVF
outcome. Anti-HSP60 IgA was present in 26.3% women who did
not become pregnant after transfer (P = 0.0007), 33.3% of women
with only transient biochemical pregnancies, 30% of women with
spontaneous abortions and only 7.3% of women with live births
(Witkin et al., 1994). No relationship existed between HSP60
antibody status and the number of oocytes retrieved or fertilized.
This suggested that some women undergoing IVF treatment were
previously sensitized to chlamydial HSP and/or a previously
undetected genital tract infection was still present in these women.
The presence of the detected HSP antibodies was correlated with
an adverse outcome after IVF treatment. The latter relationship
becomes especially obvious, if one takes a closer look at couples
where clinical sequelae of a previous infection like a hydrosalpinx
are present.
In a recent study, IVF patients with tubal occlusion, with or
without hydrosalpinges, were tested for circulating antibodies to
the chlamydial HSP10 which is known to be co-expressed with
chlamydial HSP60 (Spandorfer et al., 1999a). Sera obtained from
women whose male partners were infertile served as control. In
this study clinical pregnancies were documented in 68% of the
women with male factor infertility. This was significantly higher
than the 43.1% rate in women with tubal occlusions (P = 0.04) and
Table II. Suggested mechanism of 60 kDa heat shock protein (HSP60) immune mediated pregnancy failure
A persistent infection (e.g. Chlamydia trachomatis) sensitizes a woman to HSP60 regions present in both microbes and man.
Human (host) HSP60 is physiologically expressed during the pre- and peri-implantation stages of pregnancy by the embryo
and the maternal decidua.
Host HSP60 expression in early pregnancy reactivates lymphocytes previously sensitized to microbial (e.g. chlamydial)
HSP60.
The activated lymphocytes release pro-inflammatory cytokines, which induce also other lymphoid cells to release
inflammatory and cytotoxic mediators.
Cellular and humoral immune system activation disturbs immune regulatory mechanisms necessary to implantation and
maintenance of the embryo. Antibodies to heat shock proteins impair embryo development. Embryos are less protected from
adverse environmental conditions and are more likely to degenerate or undergo apoptosis.
The role of heat shock proteins in reproduction
the 41% rate in women with hydrosalpinx (P = 0.02).
Simultaneously, antibodies to chlamydial HSP10 were more
prevalent in women with hydrosalpinx (46.8%) than in women
with tubal occlusion (15.5%; P = 0.0009) alone or male factor
infertility (6%; P = 0.0001). Interestingly, antibodies to the human
HSP60 were also more prevalent in women with tubal occlusion
plus or minus hydrosalpinx than in women with male factor
infertility. This again provided further proof for an autoimmune
linkage between chlamydial and human HSP60.
In another study 122 IVF subjects were screened for cervical
IgA antibodies to synthetic peptides, which corresponded to the
conserved epitopes of the chlamydial HSP60 (Witkin et al., 1996).
Antibodies to a single epitope, corresponding to the amino acids
260–271 in the chlamydial HSP60 amino acid sequence, were
found to be immunodominant in these patients. More importantly
this epitope was present in both the chlamydial and human HSP60
(Yi et al., 1993). Once again women with this antibody had a
significantly increased prevalence of only transient biochemical
pregnancy (22.2%; P = 0.03) after embryo transfer than did
antibody negative women (7.4%; Witkin et al., 1996). This again
implied that cervical IgA antibody to conserved HSP60 epitopes
expressed in both the human and chlamydial heat shock proteins
and the failure of successful implantation after embryo transfer are
interrelated.
Finally, in a fourth study the detection of chlamydial IgA
antibodies in follicular fluid of IVF patients are correlated with the
presence of human HSP60 antigen in these secretions (Neuer
et al., 1997). The analysis of the clinical diagnosis of these
patients revealed that all women expressing human HSP60 had a
tubal occlusion and failed to become pregnant in their IVF cycle.
In conclusion, the summarized results of these studies revealed
that a previous infection with C.trachomatis and a resulting
immune sensitization to chlamydial heat shock protein epitopes
was associated with a poor prognosis for reproductive outcome
and, in addition, impaired IVF results.
Immunity to human HSP60 and pregnancy outcome
To further elucidate the contribution of human anti-HSP60
antibodies to reproductive failure we determined the prevalence of
IgG antibodies to the human HSP60 in maternal serum of patients
undergoing infertility treatment. The results indicated that serum
IgG antibodies to the human 60 kDa HSP were significantly more
common in patients with arrested in-vitro embryo development
than in IVF patients whose embryos continued to grow and were
transferred to the uterus (Table III, Witkin et al., 1996). This
finding seemed to be of major importance, since in IVF embryos
are sometimes cultured in medium containing maternal serum.
Thus, if a woman was already immunized to conserved HSP60
epitopes and would harbour HSP60 antibodies in her serum, this
could interfere with the in-vitro development of the embryo.
IVF culture and HSP expression
Stressful manipulation of embryos in culture is a daily event in
assisted animal reproductive technology. Embryos are transferred,
cryopreserved, cloned, or microinjected with transgene constructs.
As far as human IVF is concerned, one can also assume that
stressful culture conditions in in-vitro culture enhance HSP
expression in these embryos. Such embryos have to cope with
155
Table III. Relation between circulating immunoglobulin G antibodies to
60 kDa heat shock protein (HSP60) and the outcome of in-vitro
fertilization (IVF). Sera from 155 women were tested
IVF outcome
No. subjects
No. HSP60+ (%)
No fertilization
14
1 (7.1)
Arrested embryo development
13
6 (46.2)a
Not pregnant
75
7(9.3)
Pregnant
53
9 (17.0)
Embryo transfer
aP
= 0.004 versus all others.
handling, oxidative stress, variation of temperature and a
completely altered cellular environment. Premature transfer to the
uterus at the 4–8-cell stage may also lead to both nutritional and
environmental stress. Consequently in the mouse HSP70
expression is found to be 5–15-fold higher in cultured embryos
(Christians et al., 1995, 1997a). Induced expression of HSP due to
environmental factors and constitutive HSP expression may both
represent an essential requirement for successful embryo growth
in an adverse environment. Overexpression of HSP in this
situation is probably to the benefit of the developing embryo.
However, on the contrary failed HSP induction and immunity to
HSP could result in detrimental consequences for the growing
embryo in vitro.
In many IVF culture systems the in-vitro fertilized embryos are
cultured in medium containing maternal serum. Pre-existing HSP
antibodies in these sera at high titres could thus compromise the
growth potential of developing embryos. In a recent study,
antibodies to the most common mammalian HSP exerted a
detrimental effect on mouse embryos at unique developmental
stages (Neuer et al., 1998). In these experiments, 2-cell mouse
embryos (B6D2F1) were cultured in the presence or absence of
monoclonal antibodies specific for mammalian HSP60, HSP70
and HSP90. Embryo development was evaluated after 3, 5 and 7
days in culture by determining the number of blastocysts, hatched
blastocysts and outgrown trophoblasts at the successive time
points. Both anti-HSP60 and anti-HSP70 elicited a strong
inhibitory effect on mouse embryo growth, but at unique
development stages. At day 3, only 29% of the embryos cultured
with HSP60 antibody developed to the blastocyst stage as
compared with 67% of the embryos cultured with anti-HSP70,
72% cultured with anti-HSP90, and 79% in medium plus mouse
monoclonal IgG1, which served as a control. By day 5, hatched
embryos were present in 28% of the cultures containing antiHSP70, as opposed to 57% containing anti-HSP90 and 73%
containing IgG1. At day 7, outgrown trophoblasts were observed
in 9% of cultures containing anti-HSP70, 45% containing antiHSP90 and 66% cultured in medium plus IgG1. These results are
shown in closer detail in Figure 6.
In the presence of HSP antibodies these embryos became
growth arrested and degenerated. Gross morphology of these
embryos revealed irregular sized blastomeres and multiple
fragments. An example of the gross morphology of these embryos
is given in Figure 7. The observed embryos often contained
156
A.Neuer et al.
Figure 7. Examples of degenerated murine embryos after culture in the
presence of heat shock protein (HSP)60 immunoglobulin (Ig)G antibodies.
Figure 6. Effect of antibodies to heat shock proteins (HSP) on developmental
delay of mouse embryos after 3, 5 and 7 days in culture. Embryos were
cultured in Roswell park Memorial Institiute (RPMI) 1640 medium/10% fetal
calf serum (FCS) and monoclonal antibodies to human HSP90, HSP70 and
HSP60 (100 µg/ml). Controls consisted of RPMI/10% FCS and mouse
immunoglobulin (Ig)G1 (100 µg/ml). *P < 0.01 compared with controls; **P
< 0.0001 compared with controls
variable sized, degenerated blastomeres with multiple cellular
fragments enclosed within the zonae pellucidae. Thus, these
embryos resembled apoptotic cells. This is important to note,
since both the production of HSP and programmed cell death are
closely related to each other. Both systems are similarly
considered as supporting systems responsible for the general well
being of an organism and also as cellular responses to
environmental insults. There is a surprising overlap between
stressors inducing stress response and insults initiating apoptosis
in different experimental systems (for review, see Punyiczki and
Fesüs, 1998).
Apoptosis is detrimental to blastocyst formation and leads to
preimplantation embryo death (Jurisicova et al., 1996). The
cellular morphology of apoptosis is characterized by cell
shrinkage, chromatin condensation and membrane blebbing. In
the final stages, the apoptotic cell becomes fragmented into
apoptotic bodies, which are rapidly eliminated by phagocytes.
However in the early stages of apoptosis extensive DNA
degradation occurs. Cleavage of the DNA may yield doublestranded, low molecular weight fragments (mono-and
oligonucleosomes) as well as single strand breaks (‘nicks’) in the
high molecular weight DNA. Those DNA strand breaks can be
detected by enzymatic labelling with modified nucleotides
(dUTP). Terminal deoxynuleotidyl transferase (TdT) labels blunt
ends of double-stranded DNA breaks. The end-labelling method
has also been termed TUNEL (TdT-mediated X-dUTP nick-end
labelling). The use of fluorescein-dUTP to label the DNA strand
breaks allows the detection of the incorporated nucleotides
directly with a fluorescence microscope.
Mouse in-vitro co-culture studies
To further assess the impact of HSP antibodies on embryo
development we TUNEL stained murine embryos that had been
grown in an endometrial co-culture system in the presence of
varying concentrations (10, 50 and 100 µg/ml) of monoclonal
antibodies to HSP60. In this culture model, a total of 160 2-cell
murine embryos from B6D2F1 mice were grown under two sets of
conditions. Half of the embryos were grown using 10% fetal calf
serum (FCS) in Roswell Park Memorial Institute (RPMI) 1640
culture medium in varying concentrations of antibodies to
mammalian HSP60. The rest were grown in an endometrial coculture (ECC) system in addition to the same medium and the
same antibody concentrations. Endometrial co-culture tissue was
obtained from a fertile patient and consisted of an equal mixture of
stromal and glandular cells. Embryonic development to the
blastocyst stage (B), hatching stage (H) and outgrowth stage (O)
was analysed. The control embryos (not grown in antibodies to
HSP60) progressed to the B, H and O stages in 95, 70 and 70% of
cases respectively. In the study group without ECC, embryo
growth was inhibited at a concentration of 100 µg/ml anti-HSP60
antibodies at each stage of development (B 25%, H 15% and O
15%, P < 0.001). Utilizing the ECC system, control embryos (not
grown with antibodies to HSP60) progressed to the B, H and O
stages in 100% of cases for each stage, respectively. In this ECC
model, toxicity was only evident at a concentration of 100 µg/ml
of HSP60 antibody (B 80%, not significant; H 70%, P = 0.02; and
O 60%, P = 0.003). At the highest concentration of antibodies
used, the growth inhibition exhibited was always significantly less
in the ECC model. In addition TUNEL positivity was more
frequent in embryos exposed to antibodies to HSP60 than in
unexposed embryos (30/43 versus 6/17, P = 0.03). In Figure 8, an
example of TUNEL positivity in a murine blastocyst after
exposure to HSP60 antibodies is shown.
Recently in another set of experiments the above-described invitro model was applied to further assess the role of hydrosalpinx
fluid on embryo growth (Spandorfer et al., 1999b). This
experimental design was chosen because hydrosalpinx formation
is very prevalent in women with previous infections and tubal
occlusion. In addition it has been suggested that hydrosalpinx
formation influences pregnancy rates after IVF. Thus we
speculated that hydrosalpinx fluid might contain antibodies to
HSP60. Table IV displays the HSP antibody, cytokine and
chlamydial antibody content of hydrosalpinx fluid. Interestingly
The role of heat shock proteins in reproduction
157
Figure 8. Example of TdT-mediated X-dUTP nick-end labelling (TUNEL)-positive fluorescence. This murine blastocyst was cultured in the presence of 100 µg/
ml heat shock protein (HSP)60 antibodies. Distinct foci of TUNEL-positive fluorescence with varying intensity are visible. Figure on left shows TUNEL-stained
blastocyst; figure on right shows light microscopy. Original magnification ×200
Table IV. Detection of antibodies and cytokines in hydrosalpinx fluids
from 16 women. Values in parentheses are percentages
Compound
No. positive (pg/ml)
Human HSP60 IgG
6 (37.5)
Chlamydia HSP10 IgG
9 (56.3)a
Chlamydia IgG
2 (12.5)
Human HSP60 IgA
2 (12.5)
Chlamydia HSP10 IgA
1 (6.3)
Chlamydia IgA
5 (31.3)
Mean
11 (68.8)b
79
c
10 (62.5)
533
Interleukin-1β
9 (56.3)
24
Interleukin-6
3 (18.8)
42
Interleukin-10
3 (18.8)
10
Interferon-γ
Interleukin-1 receptor antagonist
Ig = immunoglobulin.
= 0.02 versus Chlamydia immunoglobulin (Ig)A; bP = 0.01 versus
interleukin (IL)-6, IL-10; c P = 0.02 versus IL-6, IL-10.
aP
both human HSP60 and chlamydial HSP are present in
hydrosalpinx fluid.
In a mouse model we have previously shown that anti-HSP60
and anti-HSP70 antibodies exhibited a growth inhibiting effect at
unique developmental stages of murine embryos (Neuer et al.,
1998). In this model, we used an endometrium co-culture model
that mimics the in-utero conditions of IVF patients with
hydrosalpinx. Since hydrosalpinx formation is most often the
sequela of a previous or repeated infection, antibodies to HSP60
are a possible factor in the toxicity exhibited by hydrosalpinx
fluid. Women with tubal occlusion undergoing IVF treatment with
or without hydrosalpinx harbour bacterial and human HSP
antibodies in their sera at a high rate (Spandorfer et al., 1999a).
The endometrium appears to reduce the toxicity of these
antibodies. As demonstrated by TUNEL staining, a possible
mechanism of this toxicity may involve the induction of apoptosis.
HSP are, as outlined above, essential for successful completion of
the single developmental stages of an embryo. The specific
expression pattern of HSP may thus play both an essential role in
differentiation and a protective role against apoptosis (Mailhos et
al., 1993). Since embryos exposed to anti-HSP60 stained
TUNEL-positive more often than unexposed embryos it is
possible that HSP antibodies may render an embryo more
susceptible to apoptosis. However, the precise mechanism of antiHSP related inhibition of mouse embryo development and
apoptosis is not yet clearly understood and further studies are
needed to clarify the function and clinical relevance of these
findings.
Acknowledgements
The technical assistance and encouragement of Zhing He, Carol
Mele, H.-C. Liu, Simiak Tabibzadeh, Pedro Esponda, Sergio
Olivera, Rob Soslow, Jan Jeremias, Vera Tolbert, Ann Marie
Bongiovanni, Peter Ruck, Klaus Marzusch, Ludwig Kiesel and
the critical review by H.M.Vaihinger are gratefully
acknowledged.
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Received on June 9, 1999; accepted on October 22, 1999