Journal of Assisted Reproduction and Genetics, Vol. 9, No. 4, 1992
REVIEW ically it consists of two envelopes, each being characterized by a specific microstructure, chemical composition, and developmental pattern. The inner envelope, termed the zona pellucida, is acellular, begins to be formed early during ovarian follicular development, and reaches its final thickness before the antrum formation. It is analogous to the vitelline envelope of the sea urchin egg. Studies in mice, recently reviewed by Wassarman (1), have shown that glycoproteins forming the substantial part of the zona are synthesized and secreted by the oocyte. The outer envelope, termed the cumulus oophorus, is analogous to the jelly coat of sea urchins and consists of cells and extracellular matrix surrounding the oocyte with its zona pellucida. The cumulus cells develop from undifferentiated granulosa cells, like the cells lining the follicular wall. At the beginning of antrum formation they hardly differ from the mural granulosa cells by anything other than their intimate relationship with the oocyte. However, the cumulus cells subsequently begin to produce an intercellular matrix, consisting mainly of hyaluronic acid, at much more elevated rates than the mural granulosa. In the mouse, it is just a factor(s) released by the oocyte which makes follicle stimulating hormone (FSH)-stimulated cumulus cells intensify their hyaluronic acid synthesis and secretion (2) and so promote cumulus mucification (3). In humans, cumulus cells have also been shown to secrete various proteins and glycoproteins (4), which are then deposited in the cumulus intercellular matrix but also in a peripheral layer of the zona pellucida with which they remain relatively stably associated (5). After ovulation the egg is exposed to the oviductal microenvironment and the addition of oviductal glycoproteins to preexisting zona components has been reported in some rodent species (6,7).
Control of the Fertilization Process by the Egg Coat: How Does It Work in Humans JAN TESARIK 1'2 INTRODUCTION During the last 10 years or so evidence has accumulated suggesting that mammalian fertilization is controlled mainly by interactions between spermatozoa and components of the egg coat. Most experimental data backing this conclusion originate from two types of studies. Studies of the first type address the fertilization process essentially as model with which to study general aspects of cell-cell interactions. They use gametes of laboratory animals, mostly the mouse. The other type of studies is motivated by practical demands in application spheres, such as infertility diagnosis and treatment in human medicine or improvement of reproductive capacity of farm animals in agricultural science. Data obtained so far have taught us that, though following the same general strategies, the mechanisms controlling fertilization in different species are not necessarily identical. This paper provides a brief review of the current knowledge about the function of the egg coat in human fertilization and relates it to relevant data obtained in other species.
WHAT THE EGG COAT IS AND HOW IT DEVELOPS The term egg coat is commonly used to denote the totality of somatic cells and extracellular materials associated with the ovulated oocyte. Anatom-
MOUNTAIN CHAIN AND GUIDE: THE GENERAL POLICY OF EGG COAT FUNCTION
American Hospital of Paris, Neuilly-sur-Seine, France. z To whom correspondence should be addressed at INSERM U-355, 32 Rue des Carnets, 92140 Clamart, France.
During fertilization, the two envelopes forming the egg coat act as a functional unit to regulate 313
1058-0468/92/0800-0313506.50/0 © 1992PlenumPublishingCorporation
314 sperm access to the oocyte. As they pass through, spermatozoa must overcome the physical resistance of their extracellular matrices and avoid being entrapped within them by high-affinity bonds with their components. To comply with these requirements, the spermatozoon can make use of its motility apparatus to force its forward drive and of its hydrolytic enzyme battery to diminish the egg coat resistance locally. Since both the sperm cell's energetic reserve and its enzymatic equipment are limited, it is necessary to ensure the optimal conditions of their utilization for the spermatozoon to penetrate. These conditions include (i) acquisition of a vigorous straightforward movement minimizing energy loss in the viscous environments of the egg coat, (ii) temporary immobilization of the sperm head at the zona pellucida surface, and (iii) subsequent action of acrosomal enzymes so as to diminish zona resistance locally. The change in movement pattern has been documented in capacitated human spermatozoa upon contact with the cumulus (8). Sperm head immobilization on the zona surface is effected by binding of complementary sites of sperm and zona. In the mouse this function is served by O-linked oligosaccharides of a zona glycoprotein, termed ZP3 (9), while the enzymes galactosyltransferase (10) and ~-D-mannosidase (11), a trypsin-like protease (12), and a 95-kDa protein serving a tyrosine kinase substrate (13) have been proposed as potential sperm counterparts. Of these, only oL-D-mannosidase has been demonstrated on the human sperm surface (14) and a clinical study has confirmed a relationship between the sperm capacity to bind a D-mannosylated neoglycoprotein and human male fertility (15). The proper timing of acrosomal enzymes liberation is controlled by acrosome reaction (AR) inducers contained in the cumulus oophorus and the zona pellucida. Unlike the mouse, where the ZP3 glycoprotein has also been shown to act as an AR inducer (1), the active component of the human zona pellucida has not yet been isolated, even though the stimulation of the AR by whole human zonae has been reported (16). On the other hand, two components of the human cumulus oophorus, a large glycoprotein acting as acrosin activator (4) and protein-bound progesterone (17), have been shown to induce the AR in human sperm. All three AR-inducing activities come together at the zona pellucida surface, according to a recently formulated synergistic hypothesis (18), their combined action may be required for egg penetration.
TESARIK The events outlined above illustrate the general principle of the egg coat function during fertilization. It is evident that the physical obstacle imposed by the egg coat can be overcome only with the aid of factors contained in it and guiding the sperm to make optimum use of their resources to traverse it. The obstacle is nonspecific, but the sperm-guiding factors are highly specific, as well as the sperm components with which they react. This arrangement endows the egg coat with the capacity of managing selective sperm passage, an essential requirement for its control function. Only some spermatozoa will thus be able to understand the complete instruction how to achieve penetration, while many others will ignore the message, and consequently, they will waste their resources and get blocked on the way.
PREVENTION OF POLYSPERMIC FERTILIZATION The contact of the first-arriving spermatozoon with the oolemma triggers exocytosis of specific secretory granules located in the oocyte cortical region, termed cortical granules. This event modifies the properties of both the oolemma, into which the membrane of exocytosed granules is inserted, and the zona pellucida, which is exposed to hydrolases (proteases and glycosidases) released from the granules. Both modifications have been reported to influence negatively oocyte penetrability by further spermatozoa. In humans, the zona pellucida modification is essential for block to polyspermy, as numerous spermatozoa easily penetrate oocytes after zona removal. Alteration of zona pellucida glycoproteins by cortical granule components, leading to the loss of sperm binding capacity and to increased resistance to sperm hydrolytic enzymes, is the main mechanism of the zona block in the mouse (19). Cortical granule release with subsequent zona modification can occur independently of fertilization, in response to different treatment conditions. Of practical importance is the spontaneous release of cortical granules during oocyte in vitro maturation (20) and that produced by oocyte cooling (21) or exposure to dimethyl sulfoxide at doses at which this agent is used as cryoprotectant for oocyte cryopreservation (22). Research is under way to find optimal treatment protocols reducing such early cortical granule release or minimizing its effects on zona pellucida penetrability. Journal of Assisted Reproduction and Genetics, Vol. 9, No. 4, 1992
CONTROL OF THE FERTILIZATION PROCESS BY THE EGG COAT
315
PREVENTION OF FERTILIZATION OF IMMATURE AND OVERMATURED EGGS Massive ovulation of immature oocytes has been observed in some experimental conditions, such as passive immunization of mice with antibodies against the cumulus oophorus (23). Fertilization of immature oocytes is not desirable because they are deficient in factors controlling the transformation of the sperm nucleus into the male pronucleus (24) and their own nuclear material is not ready for immediate completion of meiosis in response to sperm penetration. This situation can easily lead to aneuploidy. On the other hand, overmatured oocytes, aged in the fallopian tube, are probably more likely to bear abnormalities which can compromise embryonic development. The control of the fertilization window with prevention of fertilization in both extreme situations is another function exerted by the egg coat. The maturing human oocyte has been shown to resume its secretion into the zona pellucida, which can be documented by electron microscopic demonstration of a partial, fertilization-independent release of cortical granules (25). This secretion leads to an augmentation of the zona pellucida resistance to sperm penetration, an effect counteracted by cumulus cell secretions which impregnate the outer part of the zona (Fig. 1) and act to increase its penetrability (26). So from a physiological viewpoint,
Fig. 1. Schematic representation of the physiological and nonphysiological AR of human spermatozoa. The fertilizing spermatozoon penetrates the cumulus oophorus while still retaining the acrosomal contents (A), which is then liberated on the zona pellucida surface (C), where the spermatozoon is exposed to synergistic actions of AR inducers located in the zona pellucida (ZP) and in the cumulus oophorus matrix (CM) impregnating its pores. Spermatozoa which have lost their acrosomes before they come in contact with the zona (B) cannot penetrate.
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 4, 1992
A
zP -~
I
liiiiiiiWii':c:;~}":c;U~;:2~:i :''o';i'] iiiiiiii
IIiiiNiiiiiiiHiiiiiC'iiiiiiiii ...i.i.i.iiii]'"'"'"'"""% i.i.i.i.i.i.N.i.i.i..i.!.i.i.!.i.i..i',' .i.i;' .i'i .'2
REVIEW ically it consists of two envelopes, each being characterized by a specific microstructure, chemical composition, and developmental pattern. The inner envelope, termed the zona pellucida, is acellular, begins to be formed early during ovarian follicular development, and reaches its final thickness before the antrum formation. It is analogous to the vitelline envelope of the sea urchin egg. Studies in mice, recently reviewed by Wassarman (1), have shown that glycoproteins forming the substantial part of the zona are synthesized and secreted by the oocyte. The outer envelope, termed the cumulus oophorus, is analogous to the jelly coat of sea urchins and consists of cells and extracellular matrix surrounding the oocyte with its zona pellucida. The cumulus cells develop from undifferentiated granulosa cells, like the cells lining the follicular wall. At the beginning of antrum formation they hardly differ from the mural granulosa cells by anything other than their intimate relationship with the oocyte. However, the cumulus cells subsequently begin to produce an intercellular matrix, consisting mainly of hyaluronic acid, at much more elevated rates than the mural granulosa. In the mouse, it is just a factor(s) released by the oocyte which makes follicle stimulating hormone (FSH)-stimulated cumulus cells intensify their hyaluronic acid synthesis and secretion (2) and so promote cumulus mucification (3). In humans, cumulus cells have also been shown to secrete various proteins and glycoproteins (4), which are then deposited in the cumulus intercellular matrix but also in a peripheral layer of the zona pellucida with which they remain relatively stably associated (5). After ovulation the egg is exposed to the oviductal microenvironment and the addition of oviductal glycoproteins to preexisting zona components has been reported in some rodent species (6,7).
Control of the Fertilization Process by the Egg Coat: How Does It Work in Humans JAN TESARIK 1'2 INTRODUCTION During the last 10 years or so evidence has accumulated suggesting that mammalian fertilization is controlled mainly by interactions between spermatozoa and components of the egg coat. Most experimental data backing this conclusion originate from two types of studies. Studies of the first type address the fertilization process essentially as model with which to study general aspects of cell-cell interactions. They use gametes of laboratory animals, mostly the mouse. The other type of studies is motivated by practical demands in application spheres, such as infertility diagnosis and treatment in human medicine or improvement of reproductive capacity of farm animals in agricultural science. Data obtained so far have taught us that, though following the same general strategies, the mechanisms controlling fertilization in different species are not necessarily identical. This paper provides a brief review of the current knowledge about the function of the egg coat in human fertilization and relates it to relevant data obtained in other species.
WHAT THE EGG COAT IS AND HOW IT DEVELOPS The term egg coat is commonly used to denote the totality of somatic cells and extracellular materials associated with the ovulated oocyte. Anatom-
MOUNTAIN CHAIN AND GUIDE: THE GENERAL POLICY OF EGG COAT FUNCTION
American Hospital of Paris, Neuilly-sur-Seine, France. z To whom correspondence should be addressed at INSERM U-355, 32 Rue des Carnets, 92140 Clamart, France.
During fertilization, the two envelopes forming the egg coat act as a functional unit to regulate 313
1058-0468/92/0800-0313506.50/0 © 1992PlenumPublishingCorporation
314 sperm access to the oocyte. As they pass through, spermatozoa must overcome the physical resistance of their extracellular matrices and avoid being entrapped within them by high-affinity bonds with their components. To comply with these requirements, the spermatozoon can make use of its motility apparatus to force its forward drive and of its hydrolytic enzyme battery to diminish the egg coat resistance locally. Since both the sperm cell's energetic reserve and its enzymatic equipment are limited, it is necessary to ensure the optimal conditions of their utilization for the spermatozoon to penetrate. These conditions include (i) acquisition of a vigorous straightforward movement minimizing energy loss in the viscous environments of the egg coat, (ii) temporary immobilization of the sperm head at the zona pellucida surface, and (iii) subsequent action of acrosomal enzymes so as to diminish zona resistance locally. The change in movement pattern has been documented in capacitated human spermatozoa upon contact with the cumulus (8). Sperm head immobilization on the zona surface is effected by binding of complementary sites of sperm and zona. In the mouse this function is served by O-linked oligosaccharides of a zona glycoprotein, termed ZP3 (9), while the enzymes galactosyltransferase (10) and ~-D-mannosidase (11), a trypsin-like protease (12), and a 95-kDa protein serving a tyrosine kinase substrate (13) have been proposed as potential sperm counterparts. Of these, only oL-D-mannosidase has been demonstrated on the human sperm surface (14) and a clinical study has confirmed a relationship between the sperm capacity to bind a D-mannosylated neoglycoprotein and human male fertility (15). The proper timing of acrosomal enzymes liberation is controlled by acrosome reaction (AR) inducers contained in the cumulus oophorus and the zona pellucida. Unlike the mouse, where the ZP3 glycoprotein has also been shown to act as an AR inducer (1), the active component of the human zona pellucida has not yet been isolated, even though the stimulation of the AR by whole human zonae has been reported (16). On the other hand, two components of the human cumulus oophorus, a large glycoprotein acting as acrosin activator (4) and protein-bound progesterone (17), have been shown to induce the AR in human sperm. All three AR-inducing activities come together at the zona pellucida surface, according to a recently formulated synergistic hypothesis (18), their combined action may be required for egg penetration.
TESARIK The events outlined above illustrate the general principle of the egg coat function during fertilization. It is evident that the physical obstacle imposed by the egg coat can be overcome only with the aid of factors contained in it and guiding the sperm to make optimum use of their resources to traverse it. The obstacle is nonspecific, but the sperm-guiding factors are highly specific, as well as the sperm components with which they react. This arrangement endows the egg coat with the capacity of managing selective sperm passage, an essential requirement for its control function. Only some spermatozoa will thus be able to understand the complete instruction how to achieve penetration, while many others will ignore the message, and consequently, they will waste their resources and get blocked on the way.
PREVENTION OF POLYSPERMIC FERTILIZATION The contact of the first-arriving spermatozoon with the oolemma triggers exocytosis of specific secretory granules located in the oocyte cortical region, termed cortical granules. This event modifies the properties of both the oolemma, into which the membrane of exocytosed granules is inserted, and the zona pellucida, which is exposed to hydrolases (proteases and glycosidases) released from the granules. Both modifications have been reported to influence negatively oocyte penetrability by further spermatozoa. In humans, the zona pellucida modification is essential for block to polyspermy, as numerous spermatozoa easily penetrate oocytes after zona removal. Alteration of zona pellucida glycoproteins by cortical granule components, leading to the loss of sperm binding capacity and to increased resistance to sperm hydrolytic enzymes, is the main mechanism of the zona block in the mouse (19). Cortical granule release with subsequent zona modification can occur independently of fertilization, in response to different treatment conditions. Of practical importance is the spontaneous release of cortical granules during oocyte in vitro maturation (20) and that produced by oocyte cooling (21) or exposure to dimethyl sulfoxide at doses at which this agent is used as cryoprotectant for oocyte cryopreservation (22). Research is under way to find optimal treatment protocols reducing such early cortical granule release or minimizing its effects on zona pellucida penetrability. Journal of Assisted Reproduction and Genetics, Vol. 9, No. 4, 1992
CONTROL OF THE FERTILIZATION PROCESS BY THE EGG COAT
315
PREVENTION OF FERTILIZATION OF IMMATURE AND OVERMATURED EGGS Massive ovulation of immature oocytes has been observed in some experimental conditions, such as passive immunization of mice with antibodies against the cumulus oophorus (23). Fertilization of immature oocytes is not desirable because they are deficient in factors controlling the transformation of the sperm nucleus into the male pronucleus (24) and their own nuclear material is not ready for immediate completion of meiosis in response to sperm penetration. This situation can easily lead to aneuploidy. On the other hand, overmatured oocytes, aged in the fallopian tube, are probably more likely to bear abnormalities which can compromise embryonic development. The control of the fertilization window with prevention of fertilization in both extreme situations is another function exerted by the egg coat. The maturing human oocyte has been shown to resume its secretion into the zona pellucida, which can be documented by electron microscopic demonstration of a partial, fertilization-independent release of cortical granules (25). This secretion leads to an augmentation of the zona pellucida resistance to sperm penetration, an effect counteracted by cumulus cell secretions which impregnate the outer part of the zona (Fig. 1) and act to increase its penetrability (26). So from a physiological viewpoint,
Fig. 1. Schematic representation of the physiological and nonphysiological AR of human spermatozoa. The fertilizing spermatozoon penetrates the cumulus oophorus while still retaining the acrosomal contents (A), which is then liberated on the zona pellucida surface (C), where the spermatozoon is exposed to synergistic actions of AR inducers located in the zona pellucida (ZP) and in the cumulus oophorus matrix (CM) impregnating its pores. Spermatozoa which have lost their acrosomes before they come in contact with the zona (B) cannot penetrate.
Journal of Assisted Reproduction and Genetics, Vol. 9, No. 4, 1992
A
zP -~
I
liiiiiiiWii':c:;~}":c;U~;:2~:i :''o';i'] iiiiiiii
IIiiiNiiiiiiiHiiiiiC'iiiiiiiii ...i.i.i.iiii]'"'"'"'"""% i.i.i.i.i.i.N.i.i.i..i.!.i.i.!.i.i..i',' .i.i;' .i'i .'2
