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Millions of spermatozoa enter the female reproductive tract at coitus. In many species the spermatozoa ascend to the oviduct and bind tightly to oviductal epithelial cells (OECs) until ovulation takes place. Such sperm-oviductal epithelium binding favours the formation of a sperm storage reservoir for holding spermatozoa that are competent for fertilisation (Rodriguez-Martinez, 2007). The principal site of sperm storage is in oviduct which provides a supportive environment by producing a host of factors for both of the gametes, as well as for the maturation and development of the embryo.
Several sperm properties that are essential for successful reproduction have been shown to be maintained, regulated or improved in the oviduct. Sperm maturation known as capacitation is promptly induced and the speed of the process is finely modulated in the oviduct (Hunter and Rodriguez-Martinez, 2004, Rodriguez-Martinez, 2007). Sperm motility is also modulated in favour of formation of a sperm reservoir and oocyte penetration at ovulation (Hunter and Wilmut, 1984). Moreover, sperm- OEC interactions maintain sperm viability during the storage period in order to ensure an appropriate number of viable sperm are available at the time of ovulation (Pollard et al., 1991, Smith and Yanagimachi, 1990).
Heat Shock Proteins (HSPs) are families of molecules that exist nearly in all types of living organisms. They function as cellular chaperones and protect the cells from stressful stimuli by controlling cellular protein structure, transportation and function (Hendrick and Hartl, 1993). They also regulate cellular apoptosis and immunogenicity (Garrido et al., 2001, Schmitt et al., 2007).
HSPs are produced and present in mammalian reproductive tract, particularly in the oviduct. In mammalian reproduction, HSPs perform their protective role by protecting gametes and embryos from stressful conditions in their surrounding environment (Neuer et al., 2000). They also take part in other reproductive processes such as enhancement of sperm viability (Elliott RM, 2009), increasing the fertilisation rate and accelerating embryonic development (Neuer et al., 2000, Neuer et al., 1998).
This literature review will provide a brief overview of the biology and physiology of maternal interactions with gametes and embryos and will focus on the final maturation of gametes, fertilisation and early embryonic development. It will describe the environment in which these events are taking place and will consider HSPs and their role in maternal communication with gametes and the embryo.
2.Characters of mammalian reproduction:
2.1. Female reproductive tract:
The female reproductive tract is composed of a number of tubes with different anatomies and functions.
The Vagina serves as the copulatory segment in which the spermatozoa are deposited at intercourse.
The Cervix acts as a barrier between external and internal environment of the female reproductive tract.
The Uterus is the site for embryo implantation and development.
The Fallopian tubes, also called oviducts, are two open-ended tubes, each of them can be divided to three parts. The proximal segment is the Isthmus, which is narrow enough to trap spermatozoa to form a reservoir. The Ampulla is the middle and widest part in which fertilisation occurs. The third segment is named the Infundibulum. It opens onto the ovary and so acts as a passage for the female gamete which is released from the ovaries to reach the spermatozoa.
The Ovaries (one on each side) are the female reproductive glands that are responsible for oogenesis and storage of the oocytes. They also produce the reproductive hormones that are essential for reproduction.
Figure 1. Gross anatomy of the female reproductive tract.
The oocyte or egg is the female gamete which is produced in the female ovary via the process of oogenesis during early fetal life. Each ovary contains a definite number of eggs. Like sperm, the oocyte nucleus contains a haploid set of chromosomes (Ernst Knobil et al., 1994).In a newly born female baby each ovum is surrounded by a granulosa cell layer which provides nourishment for the ovum. The complex of the ovum and its surrounding granulosa cell layer is called a follicle. Follicles remain in their primordial state till puberty. Then begin to grow under the effect of the sex hormones (Arthur C. Guyton, 2000). At each menstrual cycle usually only one oocyte is released and fertilised by sperm in the ampulla, should sperm be present in the near vicinity.
Spermatozoa are the male gametes. Mammalian spermatozoa are characteristically tiny cells that vary in length from 28µm to 394µm in different species (Gage, 1998). Spermatozoa are formed in male reproductive gonads named testes. Each spermatozoon consists of three major parts:
The Head contains the nucleus with only one of each chromosome pair, the top of which is covered by a layer named acrosome. The acrosome contains the enzymes that are needed to penetrate the ovum (Ernst Knobil et al., 1994). The sperm head is responsible for interactions with the environment.
The Middle Piece holds a large number of mitochondria and therefore provides energy for sperm movement.
The Tail or flagellum is the device for moving the sperm towards the egg by creating forces (Ernst Knobil et al., 1994).
Figure 2. Different segments of the mammalian spermatozoon.
The embryo is the product of mitotic divisions in zygote after fertilization. The created single cell zygote starts its cleavage immediately after the gamete interaction, during which time it is transported to the uterus and becomes implanted in the uterine wall. Here, it proliferates and further develops into a complete organism (Carlson, 1999).
Figure 3. A schematic picture of the process of fertilisation, zygotic division and embryo implantation in the female reproductive tract.
The mammalian female reproductive years are characterized by rhythmical alterations in the secretion of a group of hormones and related physical changes in the female reproductive tract most notably ovaries, oviducts and the uterine endometrium. This cyclical pattern is called female monthly sexual cycle or menstrual cycle (Arthur C. Guyton, 2000).
The rhythmic property of the female sexual cycle is dependent on a complex hierarchy of hormones secreted from the hypothalamus named Gonadotropin releasing hormone or Gn Rh which regulates the anterior pituitary gland secretion of Follicular stimulating hormone (FSH) and Luteinizing hormone (LH) or sex hormones (Arthur C. Guyton, 2000). The latter two steroid hormones control production of Oestrogen and Progesterone by the ovaries. These two steroid hormones exert direct effects on the female reproductive tract.
The secretion of the hormones are not in a constant rate and fluctuate throughout the cycle which is responsible for inducing different patterns in the female reproductive tracts during the cycle (Arthur C. Guyton, 2000).
The female monthly menstrual cycle has two major results: First, usually only a single oocyte is released from the ovaries in the middle of each cycle, so that if fertilization happens only one fetus will be produced. Second, the uterine endometrium gets prepared for implantation of the fertilised ovum at the appropriate time of the cycle.
Menstrual cycle can be divided into two distinct phases by ovulation. Events in each phase are specific to each organ. During the first half, named as follicular phase in the ovaries there is an accelerated growth of a few number of primary follicles under the effect of moderately increased FSH. After several days one of the follicles begins to outgrow all the other stimulated follicles. The outgrown follicle reaches its mature size right before ovulation. In the middle of the ovarian follicular phase as the follicles continue to grow larger, oestrogen is secreted from the granulose cells surrounding the follicles and reaches to its highest level before ovulation (Arthur C. Guyton, 2000).
The first phase of the cycle in the endometrium is known as proliferative or oestrogen phase and contains two distinct parts. During the initial days of the phase most of the endometrium becomes desquamated (Arthur C. Guyton, 2000) and the cells start shedding. Covert bleeding happens only in human and close species like chimpanzees and is known as menstrual bleeding. In other female mammals, it is called estrous and shedding is totally absorbed. Shedding continues to the extent that only a thin layer deeper epithelial cells remain. As the ovarian oestrogen level rises to a certain level, desquamation stops. The endometrial epithelial cells proliferate very quickly and increase the endometrial thickness till ovulation happens.
Ovulation occurs in the middle of the menstrual cycle along with a surge in LH level in blood. The follicular out layer of the mature oocyte ruptures and the ovum is released to the ampulla. Here the ovarian luteal phase begins. The remaining follicular cells named corpus luteum after the ovum expulsion is a secretory organ and secretes large amounts of Progesterone and oestrogen (Arthur C. Guyton, 2000).
These hormones induce strong inhibitory feedback on FSH and LH and decrease their blood levels. Also they cause additional cellular proliferation as well as swelling and secretory development of the endometrium. Therefore the latter phase of the cycle is known as the secretory phase in the uterus. The main purpose of the endometrial change during the secretory period is to produce a highly secretory endometrium which contains large amounts of nutrients to provide an appropriate condition for the fertilized ovum to implant in the uterine wall (Arthur C. Guyton, 2000).
If the ovum is not fertilised, the corpus luteum in the ovaries degenerate near the end of the cycle and therefore the ovarian hormones, Oestrogen and Progesterone decrease to low levels in the blood (Arthur C. Guyton, 2000). Menstruation follows and a new cycle begins.
3. Physiology of reproduction: from sperm and oocyte to embryo:
The spermatozoa formed in the male testes are deposited inside the female reproductive tract via coitus. Although morphologically perfect, the sperm requires further maturation in order to be able to fertilise the oocyte. To acquire this full functional maturity, the sperm ascends up to the oviduct and establishes a strong bond with the oviductal epithelium to form a reservoir (Hunter et al., 1987, Rodriguez-Martinez, 2007, Suarez, 2002).
By releasing the oocyte from the ovary, the inter sperm-oviduct bond disappears and sperm becomes free to penetrate the egg. After fertilisation, the resulting zygote migrates to the uterus, into which it becomes implanted and then grows into a fully grown embryo untill the end of pregnancy.
This Literature Review focuses on the oviductal sperm reservoir and its influence on fertilisation.
3.1. Sperm in the female reproductive tract:
Of the many millions of spermatozoa that are deposited in the lower female reproductive tract at coitus, only a few thousands can get past the uterus and only one
will fertilise the egg (Petrunkina AM, 2001).This gradual reduction in sperm number is due to the sieving action of the lower parts in the female reproductive tract (Smith et al., 1987).
Sperm can spend from hours to days in the mammalian female genital tract (Rodriguez-Martinez, 2007). The female reproductive tract and particularly the caudal segment of the isthmus have a special responsibility for regulating a protective pre-ovulatory sperm reservoir prior to its interaction with oocyte.
The isthmus has been located as sperm storage site during female estrous (pre- ovulatory) period in several mammalian species including mice (Suarez, 1987), guinea pigs (Yanagimachi R, 1976), hamsters (Smith et al., 1987), rats (Shalgi and Kraicer, 1978), rabbits (Overstreet and Cooper, 1978), pigs (Hunter, 1981),sheep (Hunter and Nichol, 1983) and cows(Hunter and Wilmut, 1984).
3.2.Role of isthmic sperm reservoir in reproduction:
The isthmic sperm reservoir provides a conduit for a secure number of spermatozoa to pass through to the oocyte (Suarez, 2008). Experiments in which the isthmus has been resected, have increased the incidence of polyspermia ( the entrance of more than one spermatozoon into the ovum) (Hunter & Leglise 1971).
The isthmic sperm reservoir also functions as an environment to maintain sperm viability and motility. (Suarez ss, 2002). Bovine sperm motility and fertility are maintained longer in vitro when incubated with oviductal epithelium compared to other types of epithelial cells or in medium alone (Pollard et. Al 1991).
Thirdly, The isthmic sperm reservoir regulates the sperm maturation to ensure successful fertilisation (Smith 1998).
3.3. Mechanism of sperm reservoir formation:
Anatomically, the proximal isthmus has a very narrow lumen and thicker tunica muscularis as compared with the ampulla (Suarez et al., 1991).The lining mucosa is arranged in folds,to creating blind channels (Suarez, 2002). In addition, this narrow lumen is filled with thick sticky mucous (Suarez, 2002).Also, oedema of the isthmus wall (Boyle et al., 1987), and a decrease in sperm motility (Overstreet and Cooper, 1978, Suarez, 1987) are all known to play part.
Furthermore when spermatozoa come to close contact with oviductal epithelium, they form a strong bond with oviductal epithelial cells (OECs). This tight attachment is known as a significant contributing factor to formation of the oviductal sperm reservoir (Suarez, 2002, Hunter, 1981, Hunter and Nichol, 1983).
3.4. Direct sperm- oviductal epithelial cell interaction:
Using scanning electron micrograph, Hunter 1987 & 1991 showed direct association between the somatic oviductal epithelial cells and sperm in mated pigs and cows in vivo (Hunter et al., 1987, Hunter et al., 1991). Similar associations between sperm and oviductal epithelial explants (Pollard et al., 1991, Suarez et al., 1991) and cell monolayers (Ellington et al., 1993, Ellington et al., 1991) have been observed in vitro.
Inter epithelium- sperm contact takes place between the intact acrosomal region on the sperm head and apical segment of oviductal epithelial cells.(Pollard et al., 1991, Suarez et al., 1991)
The establishment of such an intimate contact is due to presence of ligands (lipo polysaccharides) on epithelial cells and complementary receptors (lectins) on the sperm head (Topfer-Petersen et al., 2002).
The treatment of sperm- epithelial cell co-cultures with competitive inhibitors that cross react with receptors, reduces the number of sperm that are bound to OECs (Suarez, 2001, Suarez, 2002). Apparently, species possess their unique epithelial cell sugar ligands and complementary sperm protein receptors, for example terminal sialic acid in hamster; galactose in horse and fucose in cattle oviductal epithelium (Suarez, 2001, Suarez, 2002).
Figure 4. Scanning electron micrograph of spermatozoa bound to the oviductal epithelium. (Photograph kindly provided by Dr. Edita Sostaric)
3.5.The effect of sperm- OEC binding on sperm characteristics:
The discovery that spermatozoa come into direct contact with the oviductal epithelium posed questions concerning the physiological impacts of this intimate interaction. Reports from a large number of experiments indicate that oviductal-sperm interactions are of physiologic significance rather than a mere physical attachment.
3.5.1.Effect of sperm- OEC binding on sperm maturation (capacitation):
Spermatozoa are formed via the process of spermatogenesis in the male genital tract (testes). Initially, spermatozoa do not possess thr pre-requisites that are required for fertilisation such as motility, fertilising ability or morphological differentiation (Ernst Knobil et al., 1994). Gradual transportation down the male genital tract, exposes the immature sperm to local and hormonal secretion that are partially beneficial to sperm maturation. Although ejaculated testicular sperm is morphologically complete, it is not of sufficient functional maturity for immediate egg fertilisation (Ernst Knobil et al., 1994).
About 50 years ago, CR Austin, reported that ejaculated spermatozoa are incapable of penetrating the oocyte if inseminated directly at the time and site of ovulation. Spermatozoa must spend some time in the female reproductive tract in order to achieve the ability of successful egg penetration (Austin, 1951). Therefore, the term 'capacitation' was coined in the field of reproductive biology to illustrate a process involving biochemical, biophysical and metabolic modifications of all sperm domains (Rodriguez-Martinez, 2007) for the attainment of full maturity in the female reproductive tract. Capacitated spermatozoa are principally able to fertilise the oocyte.
Sperm capacitation happens gradually in vivo during sequential exposure of spermatozoa to the different compartments of the female reproductive tract and is completed in the oviduct (Rodriguez-Martinez, 2007). The female reproductive tract induces sperm capacitation by removal of seminal plasma and epididymis proteins that coat the sperm membranes (Rodriguez-Martinez, 2007).
Oviductal isthmus is a specialized part of the female reproductive tract in modulating the capacitation process in spermatozoa (Smith, 1998, Hunter et al., 1998).
Co- incubation of sperm with isthmic cells delays capacitation (Dobrinski et al., 1997) and sperm- epithelial cell adhesion terminates as the capacitation status in attached spermatozoa completes (Lefebvre and Suarez, 1996). Since capacitated sperm dies very quickly which is an undesirable event in fertilisation process (Rodriguez-Martinez, 2007) the physiological delay prolongs sperm life till ovulation to synchronize sperm function with the ovulation time (Rodriguez-Martinez, 2007). While ovulation happens and oocyte is released in the oviduct, a chemotactic mechanism will draw the spermatozoa to the egg (Eisenbach, 1999). This is when the sperm- OEC bond disappears and viable capacitated sperm movement towards the egg gets started.
On the other hand, exposure of sperm to isthmic fluid leads to rapid sperm capacitation (Hunter and Rodriguez-Martinez, 2004).
Although sperm capacitation can be mimicked by particular capacitating media in vitro, the major difference between the laboratory and natural conditions is the rate of sperm capacitation which occurs at once in media but only very gradually inside the female reproductive tract in response to ovulation.
These observations highlight the regulatory and synchronizing role of the oviduct in the sperm capacitation process in order to ensure the presence of an appropriate number of competent sperm at the time of ovulation.
3.5.2. Effect of sperm- OEC binding on sperm motility:
Although motility is important to the transportation of spermatozoa inside the female reproductive tract, sperm are temporarily immotile temporary in the isthmic oviduct which is assumed as a contributing factor to sperm reservoir formation. The first in situ observation of sperm motility within oviduct confirmed the immotile phase for the spermatozoa which are held within mouse isthmus (Suarez, 1987).
Hunter and Wilmut 1984 reported sperm that are in a quiescent state in the oviduct(Hunter and Wilmut, 1984) for up to 20 hours in cows or 10 hours in rabbits ((Hunter and Wilmut, 1984, Smith and Nothnick, 1997) during the pre- ovulatory period.
Spermatozoa bathe in isthmic secretions and are in contact with oviduct- specific proteins, enzymes, glycol- and lipoproteins (Rodriguez-Martinez, 2007). The transient motility depression in stored sperm in the isthmus can be either due to the presence of a motility inhibitory component or lack of a motility stimulatory factors in the region (Suarez, 1987). Reprorts from Experiments in rabbits showed that most of the sperms recovered from the isthmus were immotile and the sperm regained motility when incubated with ampullar fluid (Overstreet et al., 1980).
Grippo and colleagues evaluated the effect of fluids collected from two oviductal regions at luteal and non-luteal phases on sperm motility and demonstrated the capacity of isthmic secretions to inhibit sperm motility (Grippo et al., 1995). Following these observations presence of a molecular motility inhibitor in the rabbit oviduct was reported (Overstreet et al., 1980). It is also sensible to conclude that sperm motility would be restricted whilst they form strong bonds with OECs.
Furthermore, around ovulation time, quiescent spermatozoa which are bound to isthmic epithelium exhibit a hyperactivated state in response to chemotactic mechanisms from the oocyte (Eisenbach, 1999), detach from the epithelium and swim up towards the egg. The low number of spermatozoa recovered from the ampulla during their travel to fertilization site (Smith and Nothnick, 1997, Overstreet and Cooper, 1978) explains the gradual controlled release of highly motile sperm from the reservoir.
All mentioned results suggest that the isthmus has a major role in synchronizing sperm transport and ovulation by keeping the spermatozoa quiescent prior to ovulation and sustained release of spermatozoa in response to ovulation. This way it prevents accumulation of a large number of sperm at ovulation site and consequent undesirable polyspermy.
3.5.3.Effect of sperm- OEC binding on the maintenance of sperm viability:
Mating in mammals usually occurs either around the time of, or at ovulation. The period between sperm insemination in the female reproductive tract and ovulation can be a few hours to a few days. There must therefore be a mechanism to preserve the fertile life span of the stored spermatozoa inside the female reproductive tract and particularly the isthmus (the site where accommodates the sperm for the majority of its life in the female reproductive tract).
In vivo and in vitro investigations in hamsters (Smith and Yanagimachi, 1990) and cows (Pollard et al., 1991) have illustrated that adherence of sperm to epithelial cells in isthmus has a beneficial effect on sperm viability.
The responsible mechanism can be either due to presence of epithelial secreted products at the site of interaction, or sperm's direct interaction with the apical plasma membrane of the epithelial cells.
In this regard,a 95 kDa glycoprotein component of bull oviduct secretion has been shown to enhance the viability of homologous sperm in vitro (Abe et al., 1995). However, studies in which sperm cells were brought into direct contact with vesicles isolated from apical plasma membranes of rabbit and equine oviduct (Smith and Nothnick, 1997, Dobrinski et al., 1996), supported the idea that co- incubation of sperm along with secretory products of isthmic epithelium is not sufficient to enhance cell viability (Smith and Nothnick, 1997), and that direct interaction of epithelium with spermatozoa is essential (Suarez, 2008).
A similar study in pigs showed a dose- dependent improvement of sperm viability after a 24 hour co-incubation of spermatozoa with apical plasma membranes isolated from pig oviductal epithelial cells (sAPM) (Fazeli et al., 2003). The effects on viability were abolished when sAPM proteins were denatured, thereby suggesting proteins as effectors (Fazeli et al., 2003).
Proteomics has since identified a 60kDa protein and 70kDa (HspA8) heat shock protein in bovine and porcine sAPM which are assumed to be responsible for enhancing sperm viability in vitro (Boilard et al., 2004, Elliott RM, 2009).
Figure 5. Model of sperm-oviductal epithelial cell interaction and consequent alterations in sperm characteristics.
4. Introduction to Shock Proteins:
Living organisms are in a constant dynamic interaction with their surrounding environment. Amongst all environmental factors they face, many of them can exert deleterious impacts. In order to survive stressful conditions, organisms are equipped with evolutionary defence machinery which includes a set of complex proteins with general and specific properties termed 'Stress' or 'Heat Shock Proteins'. Heat Shock Proteins (HSPs) are vital to organism growth, development and survival.
5. History of HSPs:
The discovery of heat shock proteins dates back in 1962 when Ferruccio Rittosa observed a new puffing pattern on the fruit fly's salivary gland chromosomes (Ritossa 1962) after one of his technicians accidentally shifted the incubator temperature to a non- physiological level. He repeated the experiment with normal temperature controls and observed similar puffing pattern along with new RNA synthesis. The rapidity of the new RNA synthesis, just in 2-3 minutes was impressive (Ritossa, 1996).
He concluded that the increase in temperature was responsible for immediate changes in chromosome pattern and gene expression and he called the phenomenon a heat shock response.
5.1.What is the Heat Shock Response?
The heat shock response is a remarkably conserved natural adaptive response (Moseley, 2000). Organisms acquire biologic tolerance to lethal temperatures ( thermotolerance) after brief exposure to sub-lethal temperatures via a rapid, but transient reprogramming of cellular activities. This rapid response is known as heat shock response and takes place in bacteria, plants and animals. Heat shock response ensures survival and protects crucial cellular components. In this way, stressed cells can carry on their normal function over the recovery phase.
5.2. Characterization of Heat Shock Response:
After Rittossa's report on alteration of gene activity induced by heat shock in Drosophila (Ritossa 1962), scientists started investigations to elucidate the phenomenon " Heat Shock Response".
Over the next ten years the response was studied principally at the cellular level, and several important observations were made (Lindquist, 1986):
Induction of puffs by several other stress factors (Ritossa 1962, Leenders and Berendes, 1972).
Puffs were produced within a few minutes of stress treatment (Berendes, 1968).
Appearance of puffs was associated with new RNA synthesis (Ritossa 1962, Leenders and Berendes, 1972).
Puffs were produced in other types of fruit fly and many other tissues (Ritossa, 1964, Berendes, 1965).
It took sometime before it was appreciated that the response is mediated via a transient activation of a number of genes. The genes previously silent or active at low levels were activated by stress and transcribed into a number of specific proteins (Burdon, 1986).
In 1974 for the first time, Tissieres & Mitchell reported a few number of proteins which were expressed coincidentally with chromosome puffs in Drosophila (Tissieres et al., 1974). Since the expression of the proteins were originally discovered as a consequence of exposure to thermal (heat) stress, these products of the genes were collectively termed 'Heat Shock Proteins'.
For a while induction of the heat shock response was thought to be restricted to Drosophila and certain tissues. In 1978, Schlesinger discovered an analogous response to heat shock in cultured avian and mammalian cells (Kelley and Schlesinger, 1978) . Studies in E.coli (Yamamori et al., 1978) and Tetrahymena (Fink and Zeuthen, 1980) revealed similar results. Following these observations, heat shock proteins were recognised as being present in almost every cell and tissue type, in explanted tissues and cultured cells (Lindquist, 1986) and in all organisms from bacteria to human.
5.3.Heat Shock Proteins:
Heat Shock/ Stress Proteins (HSPs), are a family of functionally-related proteins. They were initially found to be expressed intracellularly in the cells subjected to stressful conditions. Their expression is transcriptionally regulated and they are believed to be part of a stress response which has evolved to protect stressed cells from adverse insults.
Not long after their discovery, several of the major HSPs were also shown to exist in cells under natural conditions and to be essential for normal physiological cell function, growth and development (Schlesinger, 1990). For example HSP60, HSP 90 and HSC70 are constitutively expressed under normal conditions in mammalian cells, whereas, HSP70 and HSP27 are induced by various stressful stimuli (Garrido et al., 2001).
Simultaneously, most of the heat shock proteins were found to be induced by other stressful agents. Although the particular effects vary among organisms, anoxia, ethanol, certain heavy metals and chemicals and free oxygen radicals induce the expression of the proteins in almost all cells (Lindquist and Craig, 1988).
The significant common feature of HSPs is that they are highly conserved during evolution among divergent organisms (Schlesinger, 1990). It means not much change has occurred in the amino acid sequence and structure of HSPs during evolution (Neuer et al., 2000). For example, the major HSP70 protein has about 50% of its genomic sequence conserved between E.coli and human, (Schlesinger, 1990) or 73% between human and Drosophila (Hunt and Morimoto, 1985).
5.4. HSPs, extra or intracellular proteins?
Untill relatively recently, the general perception was that mammalian HSPs merely exist in intracellular compartments and the only circumstance they were found extracellularly was in pathological conditions such as necrotic cell death (Hightower and Guidon, 1989). Recently a number of scientists have reported some types of the HSPs to be present in the peripheral circulation (Pockley et al., 1998), extracellular fluid and bound to cell membrane of normal individuals (Schmitt et al., 2007) .
5.5. Categories of Heat Shock Proteins:
The HSPs are classified into families on the basis of their estimated molecular weights in kilodaltones (Garrido et al., 2001, Lindquist and Craig, 1988).
The most comprehensively studied and functionally significant families of molecules are:
This family includes two major isoforms of HSP90, Î± and Î², which are integral to cell survival. They exist constitutively in high amounts and represent about 1-2 % of the cellulr protein content (Lanneau et al., 2008). A significant function of HSP90s is their selective interaction with protein kinases transcription factors and most notably steroid receptors such as those for oestrogen and progesterone. Through binding, HSP90 keeps the receptor in their inactive state and prevents further transcriptional activity until the appropriate hormone signals interfere (Lindquist and Craig, 1988).
This family is the most highly conserved family of HSPs and contains proteins ranging from 66-78 kDa. Some are primarily localized in the cytosol such as the inducible HSP70 or constitutive HSC70, whereas others are located in mitochondria and others in the endoplasmic reticulum (Lanneau et al., 2008). Members of this family need to bind with ATP molecules in order to fulfil their protective role in cells (Lindquist and Craig, 1988).
Also called chaperonins, members of this family are primarily located in mitochondria. Although principally expressed constitutively, slight elevations of HSP60 members can be seen under stressful cinditions, particularly heat. Its ATP dependant function is regulated by binding to HSP10 (Lanneau et al., 2008).
Members of this group vary in size between 15 to 30 kDa and have sequence homologies and biochemical properties in common. The most studied member is HSP27, an ATP independent molecule whose function is to prevent proteins from aggregating. Its expression occurs very late after exposure to several stresses. It is expressed in very high amounts in cancer cells and is associated with cellular resistance to anti-cancer therapies (Lanneau et al., 2008).
6.General functions of HSPs:
Based on the incident of their discovery, HSPs were initially renowned for the protective role they play in cells against stressful stimuli. This is known as chaperone function inside the cells. According to a traditional definition chaperones are "a family of unrelated classes of protein that mediate the correct assembly of other poly peptides, but are not themselves components of the final functional structure". (Ellis and van der Vies, 1991).
In other words, chaperones are intracellular proteins that bind to and stabilise other unstable proteins. Stabilisation refers to folding, the oligomeric assembly of newly synthesised poly peptides, their transport to particular sub-cellular compartments, switching between active and inactive conformations and preventing deleterious aggregation of target proteins (Hendrick and Hartl, 1993).
HSP chaperoning activity was initially thought to be mainly limited to stressful conditions. However abundant expression of different types of functional HSPs in normal unstressed cells led to the present concept. We now certainly know that in vivo homeostatic action of HSPs covers both stressful and normal cellular situations.
7.Specific functions of HSPs:
In addition to their homeostatic and protective properties when in the intracellular environment, HSPs can elicit either innate or adaptive immune response when located in the extracellular space or on the plasma membrane (Schmitt et al., 2007).
Members of HSP70 and HSP90 families are the principal regulators of the host immune system (Schmitt et al., 2007), and this capacity is manifested via three different pathways:
HSPs have been reported to be important in the process of cross-presentation of tumor-derived antigenic peptides on antigen presenting cells (APCs) to appropriately-specific responding CD4+ and CD8+ T cell populations. Exposure of the antigen initiates an antigen specific cellular immune response . This is named as peptide carrier function. (Wells and Malkovsky, 2000)
Tumour cells have been identified as being natural source for extracellular HSPs and particularly HSP70. HSPs initiate the secretion of pro-inflammatory cytokines including TNF-Î±, IL-1, 2, 6 and...(Asea et al., 2000) via interactions with TLRs and CD-14 on APCs. This is the cytokine inducing effect of HSPs
(Schmitt et al., 2007).
Tumour cells have been found to have the capacity of specific expression of HSP72, a member of HSP70 family, on their surface (Multhoff et al., 1995). HSP70 selectively expressed on the membrane of tumour cells acts as a target recognition structure for activated NK cells. The interaction of membrane HSP70 positive tumour cells with activated NK cells result in a perforin independent, granzyme B-mediated killing of the former by the latter (Schmitt et al., 2007).
7.2. Regulation of cell apoptosis:
Apoptosis is a natural process of cell death which results from a series of genetically programmed events that removes old or unhealthy cells.
Two pathways lead to apoptosis: the intrinsic pathway in which the mitochondria acts as a coordinator of the catabolic reactions leading in apoptosis, and the extrinsic pathway in which plasma membrane death receptors initiate the process via interaction with intracytoplasmic apoptotic effectors (Garrido et al., 2001).
Figure 6. Schematic model of intrinsic cell apoptosis.
Figure 7. Schematic model of extrinsic cell apoptosis.
Some HSP members regulate apoptosis pathway at multiple stages in the cell.
HSP27 exerts anti-apoptotic effect by neutralising the toxic effects of oxidised proteins (Rogalla et al., 1999) and cooperating with other intrinsic anti-apoptotic members (Garrido et al., 1999) . It has also been shown to block death receptor pathway by deactivating the associated receptors (Garrido et al., 2001).
On the other hand, HSP70 increases the survival of cells by protecting them from energy deprivation related to cell death (Wong et al., 1998), or by reducing mitochondrial damage, nuclear fragmentation (Buzzard et al., 1998) and blocking the apoptotic activator factors in intrinsic pathway. Extracellular HSP70 promotes the killing of tumour cells by interacting with lipid complexes on their membrane (Schilling et al., 2009).
In contrast, HSP60 and HSP90 at some circumstances appear to act as pro-apoptotic effectors in cells along with their original cytoprotective role.
Categories Main Members Co-chaperones Location Type of Expression Main Function
HSP90 HSP90Î± p50/Cdc37, Cytoplasm Constitutive Regulation of tyrosine
P23, Aha1 kinase & steroid hormone
HSP90Î² Cytoplasm Constitutive transcriptional activities,
HSP70 HSP70 HSP40,110 Cytosol/Nucleus Stress Induced Protein transport, repair&
HSC70 HSP40,110 Cytosol/Nucleus Constitutive assembly, Anti-apoptosis
GRP75(Mortalin) DnaJ Mitochondria Constitutive
Bip(GRP78) DnaJ ER
HSP60 HSP60(Chaperonin) HSP10 Mitochondria Constiutive Prevention of protein
TCP1 HSP10 Mitochondria Induced aggregation& misfolding,
sHSP HSP27 Cytosol / Constitutive Anti-apoptosis ,
Î±Crystaline Membrane / & Prevention of protein
Î²Crystaline Nucleus Induced aggregation
Table 1. Families of the Heat Shock Proteins, their related properties and main functions.
8. HSPs and Reproduction:
8.1. Effect of HSPs on spermatozoa:
Although somatic cells are capable of producing HSPs in response to stress, spermatozoa lose their protein translational machinery during spermatogenesis and cannot therefore undertake the synthesis of new proteins.
However, high resolution microscopic and immunolocalisation methods have revealed the existence of HSP60, constitutive Hsc70, inducible HSP70 and HSP90 in a number of mammalian spermatozoa (Huang et al., 1999, Kamaruddin et al., 2004, Lachance et al., 2009, Spinaci et al., 2005) and constant dynamic changes in their localisation over consequent maturational stages. Therefore, HSPs in mature sperm are produced during spermatogenesis (rather than newly, stress-induced) (Spinaci et al., 2005), or are absorbed from their local environment such as seminal plasma (Kamaruddin et al., 2004) or the female reproductive tract.
Low levels of intracellular HSP60 expression in spermatogonia (primary spermatozoa) is associated with a reduced protection and spermatogenic efficiency (Neuer et al., 2000). HSP70 in sperm seems to be involved in reducing the deleterious effects of high temperature on sperm quality and development, sperm-oocyte interactions (Kamaruddin et al., 2004) and the preservation of sperm membrane integrity via direct interactions with lipids and protein components of sperm membrane (Spinaci et al., 2005). By enhancing ATP metabolism in sperm, intracellular HSP90 protects sperm against oxidative stress and maintains its motility (Huang et al., 1999).
HSP60, HSP70 and HSP90 have also been identified in other regions of the reproductive system such as the mammalian endometrium (Tabibzadeh and Broome, 1999) , oviductal epithelium (Boilard et al., 2004, Elliott RM, 2009) and the seminal plasma (in the case of HSP90).They are all associated with maintaining sperm membrane integrity and signalling pathways (Boilard et al., 2004), viability (Boilard et al., 2004, Elliott RM, 2009) and motility (Boilard et al., 2004, Huang et al., 2000).
8.2. Effect of HSPs on Oocyte:
Similar to spermatogenesis, oogenesis in mammals is also accompanied by HSP expression, although this area is little studied. Fully developed oocytes are unable to synthesise inducible HSPs and therefore are very sensitive to high temperatures (Neuer et al., 2000). This reveals the important function of HSPs in the female gamete. However, forms of HSP particularly HSC70 are expressed in high amounts during oocyte growth and early stages of development, the synthesis of which disappears completely as the gamete approaches the end of its maturation process. Hence, HSPs seem to play roles in preservation of oocyte metabolic activity, survival, and ovulation process (Neuer et al., 2000).
8.3. Effect of HSPs on fertilisation and embryo development:
Fertilisation starts from the point of sperm-oocyte recognition, attachment of the two cells together and zygote formation . HSPs are among first proteins synthesised in the growing mammalian embryo (Neuer et al., 2000). Constitutive HSC70 is expressed as soon as zygotic cleavage begins whereas expression of the inducible form is delayed until blastocyst (16 cell) stage.
A dynamic relocalisation and distribution of HSP70 on bovine spermatozoa indicates its role in gamete interaction (Matwee et al., 2001). The supporting evidence is that exposure to HSP70 monoclonal antibodies disrupts gamete interaction, fusion and fertilisation (Matwee et al., 2001). It is also very likely that the protein exerts significant effects on early embryonic development since antibodies to HSP70 in the 3 to 9 day bovine embryo increases apoptosis and hinders the embryo reaching stages with higher number of cells (Matwee et al., 2001). Another group tested the effect of three anti- HSP60, 70 and 90 anti-bodies on 2-day mouse embryos. They observed disruptive effects of each antibody to unique developmental stages, with anti- HSP60 exerting most detrimental effect on the third day, anti- HSP70 on the fifth day and anti-HSP90 at later times (Neuer et al., 1998).
Experiments exposing early embryos to high temperatures, have revealed lower proportion of apoptotic cells in them (Matwee et al., 2001). As it is clear, heat induces HSP synthesis through heat shock response and the newly-induced protein protects the embryo.
Heat Shock Proteins are essential components of life. They guard living cells from harmful stimuli, regulate protein structure and location and prevent cell death when necessary. Like all other organs, mammalian reproduction relies on the beneficial action of HSPs in various stages. HSPs terminate stressful effects to gametes and maintain their viability as well as motility. Also fertilisation and embryo development heavily depend on the presence of these proteins. Taken together, growth, development, reproduction and life are not possible without HSPs.