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Reproduction and love: strategies of the organism’s cellular defense system?
Comparative Biochemistry and Physiology Part C 120 (1998) 167 – 176
Review
Reproduction and love: strategies of the organism’s cellular defense system? A. De Loof *, R. Huybrechts, S. Kotanen Zoological Institute of the K.U. Leu6en, Naamsestraat 59, 3000 Leu6en, Belgium Received 19 August 1997; received in revised form 02 February 1998; accepted 26 February 1998
Abstract A novel view is presented which states that primordial germ cells and their descendants can be regarded as ‘cancerous cells’ which emit signals that activate a whole array of cellular defensive mechanisms by the somatoplasm. These cells have become unrestrained in response to the lack of typical cell adhesion properties of epithelial cells. From this point of view: (1) the encapsulation of oocytes by follicle cells, vitelline membrane and egg shell; (2) the suppression of gonadal development in larval life; (3) the production of sex steroid hormones and of vitellogenin; and (4) the expulsion of the gametes from the body fit into a general framework for a defense strategy of the somatoplasm against germ line cells. Accordingly, the origin of sexual reproduction appears to be a story of failure and intercellular hostility rather than a ‘romantic’ and altruistic event. Yet, it has resulted in evolutionary success for the system in which it has evolved; probably through realizing feelings of ‘pleasure’ associated with reproduction. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Cytoskeleton; Cancer; Endocrinology; Ecdysone; Estrogen; Germ cells; Pole plasm; Sex
1. Introduction After living matter came into being about 4 billion years ago, some organisms started to divide. By doing so, they generated offspring and became successful in terms of evolution. It is often overlooked that a cell, while engaging in splitting off a daughter compartment, risks death. Although we do not have an idea about ‘emotions’ in other organisms, one cannot escape the impression that despite the inherent risks involved, reproduction is always associated with ‘well-being’, with ‘success’ and with ‘good communication’ [11]. A much less romantic picture appears, however, if one analyzes, at the cellular level, a variety of mechanisms which are operational during reproduction and than tries to understand how they could have originated. * Corresponding author. Tel.: +32 16 323912; fax: + 32 16 323902; e-mail: [email protected] 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0742-8413(98)10007-5
2. Speculations about the origin of cell division and reproduction Let us assume that the first living organism on earth, that appeared about 3.7 billion years ago, was a bacterium-like cell surrounded by a plasma membrane and devoid of membrane-bound cell organelles. Later, this cell, often referred to as the progenote, pinched off an identical daughter cell that contained the whole machinery needed for growth, metabolism and generation of other daughter cells and thereby initiated a continuous propagation of living cells. It is not likely, that this cell division occurred ‘intentionally’ but probably by physiological necessity. Which physiological process initiated such a division into two halves in the progenote, with the inherent danger of not surviving such a risky operation? Did an external substance enter the primordial cell and trigger a division process? Or
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did this cell synthesize a molecule(s) that triggered the division process? Since no records exist dating 4 billion years ago, we can only propose mechanisms which operate in present day bacteria. The fission process was probably initiated once a critical level of an inhibitor or stimulator of growth was reached [24]. For a long time there was a lack of definitive evidence for cell cycle-dependent synthesis of particular molecules in bacteria. Thus, it was thought that their cell cycle-control system was different from that in eukaryotes with respect to the involvement of cyclins [26] (see further). It has been known for a few years that the DnaA protein plays an essential role in an early step of DNA initiation by binding to three Dna boxes at oriC on the chromosome of Escherichia coli [26]. Substantial progress in understanding bacterial cell cycle control has recently been made in the bacterium Caulobacter crescentus which produces two different cell types at each cell division, a sessile stalked cell and a motile swarmer cell. In this prokaryote, a protein, called tubulin-like GTPase FtsZ (Fts, filamenting temperature sensitive) is critical for the initiation of cell division. Its intracellular concentration is highest at the beginning of cell division. Later, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is most probably due to the degradation of FtsZ in the swarmer compartment of the pre-divisional cell [27]. Bacterial ‘sex life’ is limited to the formation of cytoplasmic bridges through which DNA can be exchanged. This process is called conjugation, in which the donor cell’s sex pili (male) are attached to the recipient cell (female) [7]. If the mechanism involved in cell division which is universally used by contemporary eukaryotes was used by the primordial eukaryote, then we can speculate upon similarity to the mitosis-oscillator in eukaryotes which is based upon synthesis and degradation of specific proteins that are called cyclins. A high concentration of cyclins prevents cell division and keeps the cells in metaphase. Breakdown of cyclins, triggered by an increase in the levels of free Ca2 + , enables the cells to return to interphase and cell division to restart [13]. There are only two types of inorganic ions which are widely used as triggers in a variety of biochemical processes, namely Ca2 + and H + . High concentrations of both are toxic because they cause conformational changes in a variety of proteins. The concentration of intracellular calcium is usually kept very low, at about 10 − 7 –10 − 8 M while its concentration in the extracellular fluid is often several orders of magnitude higher. If the plasma membrane of a cell becomes more permeable to Ca2 + , it will enter the cytoplasm and trigger a variety of biochemical processes. High H + concentration causes acidity, which in turn induces conformational changes in proteins. This means that the two
inorganic ionic species which are frequently used in signaling are in fact toxic to cells. Thus, the mechanism which triggered cell division could have been either a selective change in the permeability to Ca2 + or H + , or a change in the activity of ion pumps present in the plasma membrane.
3. Basic physiological cellular processes underlying reproduction in animals As a result of cellular physiology of reproduction is best documented in animals, analogies will be drawn from animal model systems, such as insects and mammals. We will discuss defense strategies in pre- and postvitellogenic females, in males, at fertilization and during intra-uterine development.
4. Defense mechanisms at work before vitellogenesis
4.1. The origin of the germ cell line One of the best documented models in the study of animal embryonic development is the fruitfly Drosophila melanogaster. At its posterior pole, its newly laid egg contains a distinct class of granules, the polar granules, which are important in segregating and maintaining the germ cell line. At least eight genes and probably a mitochondrial factor as well, are required for the formation of a functional polar plasm. Fertilization causes an increase in the cytoplasmic Ca2 + concentration. Next, the zygote nucleus starts to divide without concomitant formation of plasma membranes. Each nucleus is associated with a centrosome that plays an important role in nuclear division. Thus, early in embryogenesis of insects (but not in all animal species) only nuclei-centrosome complexes are formed during each mitotic cycle, not whole cells. The third mitotic cycle yields eight of these complexes. Depending upon the species, from this eight complex stage on, a centrosome, which usually has a nucleus in tow, comes in the immediate neighborhood of the polar granules. Next, an as yet unidentified mechanism is activated which makes the nucleus-centrosome-polar plasm complex migrate towards the boundary of the egg and it causes this complex to be pinched off from the rest of the egg in the form of the first cell with plasma membranes (Fig. 1). This ejected pole cell will give rise to the future gametes while all cells that will eventually be formed inside the egg will turn into somatic cells and form all the tissues of the body: integument, muscles, gut, nervous tissue etc.. Nuclear divisions without plasma membrane formation continue in the egg. The germ cell precursor cell also undergoes a few mitotic rounds
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Fig. 1. The definition of an animal is that it is an organism which develops from a blastula [8]. This means that the young embryo passes through a stage in which it organizes itself as an epithelium. At some early stage in development, some cells escape from being integrated in an epithelium and form the germ cell anlage. In dipteran insects (Drosophila has been well studied in this respect), the segregation of this anlage happens already before the blastoderm is formed. At one end of the egg, there is a group of particles with as yet poorly defined chemical composition (anyhow rich in RNA), called the polar granules. When a nucleus comes in the vicinity of these granules, segregation is induced. The isolated cell will undergo several rounds of mitosis, thereby forming the germ cell anlage.
which include plasma membrane formation. At a given developmental stage plasma membranes form around the nuclei which in the mean time have aligned themselves in the cortical layer underneath the plasma membrane. In this way, a blastula is formed. The pole cells remain clustered together at the posterior pole of the blastoderm until gastrulation (gut formation) movements carry them into the lumen of the posterior midgut primordium. The pole cells then migrate freely through the walls of the midgut into the body cavity, interact with mesodermal cells that encapsulate them, as if they were foreign intruders that have to be inactivated. During this time, the pole cells do not divide whereas somatic cells continue to divide, indicating that germ cells are selectively inhibited. Early in the embryonic life of invertebrates, the germ cells with their big
Fig. 2. In a human embryo of 3-weeks-old, the primordial germ cells are found in the wall of the yolk sac, close to the site where the future umbilical cord will be attached (redrawn after Langman [23]). Here again, the primordial germ cells are not epithelially organized. By ameboid movements, these cells migrate towards the gonad primordia which they invade.
nuclei migrate towards the anlage of the gonads (Fig. 2). Once there, they go through a number of rounds of mitotic divisions. However, not all the germ cells survive [23]. With respect to the origin of meiosis, one can only speculate about the cellular mechanism(s) that initiated this complex process.
4.2. Cell adhesion differences between somatoplasm and germ plasm All somatic cells are the progeny of stem cells which were once part of the blastular epithelium. Depending upon the species, the stem cells of the germ cell line either did not integrate in this epithelium or did escape from an epithelial environment early in development, leaving them solitary. This means that the normal cell adhesion phenomena which are typical for epithelia are not functioning in the primordial germ cells. This aspect is important because it is closely linked to the very nature of being an animal. The Five Regna classification system of Whittaker [33], defines an animal as an organism which develops from a blastula. Histologically, it means that an animal is an organism that in its early development is an epithelium enclosing a fluidfilled cavity [8]. Thus, the basic principle underlying animal development is epithelial formation, folding and segregation. In diblastic animals (Coelenterates), all somatic cells are organized as folded epithelia, but the germ cells are not. In triblastic animals, the ecto- and endoderm are usually epithelia. The mesoderm gives rise to tissues, some of which have an epithelial structure, others, for example, muscle cells, the endoskeleton of vertebrates and blood cells have not. The major characteristic of an epithelium is that its cells are tightly interconnected by specific cytoskeletal elements and cell adhesion molecules. The fact that primordial germ cells are solitary indicates that their cytoskeleton is not able to form tight junctions with their neighbors. Cells
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which loose their cell adhesion properties and start wandering around in the body and proliferate, are considered as having properties of cancer cells. Primordial germ cells have such properties. Female germ cells are aberrant because they grow so big and male primordial germ cells are aberrant because of their frequent divisions. Blood cells too are solitary and mobile cells which, in the case of erythrocytes, have a fast proliferation rate. The body can cope with them by either eliminating them at a fast rate (erythrocytes) or by controlling their growth (leucocytes). Many germ cells are also killed soon after they are formed. The ones that survive are restrained in a variety of ways.
4.3. Programmed cell death in part of the germ cell line population In Drosophila, less than half of the approximately 40 pole cells will succeed in establishing themselves in the forming gonads; the others die. In the human female fetus, during the fifth month of embryonic development there are about 6 million egg cells. They are all arrested in early first meiotic division, apparently a stage in which human egg cells can survive for approximately 50 years. After this arrest, no more cells will be formed during life, on the contrary, quite a lot will degenerate. By the time of birth, only 700000 to 2 million out of the original 6 million survive. By the time a girl reaches puberty, this number further shrinks to about 40000 [23]. The cause(s) of disappearance of so many germ cells is unknown. Perhaps they are destroyed by somatic cells or tissues, or by apoptosis.
4.4. A role for the cytoskeleton in defining the essence of being male or female Reproduction started with the introduction of cell division. In eukaryotes, the cytoskeleton plays a key role in this process. In prokaryotes, the properties and role of the cytoskeleton are not so well documented. The primordial difference between male and female resides in the genes and this is evidently true in all species in which there is no sex reversibility under the influence of environmental factors, as was found in some fishes. A master gene can be absent in one of the sexes or its mRNA can be processed differentially in males and females as was found in Drososphila. One of the sexes can be haploid while the other is diploid as in bees. There are still other possibilities for sex determination. Whatever mechanism is used, there must be master proteins activating cellular mechanisms in a given sex-specific way. Vertebrate females have larger amounts of estrogens than males whereas males have more androgens than females (Fig. 3). Estrogens are made from androgens through the action of specific enzymes. For example, aromatase converts testosterone
into estradiol. In adult flies, the steroid moulting hormone 20-OH ecdysone seems to act as the equivalent to the estrogens of vertebrates. It induces vitellogenin synthesis if injected into adult males [20,21]. The difference between adult male and female flies is that females have a higher titer of 20-OH ecdysone in their hemolymph than males [2]. Besides the hormonal difference, there is also a difference in cytokinesis in the germ cells. During meiotic divisions, four functional spermatozoa form from one spermatogonium as compared to one oocyte and to three non-functional polar cells stem from one oogonium. The typical textbook explanation to this phenomenon is that the oocyte needs to store as much cytoplasm as possible in order to accumulate the greatest reserves possible for embryogenesis, while the male germ cells need only to be able to swim for a short while. The underlying principle is that cytokinesis is asymmetrical in the female germ cells which enter meiosis whereas in males it is symmetrical. The cytoskeleton plays an important role in cytokinesis. The major difference between the cytoskeleton of oocytes and spermatozoa is that the oocytes are actin-rich and the spermatozoa are tubulin-rich. The combination of asymmetry in cytokinesis and differences in sex hormones constitute the very nature of sexual differentiation.
4.5. Inhibition of gonadal de6elopment during lar6al life There is no a priori reason that the germ-line (stem) cells which are always formed very early in development should be developmentally arrested during fetal and larval life. It appears that it is not due to an internal clock present in the oocytes but to an inhibitory action of the somatoplasm. In mammals the arrest of oocyte development and sperm production is thought to be primarily due to a low production rate of two gonadotropic hormones: follicle stimulating hormone (FSH) and luteinizing hormone (LH). Although this could explain the arrest of gonadal development, it is probably only one of several mechanisms that are involved. The low production rates of FSH and LH, which are controlled by an unknown mechanism, come to an end at puberty. In adults of the fleshfly, Neobellieria bullata, a different mechanism has been detected. Adult females have to ingest a protein meal (e.g. meat) in order to produce substantial amounts of yolk proteins. Each ovary contains several dozens of ovarioles in which only one follicle at a time enters vitellogenesis. Once the ripe eggs have been expelled from the body, after having been retained in the uterus for about 4 days to complete embryogenesis, a second batch of ovarian follicles will enter vitellogenesis. The reason why the penultimate follicles do not start vitellogenesis is that the ovary releases colloostatin, a 19-mer peptide, which inhibits
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Fig. 3. Major physiological differences between the female and male sex which result from the genetic difference, whatever its nature, between the sexes. A first difference is that only the oocyte is capable of accumulating, by pinocytosis, vitellogenins from extra-oocytic origin. Actin plays an important role in this process. A second difference is that during meiosis cytokinesis is symmetrical in males while it is asymmetrical in females. This reflects a differential organization of the cytoskeleton in both sexes. A third difference is that the cytoskeleton of oocytes is actin-rich, while that of spermatozoa is rich in tubulin. Microtubules which are organized in the typical 9 +2 configuration allow fast movement. Actin can bind a variety of different molecules, for example some maternal messenger RNA’s and proteins. A fourth difference concerns sex hormones, when present. In vertebrates the oestrogen-androgen situation is well understood. In the fleshfly Neobellieria, the best documented species in this respect, the moulting hormone 20-OH-ecdysone induces vitellogenin synthesis. Its titer is much higher in females than in males. In many invertebrates, the nature of sex hormones, if present, is not yet known.
growth of the penultimate oocytes but not of vitellogenic ones [4]. The strong resemblance of colloostatin to the sequence 468 – 480 of preprocollagen a1 (IV) from Drosophila, indicates that colloostatin is probably a degradation product of a collagen-like molecule present in the sheath which surrounds the individual ovarioles. This offers an interesting model for growth
control [9]. Indeed, it is the expansion of the ovary itself which triggers the release of an inhibitor which prevents younger follicles from becoming pinocytotic and competing with older oocytes for vitellogenin uptake. During periods of fast growth, any tissue sheathed in a basement membrane is probably capable of using such a mechanism. A similar mechanism may also operate in
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Fig. 4. Schematic representation of a mammalian ovarian follicle. The oocyte releases signals which govern the physiological behavior of the follicular epithelium. The only factor of the chemical identity has as yet been established is growth differentiation factor-9 (GFD-9). This factor is required for follicular development beyond the primary one-layer follicle stage [12]. Redrawn after Gosden [16].
vertebrates before reaching sexual maturity. Growing vertebrates degrade and rebuild their collagen every day. Some degradation products resulting from this process could perhaps act as inhibitors of gonadal development. It cannot be excluded that in insects, a brain gonadotropin-based mechanism could also play a role in inhibiting gonadal development in larvae. Presently, experimental evidence is lacking. Although a receptor, structurally related to the FSH/LH receptor family of vertebrates has been cloned in Drosophila [18] and although FSH- and LH-immunoreactivity has been demonstrated in cockroach brains [31], peptides with similar amino acid sequence as GnRH, LH and FSH have not yet been found in insects [10]. In Locusta migratoria, an ovary maturating parsin (Lom-OMP) [15] has been isolated and identified in the brain-corpus cardiacum complex. This gonadotropin is the physiological equivalent of FSH/LH. Lom-OMP is not homologous to any known vertebrate peptide. It stimulates the production of the steroid hormone ecdysone [14], which acts as the equivalent of estrogens in some insects like flies [20,21]. As this is the case for FSH/LH in vertebrates, Lom-OMP immunoreactivity is not restricted to neurosecretory cells in the pars intercerebralis of adult females only. It is present in larvae and males as well. Injection of Lom-OMP into larvae does not make the gonads develop, just like injections of FSH/LH fail to do so in immature vertebrates. Apparently, an inhibitory mechanism(s) is at work. In Neobellieria, the N-terminus sequence 1 – 17 of the cAMP generating peptide [28] (a 48-mer, not related to any known vertebrate or invertebrate hormone: [28]) stimulates vitellogenesis (unpublished results). In the moth Lymantria dispar, a gonadotropin (testis ecdysiotropin) has been identified which stimulates ecdysone biosynthesis in the testis [25,32]. In dipteran insects, a second hormone capable of inhibiting ovarian follicle development has been discov-
ered. It has been named trypsin modulating oostatic factor (TMOF). Since it acts on vitellogenic follicles it will be discussed later. In plants, a germ-line cell anlage is not yet present in the plant seed (which is a baby plant): the primordia only start developing once the flower primordium forms. In fungi, ferns and mosses, meiosis is also a late phenomenon. This means that during the immature stages, there must be a factor(s) which inhibits the development of sexual organs and the production of ripe gametes.
4.6. Oocyte-follicle cell interactions Oocytes become ensheathed by one or a few layers of follicle cells. The typical textbook states that the functions which are attributed to these cells are in providing information and nourishment to the oocyte [16]. Our view upon this phenomenon is different. If a relatively large foreign body enters the body, one of the possible defense strategies is that it will be encapsulated by specialized cells of the defense system. In this case the encapsulation process is a hostile reaction, intended to neutralize the invader. We think that the ensheating of the oocyte could be a reaction of the somatoplasm to the oocyte which grows much bigger than a normal somatic cell and perhaps, releases (or causes surrounding cells or membranes to release) signaling molecules which are recognized by somatic cells as ‘alarm’ signals. Encapsulation is then ensured to restrain the oocyte. Since the oocyte does not appear to ‘suffer’ much from this encapsulation, follicle cell overlayering has usually been explained as an act of protection by the follicle cells. The oocyte and the follicle cells communicate using various signals (Fig. 4). Mammalian oocytes affect the growth and differentiation of the granulosa cells around it [16]. They produce a factor(s) which increases the rate of synthesis of estrogens and decrease the
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synthesis of progesterone by granulosa cells that have been stimulated by FSH in vitro. In mice, oocytes secrete a factor(s) that stimulates the production of hyaluronic acid which is involved in the process of mucification. Removal of the oocyte, stops the secretion of hyaluronic acid from the granulosa cells after FSH stimulation. Another factor prevents the breakdown of the extracellular matrix by inhibiting the production of urokinase plasminogen activator. The oocyte can also inhibit the proliferation of the granulosa cells throughout growth of the follicle. Knock-out technology has shown that GFD-9 deficient mice formed only one layer of granulosa cells and their theca failed to differentiate. It has been suggested that GDF-9 might be the mucification factor [12]. However, no definitive evidence is available to prove communication between oocyte and follicle cells in invertebrates.
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lifted. After gonadotropic hormones are secreted and oocytes start to grow, cell layers surrounding the oocyte secrete large amounts of sex steroid hormones, namely estrogens in vertebrates and ecdysone in insects. These hormones signal the development of female gonads. The liver of egg laying vertebrates and the fat body of insects start to synthesize large amounts of vitellogenins, which are secreted and circulated in the blood or hemolymph. Vitellogenins bind to specific receptors on the surface of the oocyte and the oocyte internalizes the vitellogenins by pinocytosis. The process of vitellogenin biosynthesis resembles mobilization of antibodies against a foreign intruder (bacterium, virus etc.). However, vitellogenins do not share structural similarities with the immunoglobulins of vertebrates, they are agglutinins.
5.3. Agglutinins and lectins 5. Defense strategies from vitellogenesis onwards
5.1. Steroid hormone production by the follicle cells Production of estrogens by the ovary of vertebrates, the major site of sex hormone synthesis, makes the body of females competent for reproduction. In our view, the sex steroids that are released by the follicles might be a warning signal targeted at the somatoplasm in order to incite it to suppress the growing oocyte(s). In flies, the equivalent of estrogens is the steroid moulting hormone 20-OH-ecdysone [20,21]. The follicle cells are a major site of synthesis. Ecdysone’s titer elevation during vitellogenesis is the major stimulating factor of vitellogenin synthesis in many insect species.
5.2. The mechanism of fast growth of yolky eggs The majority of animals produce yolky eggs which are expelled from the female’s body. General biology textbooks may explain the difference between the big egg cell and the small, mobile spermatozoon from a teleological point of view. In order to allow embryogenesis, egg cells should accumulate nutrients. The spermatozoa should specialize for swimming. Yet, physiologists think in another way. Oocyte growth is accomplished by the uptake of specific egg yolk proteins from the hemolymph, called vitellogenins. To find out why oocytes accumulate specific blood proteins, an insight in the physiological principles underlying yolk formation is necessary. Yolk proteins are not formed by the oocytes themselves, they are synthesized elsewhere. Distant tissues such as the liver in vertebrates and the fat body in insects are major sites for this synthesis. In some species, the follicle cells also contribute a relatively small amount. When females become sexually mature, the block on oocyte growth is
Lectins are proteins with specific recognition and reversible binding to carbohydrate moieties without altering the covalent structure of the glycosylated ligands [22]. A few years ago, the name lectins was used for proteins that are present in plants, whereas the term agglutinins was used for proteins of animal origin. At present the term lectins is used for proteins from plants and animals. Well known agglutinins are those that are present in the plasma of persons with blood group A (these have agglutinins b) and blood group B (which have agglutinins a). Some lectins that recognize specific carbohydrate moieties on red blood cells can agglutinate the cells. Agglutination will occur when nonmatching blood groups are mixed (e.g. when a donor of blood group A would receive blood from a donor with group B). Agglutinins (lectins) in the plasma of a person with blood group A, will bind to the carbohydrate moieties on the surface of the erythrocytes of the donor with blood type B causing agglutination. Agglutination is not a defense mechanism against invading red blood cells from other individuals because this rarely happens in nature. The fact that agglutinins are found in both vertebrates and invertebrates suggests that such molecules were present early in the evolution of all animals. Lectins/agglutinins might be a primitive mechanism against micro-organisms before genes coding for antibodies became functional in some groups of animals. This view, however, is not shared by everybody. ‘Lower’ animals do not have an immunoglobulin-based defense system. They use other defense mechanisms, for example antibacterial and antifungal peptides, apotopsis, encapsulation and agglutinin/lectin production. These might have been the defense mechanisms when gametes were first produced. Stynen and De Loof [29,30] first described the agglutinin properties of two major vitellogenic hemolymph proteins from the Colorado potato beetle, Leptinotarsa
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decemlineata. Lectins labeled with a fluorescencent tag, strongly stain developing oocytes of Drosophila, suggesting a correlation between lectin activity and yolk uptake [5,6]. Hafer and Ferenz [17] reported that the locust vitellogenin receptor is an acidic glycoprotein with 54% of the apparent molecular mass contributed by O- or N-linked carbohydrates and that neuraminic acid is essential for binding vitellogenins. If the binding of vitellogenins to the surface of the oocyte was to inactivate and encapsulate the oocyte, the opposite happened; the oocyte internalized vitellogenins and stored them as vitellins in yolk vesicles. The seeds of many species of plants contain large amounts of lectins: soybean lectin, wheat lectin etc. The exact role of these lectins in plants is not known. Lectins in seeds may be used as a nutrient reserve and as antimicrobial agents preventing degradation by bacteria, fungi and insects of stored reserves. Thus, vitellogenins stored in the yolk of animal eggs are analogous to the lectins stored in plant seeds.
5.4. Encapsulation by coating layers Another type of interaction of the follicle cells with the oocyte is the deposition by the follicle cells of material which encapsulates the oocyte, for example the vitelline membrane and chorion of the insect egg. The formation of a layer of albumen by the oviduct and of egg membrane and egg shell by the uterus of reptiles and birds are other examples of protective layers. Encapsulation by a rigid layer may block oocyte expansion and vitellogenin or other molecules transport to the surface of the oocyte. Thus, oocyte growth will be halted. The major components of the vitelline membrane form a fibrous mat with holes that allow blood proteins to move in. During oocyte growth, it is probably remodeled by the combined action of proteases and deposition of newly formed material originating from the follicle cells. In placental mammals, the equivalent of the vitelline membrane of the insect egg is a thick extracellular matrix called the zona pellucida [13]. The granulosa cells-zona pellucida-oocyte complex is released from the ovary as a Graafian follicle. The oviducts do not produce thick layers of albumen and the uterus does not make an egg shell. However, in animals which oviposit in a dry environment, an egg shell is made. For example, in insects the follicles synthesize chorion proteins shielding the oocyte. Then, the follicle cells die. In reptiles and birds, the uterus synthesizes the egg’s shell. Making a shell can be regarded as the last option left for permanently encapsulating an ‘unwanted’ entity in the body. After this process is over, the female carries in its interior a large egg(s) which does not contribute any-
more to her internal physiology, and the best the female can do is to expel the egg(s) from her body. This, however, involves the action of myotropic egg laying hormones such as oxytocin in vertebrates and egg laying hormone in molluscs and annelids, etc. In some species, egg laying seems to be a big relief for the female, as might be guessed from the sound that a hen makes after laying an egg. In the language of cell physiology this might read as: ‘Hurah, the cancerous cell is conquered!’
5.5. Inhibition of o6arian de6elopment by TMOF In the mosquito, Aedes aegypti the follicle cells synthesize and release a decapeptide (H-Tyr-Asp-Pro-AlaPro-Pro-Pro-Pro-Pro-Pro-OH) which binds to the gut receptor and shuts down the trypsin biosynthesis in the gut. Thus, blood digestion is arrested and the supply of nutrients for vitellogenin biosynthesis stops. The peptide has been named Aedes-trypsin modulating oostatic factor or Aea-TMOF [1]. From the fleshfly Neobellieria, a hexapeptide (H-Asn-Pro-ThrAsn-Leu-His-OH) with similar functions has been isolated (Neb-TMOF: [3]). Neb-TMOF has been found to be a potent inhibitor of ecdysone biosynthesis in larvae of the fleshfly Calliphora [19] and to inhibit vitellogenin production in adult females [3]. Although the coding gene has not yet been identified, there are good indications that TMOF is cleaved from a vitelline membrane protein.
5.6. Males: fertilization If estrogens are alarm hormones, at high titer, what are androgens? These hormones are produced by Leydig cells in the testis. When sperm production starts, the titer of androgens is high. This drives the male to ejaculate sperm cells into a hostile environment in which their survival is limited. Thus, the interaction between the somatoplasm and the sperm cells is hostile. Spermatozoa are armed with a vesicle that contains lysosomal enzymes that can make a hole in the plasma membrane of the oocyte. Too big a hole in a membrane will result in cell death. When are females ready for copulation? Our answer to this question is when all their own defense strategies against neutralizing the oocytes have failed. The final strategy is to call for help from a male which, in a watery environment, brings killer cells in the immediate vicinity of the egg(s), or in a dry environment brings these cells inside the body of a female to specifically attack the oocyte. The oocytes survive because they close the hole immediately after they swallowed the nucleus of the spermatozoon.
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5.7. Intrauterine de6elopment: parasitism. Brood care: an act of ensla6ing? In mammals with placenta, the oocyte is devoid of yolk. Their ancestors, the reptiles had yolky eggs. Apparently, some mutation that occurred, perhaps in the starting codon of the vitellogenin gene, ‘wiped out’ vitellogenin biosynthesis. An embryo that develops from such an egg can survive only if it is ‘hooked’ to a constant food supply. This was achieved by invading the epithelial lining of the uterus and developing in the uterus. Nutrition-wise these embryos are parasites of their mother. The developing embryo contains many proteins, coded for by paternal genes, which should be recognized as foreign by the immunological system of the mother. However, the developing embryo has mechanisms which prevent the mother from making antibodies against it. At least in humans, nobody will question that parents who foster their offspring display ‘love’. But is this love a deliberate strategy from the side of the parents themselves, or is this behavior imposed by the offspring in which the germ cell anlage of the next generation is already at work? To a large extent, the offspring enslaves one or both of the parents who would have had an easier life without the offspring. Yet, having the offspring means transmitting 50% of the genes of each parent to the next generation, thereby ensuring genetic success.
6. Conclusion Humans find that the opposite of aggression is reproduction in all its aspects: courtship, copulation, pregnancy, love, care for partner and offspring etc. Therefore, the view we presented here may elicit rejection at first reading. After a careful analysis of the interactions between the different cell types involved in animal reproduction, we came to the apparent conclusion that the somatoplasm treats the germ cells as if they were cancer- or foreign cells that have to be destroyed with all means. From our approach, it follows that the origin of ‘love’ in the course of evolution, might have been actually due to the ‘nasty’ behavior of germ cells which elicited a whole array of hostile defense mechanisms by somatic cells. Thus, our story turns the ‘romance of reproduction’ into a story of cellular aggression and hostility. Should one be shocked? Not at all. The oocytes, these ‘obese’ beauties with their ‘dominant’ and ‘nasty’ character succeed in making both the male and female somatoplasm believe that caring for them is a joy, which cleverly ensures their survival and development. The final outcome of this story of hostility is genetic/evolutionary success. Reproduction at the or-
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ganismal level is a mechanism that ensures that genes survive longer than the organisms themselves; it tries to attain immortality. At the level of the population, reproduction can be considered as regeneration of the population [11]. We are evidently aware that in describing physiological/biochemical phenomena it is not common practice to use terms referring to human feelings, such as hostility, pleasure and love. No doubt, some readers will find that cell biologists and physiologists should keep far away from such an antropomorphic approach. Our view is that this is a too narrow approach for biology in general. Communication is essential to life. In fact, what we call ‘life’ is the communication activity of a communicating compartment [11]. In communication, feelings play a very important role, which is ignored in contemporary biology. Finally, we would not be surprised if some of the cell’s strategies that are used to inhibit oocyte growth might one day lead to practical applications in treating cancer. Acknowledgements The authors thank the Fonds voor Wetenschappelijk Onderzoek, the Onderzoeksraad of the K.U.Leuven and NATO for supporting their research, Dr Anne´ for providing data on bacterial reproduction and Julie Puttemans for the drawings. References [1] Borovsky D, Carlson DA, Griffin PR, Shabanowitz J, Hunt DF. Mosquito factor: a novel decapeptide modulating trypsin enzyme like biosynthesis in the midgut. FASEB J 1990;4:3015 –20. [2] Briers T, De Loof A. The molting hormone activity in Sarcophaga bullata in relation to metamorphosis and reproduction. Int J Invertebrate Reprod 1980;2:362 – 72. [3] Bylemans D, Borovsky D, Hunt DF, Shabanowitz J, Grauwels L, De Loof A. Sequencing and characterization of trypsin like oostatic (TMOF) from the ovaries of the grey fleshfly, Neobellieria (Sarcophaga) bullata. Regul Pept 1994;50:61 – 72. [4] Bylemans D, Proost P, Samyn B, Borovsky D, Grauwels R, Huybrechts R, Van Damme J, Van Beeumen J, De Loof A. Neb-colloostatin, a second folliculostatin of the grey fleshfly, Neobellieria bullata. Eur J Biochem 1995;228:45 – 9. [5] Callaerts P. Lectin binding sites during Drosophila embryogenesis. PhD thesis. Belgium: K.U. Leuven, 1992. [6] Callaerts P, Vulsteke V, Peumans W, De Loof A. Lectin binding sites during Drosophila embryogenesis. Roux’s Arch Dev Biol 1995;204:229 – 43. [7] Campbell NA, Mitchell LG, Reece JB. Biology. Concepts and Connections. Redwood City, CA: Benjamin-Cummings, 1994. [8] De Loof A. All animals develop from a blastula: consequences of an undervalued definition for thinking on development. Bioessays 1992;14:573 – 5. [9] De Loof A, Bylemans D, Schoofs L, Janssen I, Huybrechts R. The folliculostatins of two dipteran insect species, their relation to matrix proteins and prospects for practical applications. Entomol Exp Appl 1995a;77:1 – 9.
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[10] De Loof A, Bylemans D, Schoofs L, Janssen I, Spittaels K, Vanden Broeck J, Huybrechts R, Borovsky D, Hua Y-J, Koolman J, Sower S. Folliculostatins, gonadotropins and a model for control of growth in the grey fleshfly, Neobellieria (Sarcophaga) bullata. Insect Biochem Mol Biol 1995b;25:661–7. [11] De Loof A, Vanden Broeck J. Communication: the key to defining ‘life’, ‘death’ and the force driving evolution. ‘Organic chemistry-based’ versus ‘artificial life’. Belgian J Zool 1995;125:5 – 28. [12] Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 1996;383:531–5. [13] Gilbert SF. Developmental Biology. Sunderland, Massachusetts: Sinauer Associates, 1994. [14] Girardie J, Girardie A. Lom-OMP, a putative ecdysiotropic factor for the ovary of Locusta migratoria. J Insect Physiol 1995;42:215 – 21. [15] Girardie J, Richard O, Huet JC, Nespoulos C, Van Dorsselaer A, Pernollet JC. Physical characterization and sequence identification of the ovary maturating parsin. A new neurohormone purified from the nervous corpora cardiaca of the African locust Locusta migratoria migratorioides. Eur J Biochem 1991;202:1121– 6. [16] Gosden R. The vocabulary of the egg. Nature 1996;383:485 – 6. [17] Hafer J, Ferenz HJ. Locust vitellogenin receptor: an acidic glycoprotein with N-and O-linked oligosaccharides. Comp Biochem Physiol 1991;100B:579–86. [18] Hauser F, Nothacker HP, Grimmelikhuijzen CPJ. Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drososphila melanogaster, structurally related to members of the thyroid-stimulating hormone, folliclestimulating hormone, luteinising hormone/choriogonadotropin receptor family from mammals. J Biol Chem 1997;272:1002– 10. [19] Hua Y-J, Bylemans D, De Loof A, Koolman J. Inhibition of ecdysone biosynthesis in flies by a hexapeptide isolated from vitellogenic ovaries. Mol Cell Endocrinol 1994;104:R1–4. [20] Huybrechts R, De Loof A. Induction of vitellogenin synthesis in male Sarcophaga bullata by ecdysterone. J Insect Physiol 1977;23:1359 – 62. [21] Huybrechts R, De Loof A. Similarities in vitellogenin and control of vitellogenin synthesis within the genera Sarcophaga, Cal-
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Review
Reproduction and love: strategies of the organism’s cellular defense system? A. De Loof *, R. Huybrechts, S. Kotanen Zoological Institute of the K.U. Leu6en, Naamsestraat 59, 3000 Leu6en, Belgium Received 19 August 1997; received in revised form 02 February 1998; accepted 26 February 1998
Abstract A novel view is presented which states that primordial germ cells and their descendants can be regarded as ‘cancerous cells’ which emit signals that activate a whole array of cellular defensive mechanisms by the somatoplasm. These cells have become unrestrained in response to the lack of typical cell adhesion properties of epithelial cells. From this point of view: (1) the encapsulation of oocytes by follicle cells, vitelline membrane and egg shell; (2) the suppression of gonadal development in larval life; (3) the production of sex steroid hormones and of vitellogenin; and (4) the expulsion of the gametes from the body fit into a general framework for a defense strategy of the somatoplasm against germ line cells. Accordingly, the origin of sexual reproduction appears to be a story of failure and intercellular hostility rather than a ‘romantic’ and altruistic event. Yet, it has resulted in evolutionary success for the system in which it has evolved; probably through realizing feelings of ‘pleasure’ associated with reproduction. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Cytoskeleton; Cancer; Endocrinology; Ecdysone; Estrogen; Germ cells; Pole plasm; Sex
1. Introduction After living matter came into being about 4 billion years ago, some organisms started to divide. By doing so, they generated offspring and became successful in terms of evolution. It is often overlooked that a cell, while engaging in splitting off a daughter compartment, risks death. Although we do not have an idea about ‘emotions’ in other organisms, one cannot escape the impression that despite the inherent risks involved, reproduction is always associated with ‘well-being’, with ‘success’ and with ‘good communication’ [11]. A much less romantic picture appears, however, if one analyzes, at the cellular level, a variety of mechanisms which are operational during reproduction and than tries to understand how they could have originated. * Corresponding author. Tel.: +32 16 323912; fax: + 32 16 323902; e-mail: [email protected] 0742-8413/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0742-8413(98)10007-5
2. Speculations about the origin of cell division and reproduction Let us assume that the first living organism on earth, that appeared about 3.7 billion years ago, was a bacterium-like cell surrounded by a plasma membrane and devoid of membrane-bound cell organelles. Later, this cell, often referred to as the progenote, pinched off an identical daughter cell that contained the whole machinery needed for growth, metabolism and generation of other daughter cells and thereby initiated a continuous propagation of living cells. It is not likely, that this cell division occurred ‘intentionally’ but probably by physiological necessity. Which physiological process initiated such a division into two halves in the progenote, with the inherent danger of not surviving such a risky operation? Did an external substance enter the primordial cell and trigger a division process? Or
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did this cell synthesize a molecule(s) that triggered the division process? Since no records exist dating 4 billion years ago, we can only propose mechanisms which operate in present day bacteria. The fission process was probably initiated once a critical level of an inhibitor or stimulator of growth was reached [24]. For a long time there was a lack of definitive evidence for cell cycle-dependent synthesis of particular molecules in bacteria. Thus, it was thought that their cell cycle-control system was different from that in eukaryotes with respect to the involvement of cyclins [26] (see further). It has been known for a few years that the DnaA protein plays an essential role in an early step of DNA initiation by binding to three Dna boxes at oriC on the chromosome of Escherichia coli [26]. Substantial progress in understanding bacterial cell cycle control has recently been made in the bacterium Caulobacter crescentus which produces two different cell types at each cell division, a sessile stalked cell and a motile swarmer cell. In this prokaryote, a protein, called tubulin-like GTPase FtsZ (Fts, filamenting temperature sensitive) is critical for the initiation of cell division. Its intracellular concentration is highest at the beginning of cell division. Later, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is most probably due to the degradation of FtsZ in the swarmer compartment of the pre-divisional cell [27]. Bacterial ‘sex life’ is limited to the formation of cytoplasmic bridges through which DNA can be exchanged. This process is called conjugation, in which the donor cell’s sex pili (male) are attached to the recipient cell (female) [7]. If the mechanism involved in cell division which is universally used by contemporary eukaryotes was used by the primordial eukaryote, then we can speculate upon similarity to the mitosis-oscillator in eukaryotes which is based upon synthesis and degradation of specific proteins that are called cyclins. A high concentration of cyclins prevents cell division and keeps the cells in metaphase. Breakdown of cyclins, triggered by an increase in the levels of free Ca2 + , enables the cells to return to interphase and cell division to restart [13]. There are only two types of inorganic ions which are widely used as triggers in a variety of biochemical processes, namely Ca2 + and H + . High concentrations of both are toxic because they cause conformational changes in a variety of proteins. The concentration of intracellular calcium is usually kept very low, at about 10 − 7 –10 − 8 M while its concentration in the extracellular fluid is often several orders of magnitude higher. If the plasma membrane of a cell becomes more permeable to Ca2 + , it will enter the cytoplasm and trigger a variety of biochemical processes. High H + concentration causes acidity, which in turn induces conformational changes in proteins. This means that the two
inorganic ionic species which are frequently used in signaling are in fact toxic to cells. Thus, the mechanism which triggered cell division could have been either a selective change in the permeability to Ca2 + or H + , or a change in the activity of ion pumps present in the plasma membrane.
3. Basic physiological cellular processes underlying reproduction in animals As a result of cellular physiology of reproduction is best documented in animals, analogies will be drawn from animal model systems, such as insects and mammals. We will discuss defense strategies in pre- and postvitellogenic females, in males, at fertilization and during intra-uterine development.
4. Defense mechanisms at work before vitellogenesis
4.1. The origin of the germ cell line One of the best documented models in the study of animal embryonic development is the fruitfly Drosophila melanogaster. At its posterior pole, its newly laid egg contains a distinct class of granules, the polar granules, which are important in segregating and maintaining the germ cell line. At least eight genes and probably a mitochondrial factor as well, are required for the formation of a functional polar plasm. Fertilization causes an increase in the cytoplasmic Ca2 + concentration. Next, the zygote nucleus starts to divide without concomitant formation of plasma membranes. Each nucleus is associated with a centrosome that plays an important role in nuclear division. Thus, early in embryogenesis of insects (but not in all animal species) only nuclei-centrosome complexes are formed during each mitotic cycle, not whole cells. The third mitotic cycle yields eight of these complexes. Depending upon the species, from this eight complex stage on, a centrosome, which usually has a nucleus in tow, comes in the immediate neighborhood of the polar granules. Next, an as yet unidentified mechanism is activated which makes the nucleus-centrosome-polar plasm complex migrate towards the boundary of the egg and it causes this complex to be pinched off from the rest of the egg in the form of the first cell with plasma membranes (Fig. 1). This ejected pole cell will give rise to the future gametes while all cells that will eventually be formed inside the egg will turn into somatic cells and form all the tissues of the body: integument, muscles, gut, nervous tissue etc.. Nuclear divisions without plasma membrane formation continue in the egg. The germ cell precursor cell also undergoes a few mitotic rounds
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Fig. 1. The definition of an animal is that it is an organism which develops from a blastula [8]. This means that the young embryo passes through a stage in which it organizes itself as an epithelium. At some early stage in development, some cells escape from being integrated in an epithelium and form the germ cell anlage. In dipteran insects (Drosophila has been well studied in this respect), the segregation of this anlage happens already before the blastoderm is formed. At one end of the egg, there is a group of particles with as yet poorly defined chemical composition (anyhow rich in RNA), called the polar granules. When a nucleus comes in the vicinity of these granules, segregation is induced. The isolated cell will undergo several rounds of mitosis, thereby forming the germ cell anlage.
which include plasma membrane formation. At a given developmental stage plasma membranes form around the nuclei which in the mean time have aligned themselves in the cortical layer underneath the plasma membrane. In this way, a blastula is formed. The pole cells remain clustered together at the posterior pole of the blastoderm until gastrulation (gut formation) movements carry them into the lumen of the posterior midgut primordium. The pole cells then migrate freely through the walls of the midgut into the body cavity, interact with mesodermal cells that encapsulate them, as if they were foreign intruders that have to be inactivated. During this time, the pole cells do not divide whereas somatic cells continue to divide, indicating that germ cells are selectively inhibited. Early in the embryonic life of invertebrates, the germ cells with their big
Fig. 2. In a human embryo of 3-weeks-old, the primordial germ cells are found in the wall of the yolk sac, close to the site where the future umbilical cord will be attached (redrawn after Langman [23]). Here again, the primordial germ cells are not epithelially organized. By ameboid movements, these cells migrate towards the gonad primordia which they invade.
nuclei migrate towards the anlage of the gonads (Fig. 2). Once there, they go through a number of rounds of mitotic divisions. However, not all the germ cells survive [23]. With respect to the origin of meiosis, one can only speculate about the cellular mechanism(s) that initiated this complex process.
4.2. Cell adhesion differences between somatoplasm and germ plasm All somatic cells are the progeny of stem cells which were once part of the blastular epithelium. Depending upon the species, the stem cells of the germ cell line either did not integrate in this epithelium or did escape from an epithelial environment early in development, leaving them solitary. This means that the normal cell adhesion phenomena which are typical for epithelia are not functioning in the primordial germ cells. This aspect is important because it is closely linked to the very nature of being an animal. The Five Regna classification system of Whittaker [33], defines an animal as an organism which develops from a blastula. Histologically, it means that an animal is an organism that in its early development is an epithelium enclosing a fluidfilled cavity [8]. Thus, the basic principle underlying animal development is epithelial formation, folding and segregation. In diblastic animals (Coelenterates), all somatic cells are organized as folded epithelia, but the germ cells are not. In triblastic animals, the ecto- and endoderm are usually epithelia. The mesoderm gives rise to tissues, some of which have an epithelial structure, others, for example, muscle cells, the endoskeleton of vertebrates and blood cells have not. The major characteristic of an epithelium is that its cells are tightly interconnected by specific cytoskeletal elements and cell adhesion molecules. The fact that primordial germ cells are solitary indicates that their cytoskeleton is not able to form tight junctions with their neighbors. Cells
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which loose their cell adhesion properties and start wandering around in the body and proliferate, are considered as having properties of cancer cells. Primordial germ cells have such properties. Female germ cells are aberrant because they grow so big and male primordial germ cells are aberrant because of their frequent divisions. Blood cells too are solitary and mobile cells which, in the case of erythrocytes, have a fast proliferation rate. The body can cope with them by either eliminating them at a fast rate (erythrocytes) or by controlling their growth (leucocytes). Many germ cells are also killed soon after they are formed. The ones that survive are restrained in a variety of ways.
4.3. Programmed cell death in part of the germ cell line population In Drosophila, less than half of the approximately 40 pole cells will succeed in establishing themselves in the forming gonads; the others die. In the human female fetus, during the fifth month of embryonic development there are about 6 million egg cells. They are all arrested in early first meiotic division, apparently a stage in which human egg cells can survive for approximately 50 years. After this arrest, no more cells will be formed during life, on the contrary, quite a lot will degenerate. By the time of birth, only 700000 to 2 million out of the original 6 million survive. By the time a girl reaches puberty, this number further shrinks to about 40000 [23]. The cause(s) of disappearance of so many germ cells is unknown. Perhaps they are destroyed by somatic cells or tissues, or by apoptosis.
4.4. A role for the cytoskeleton in defining the essence of being male or female Reproduction started with the introduction of cell division. In eukaryotes, the cytoskeleton plays a key role in this process. In prokaryotes, the properties and role of the cytoskeleton are not so well documented. The primordial difference between male and female resides in the genes and this is evidently true in all species in which there is no sex reversibility under the influence of environmental factors, as was found in some fishes. A master gene can be absent in one of the sexes or its mRNA can be processed differentially in males and females as was found in Drososphila. One of the sexes can be haploid while the other is diploid as in bees. There are still other possibilities for sex determination. Whatever mechanism is used, there must be master proteins activating cellular mechanisms in a given sex-specific way. Vertebrate females have larger amounts of estrogens than males whereas males have more androgens than females (Fig. 3). Estrogens are made from androgens through the action of specific enzymes. For example, aromatase converts testosterone
into estradiol. In adult flies, the steroid moulting hormone 20-OH ecdysone seems to act as the equivalent to the estrogens of vertebrates. It induces vitellogenin synthesis if injected into adult males [20,21]. The difference between adult male and female flies is that females have a higher titer of 20-OH ecdysone in their hemolymph than males [2]. Besides the hormonal difference, there is also a difference in cytokinesis in the germ cells. During meiotic divisions, four functional spermatozoa form from one spermatogonium as compared to one oocyte and to three non-functional polar cells stem from one oogonium. The typical textbook explanation to this phenomenon is that the oocyte needs to store as much cytoplasm as possible in order to accumulate the greatest reserves possible for embryogenesis, while the male germ cells need only to be able to swim for a short while. The underlying principle is that cytokinesis is asymmetrical in the female germ cells which enter meiosis whereas in males it is symmetrical. The cytoskeleton plays an important role in cytokinesis. The major difference between the cytoskeleton of oocytes and spermatozoa is that the oocytes are actin-rich and the spermatozoa are tubulin-rich. The combination of asymmetry in cytokinesis and differences in sex hormones constitute the very nature of sexual differentiation.
4.5. Inhibition of gonadal de6elopment during lar6al life There is no a priori reason that the germ-line (stem) cells which are always formed very early in development should be developmentally arrested during fetal and larval life. It appears that it is not due to an internal clock present in the oocytes but to an inhibitory action of the somatoplasm. In mammals the arrest of oocyte development and sperm production is thought to be primarily due to a low production rate of two gonadotropic hormones: follicle stimulating hormone (FSH) and luteinizing hormone (LH). Although this could explain the arrest of gonadal development, it is probably only one of several mechanisms that are involved. The low production rates of FSH and LH, which are controlled by an unknown mechanism, come to an end at puberty. In adults of the fleshfly, Neobellieria bullata, a different mechanism has been detected. Adult females have to ingest a protein meal (e.g. meat) in order to produce substantial amounts of yolk proteins. Each ovary contains several dozens of ovarioles in which only one follicle at a time enters vitellogenesis. Once the ripe eggs have been expelled from the body, after having been retained in the uterus for about 4 days to complete embryogenesis, a second batch of ovarian follicles will enter vitellogenesis. The reason why the penultimate follicles do not start vitellogenesis is that the ovary releases colloostatin, a 19-mer peptide, which inhibits
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Fig. 3. Major physiological differences between the female and male sex which result from the genetic difference, whatever its nature, between the sexes. A first difference is that only the oocyte is capable of accumulating, by pinocytosis, vitellogenins from extra-oocytic origin. Actin plays an important role in this process. A second difference is that during meiosis cytokinesis is symmetrical in males while it is asymmetrical in females. This reflects a differential organization of the cytoskeleton in both sexes. A third difference is that the cytoskeleton of oocytes is actin-rich, while that of spermatozoa is rich in tubulin. Microtubules which are organized in the typical 9 +2 configuration allow fast movement. Actin can bind a variety of different molecules, for example some maternal messenger RNA’s and proteins. A fourth difference concerns sex hormones, when present. In vertebrates the oestrogen-androgen situation is well understood. In the fleshfly Neobellieria, the best documented species in this respect, the moulting hormone 20-OH-ecdysone induces vitellogenin synthesis. Its titer is much higher in females than in males. In many invertebrates, the nature of sex hormones, if present, is not yet known.
growth of the penultimate oocytes but not of vitellogenic ones [4]. The strong resemblance of colloostatin to the sequence 468 – 480 of preprocollagen a1 (IV) from Drosophila, indicates that colloostatin is probably a degradation product of a collagen-like molecule present in the sheath which surrounds the individual ovarioles. This offers an interesting model for growth
control [9]. Indeed, it is the expansion of the ovary itself which triggers the release of an inhibitor which prevents younger follicles from becoming pinocytotic and competing with older oocytes for vitellogenin uptake. During periods of fast growth, any tissue sheathed in a basement membrane is probably capable of using such a mechanism. A similar mechanism may also operate in
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Fig. 4. Schematic representation of a mammalian ovarian follicle. The oocyte releases signals which govern the physiological behavior of the follicular epithelium. The only factor of the chemical identity has as yet been established is growth differentiation factor-9 (GFD-9). This factor is required for follicular development beyond the primary one-layer follicle stage [12]. Redrawn after Gosden [16].
vertebrates before reaching sexual maturity. Growing vertebrates degrade and rebuild their collagen every day. Some degradation products resulting from this process could perhaps act as inhibitors of gonadal development. It cannot be excluded that in insects, a brain gonadotropin-based mechanism could also play a role in inhibiting gonadal development in larvae. Presently, experimental evidence is lacking. Although a receptor, structurally related to the FSH/LH receptor family of vertebrates has been cloned in Drosophila [18] and although FSH- and LH-immunoreactivity has been demonstrated in cockroach brains [31], peptides with similar amino acid sequence as GnRH, LH and FSH have not yet been found in insects [10]. In Locusta migratoria, an ovary maturating parsin (Lom-OMP) [15] has been isolated and identified in the brain-corpus cardiacum complex. This gonadotropin is the physiological equivalent of FSH/LH. Lom-OMP is not homologous to any known vertebrate peptide. It stimulates the production of the steroid hormone ecdysone [14], which acts as the equivalent of estrogens in some insects like flies [20,21]. As this is the case for FSH/LH in vertebrates, Lom-OMP immunoreactivity is not restricted to neurosecretory cells in the pars intercerebralis of adult females only. It is present in larvae and males as well. Injection of Lom-OMP into larvae does not make the gonads develop, just like injections of FSH/LH fail to do so in immature vertebrates. Apparently, an inhibitory mechanism(s) is at work. In Neobellieria, the N-terminus sequence 1 – 17 of the cAMP generating peptide [28] (a 48-mer, not related to any known vertebrate or invertebrate hormone: [28]) stimulates vitellogenesis (unpublished results). In the moth Lymantria dispar, a gonadotropin (testis ecdysiotropin) has been identified which stimulates ecdysone biosynthesis in the testis [25,32]. In dipteran insects, a second hormone capable of inhibiting ovarian follicle development has been discov-
ered. It has been named trypsin modulating oostatic factor (TMOF). Since it acts on vitellogenic follicles it will be discussed later. In plants, a germ-line cell anlage is not yet present in the plant seed (which is a baby plant): the primordia only start developing once the flower primordium forms. In fungi, ferns and mosses, meiosis is also a late phenomenon. This means that during the immature stages, there must be a factor(s) which inhibits the development of sexual organs and the production of ripe gametes.
4.6. Oocyte-follicle cell interactions Oocytes become ensheathed by one or a few layers of follicle cells. The typical textbook states that the functions which are attributed to these cells are in providing information and nourishment to the oocyte [16]. Our view upon this phenomenon is different. If a relatively large foreign body enters the body, one of the possible defense strategies is that it will be encapsulated by specialized cells of the defense system. In this case the encapsulation process is a hostile reaction, intended to neutralize the invader. We think that the ensheating of the oocyte could be a reaction of the somatoplasm to the oocyte which grows much bigger than a normal somatic cell and perhaps, releases (or causes surrounding cells or membranes to release) signaling molecules which are recognized by somatic cells as ‘alarm’ signals. Encapsulation is then ensured to restrain the oocyte. Since the oocyte does not appear to ‘suffer’ much from this encapsulation, follicle cell overlayering has usually been explained as an act of protection by the follicle cells. The oocyte and the follicle cells communicate using various signals (Fig. 4). Mammalian oocytes affect the growth and differentiation of the granulosa cells around it [16]. They produce a factor(s) which increases the rate of synthesis of estrogens and decrease the
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synthesis of progesterone by granulosa cells that have been stimulated by FSH in vitro. In mice, oocytes secrete a factor(s) that stimulates the production of hyaluronic acid which is involved in the process of mucification. Removal of the oocyte, stops the secretion of hyaluronic acid from the granulosa cells after FSH stimulation. Another factor prevents the breakdown of the extracellular matrix by inhibiting the production of urokinase plasminogen activator. The oocyte can also inhibit the proliferation of the granulosa cells throughout growth of the follicle. Knock-out technology has shown that GFD-9 deficient mice formed only one layer of granulosa cells and their theca failed to differentiate. It has been suggested that GDF-9 might be the mucification factor [12]. However, no definitive evidence is available to prove communication between oocyte and follicle cells in invertebrates.
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lifted. After gonadotropic hormones are secreted and oocytes start to grow, cell layers surrounding the oocyte secrete large amounts of sex steroid hormones, namely estrogens in vertebrates and ecdysone in insects. These hormones signal the development of female gonads. The liver of egg laying vertebrates and the fat body of insects start to synthesize large amounts of vitellogenins, which are secreted and circulated in the blood or hemolymph. Vitellogenins bind to specific receptors on the surface of the oocyte and the oocyte internalizes the vitellogenins by pinocytosis. The process of vitellogenin biosynthesis resembles mobilization of antibodies against a foreign intruder (bacterium, virus etc.). However, vitellogenins do not share structural similarities with the immunoglobulins of vertebrates, they are agglutinins.
5.3. Agglutinins and lectins 5. Defense strategies from vitellogenesis onwards
5.1. Steroid hormone production by the follicle cells Production of estrogens by the ovary of vertebrates, the major site of sex hormone synthesis, makes the body of females competent for reproduction. In our view, the sex steroids that are released by the follicles might be a warning signal targeted at the somatoplasm in order to incite it to suppress the growing oocyte(s). In flies, the equivalent of estrogens is the steroid moulting hormone 20-OH-ecdysone [20,21]. The follicle cells are a major site of synthesis. Ecdysone’s titer elevation during vitellogenesis is the major stimulating factor of vitellogenin synthesis in many insect species.
5.2. The mechanism of fast growth of yolky eggs The majority of animals produce yolky eggs which are expelled from the female’s body. General biology textbooks may explain the difference between the big egg cell and the small, mobile spermatozoon from a teleological point of view. In order to allow embryogenesis, egg cells should accumulate nutrients. The spermatozoa should specialize for swimming. Yet, physiologists think in another way. Oocyte growth is accomplished by the uptake of specific egg yolk proteins from the hemolymph, called vitellogenins. To find out why oocytes accumulate specific blood proteins, an insight in the physiological principles underlying yolk formation is necessary. Yolk proteins are not formed by the oocytes themselves, they are synthesized elsewhere. Distant tissues such as the liver in vertebrates and the fat body in insects are major sites for this synthesis. In some species, the follicle cells also contribute a relatively small amount. When females become sexually mature, the block on oocyte growth is
Lectins are proteins with specific recognition and reversible binding to carbohydrate moieties without altering the covalent structure of the glycosylated ligands [22]. A few years ago, the name lectins was used for proteins that are present in plants, whereas the term agglutinins was used for proteins of animal origin. At present the term lectins is used for proteins from plants and animals. Well known agglutinins are those that are present in the plasma of persons with blood group A (these have agglutinins b) and blood group B (which have agglutinins a). Some lectins that recognize specific carbohydrate moieties on red blood cells can agglutinate the cells. Agglutination will occur when nonmatching blood groups are mixed (e.g. when a donor of blood group A would receive blood from a donor with group B). Agglutinins (lectins) in the plasma of a person with blood group A, will bind to the carbohydrate moieties on the surface of the erythrocytes of the donor with blood type B causing agglutination. Agglutination is not a defense mechanism against invading red blood cells from other individuals because this rarely happens in nature. The fact that agglutinins are found in both vertebrates and invertebrates suggests that such molecules were present early in the evolution of all animals. Lectins/agglutinins might be a primitive mechanism against micro-organisms before genes coding for antibodies became functional in some groups of animals. This view, however, is not shared by everybody. ‘Lower’ animals do not have an immunoglobulin-based defense system. They use other defense mechanisms, for example antibacterial and antifungal peptides, apotopsis, encapsulation and agglutinin/lectin production. These might have been the defense mechanisms when gametes were first produced. Stynen and De Loof [29,30] first described the agglutinin properties of two major vitellogenic hemolymph proteins from the Colorado potato beetle, Leptinotarsa
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decemlineata. Lectins labeled with a fluorescencent tag, strongly stain developing oocytes of Drosophila, suggesting a correlation between lectin activity and yolk uptake [5,6]. Hafer and Ferenz [17] reported that the locust vitellogenin receptor is an acidic glycoprotein with 54% of the apparent molecular mass contributed by O- or N-linked carbohydrates and that neuraminic acid is essential for binding vitellogenins. If the binding of vitellogenins to the surface of the oocyte was to inactivate and encapsulate the oocyte, the opposite happened; the oocyte internalized vitellogenins and stored them as vitellins in yolk vesicles. The seeds of many species of plants contain large amounts of lectins: soybean lectin, wheat lectin etc. The exact role of these lectins in plants is not known. Lectins in seeds may be used as a nutrient reserve and as antimicrobial agents preventing degradation by bacteria, fungi and insects of stored reserves. Thus, vitellogenins stored in the yolk of animal eggs are analogous to the lectins stored in plant seeds.
5.4. Encapsulation by coating layers Another type of interaction of the follicle cells with the oocyte is the deposition by the follicle cells of material which encapsulates the oocyte, for example the vitelline membrane and chorion of the insect egg. The formation of a layer of albumen by the oviduct and of egg membrane and egg shell by the uterus of reptiles and birds are other examples of protective layers. Encapsulation by a rigid layer may block oocyte expansion and vitellogenin or other molecules transport to the surface of the oocyte. Thus, oocyte growth will be halted. The major components of the vitelline membrane form a fibrous mat with holes that allow blood proteins to move in. During oocyte growth, it is probably remodeled by the combined action of proteases and deposition of newly formed material originating from the follicle cells. In placental mammals, the equivalent of the vitelline membrane of the insect egg is a thick extracellular matrix called the zona pellucida [13]. The granulosa cells-zona pellucida-oocyte complex is released from the ovary as a Graafian follicle. The oviducts do not produce thick layers of albumen and the uterus does not make an egg shell. However, in animals which oviposit in a dry environment, an egg shell is made. For example, in insects the follicles synthesize chorion proteins shielding the oocyte. Then, the follicle cells die. In reptiles and birds, the uterus synthesizes the egg’s shell. Making a shell can be regarded as the last option left for permanently encapsulating an ‘unwanted’ entity in the body. After this process is over, the female carries in its interior a large egg(s) which does not contribute any-
more to her internal physiology, and the best the female can do is to expel the egg(s) from her body. This, however, involves the action of myotropic egg laying hormones such as oxytocin in vertebrates and egg laying hormone in molluscs and annelids, etc. In some species, egg laying seems to be a big relief for the female, as might be guessed from the sound that a hen makes after laying an egg. In the language of cell physiology this might read as: ‘Hurah, the cancerous cell is conquered!’
5.5. Inhibition of o6arian de6elopment by TMOF In the mosquito, Aedes aegypti the follicle cells synthesize and release a decapeptide (H-Tyr-Asp-Pro-AlaPro-Pro-Pro-Pro-Pro-Pro-OH) which binds to the gut receptor and shuts down the trypsin biosynthesis in the gut. Thus, blood digestion is arrested and the supply of nutrients for vitellogenin biosynthesis stops. The peptide has been named Aedes-trypsin modulating oostatic factor or Aea-TMOF [1]. From the fleshfly Neobellieria, a hexapeptide (H-Asn-Pro-ThrAsn-Leu-His-OH) with similar functions has been isolated (Neb-TMOF: [3]). Neb-TMOF has been found to be a potent inhibitor of ecdysone biosynthesis in larvae of the fleshfly Calliphora [19] and to inhibit vitellogenin production in adult females [3]. Although the coding gene has not yet been identified, there are good indications that TMOF is cleaved from a vitelline membrane protein.
5.6. Males: fertilization If estrogens are alarm hormones, at high titer, what are androgens? These hormones are produced by Leydig cells in the testis. When sperm production starts, the titer of androgens is high. This drives the male to ejaculate sperm cells into a hostile environment in which their survival is limited. Thus, the interaction between the somatoplasm and the sperm cells is hostile. Spermatozoa are armed with a vesicle that contains lysosomal enzymes that can make a hole in the plasma membrane of the oocyte. Too big a hole in a membrane will result in cell death. When are females ready for copulation? Our answer to this question is when all their own defense strategies against neutralizing the oocytes have failed. The final strategy is to call for help from a male which, in a watery environment, brings killer cells in the immediate vicinity of the egg(s), or in a dry environment brings these cells inside the body of a female to specifically attack the oocyte. The oocytes survive because they close the hole immediately after they swallowed the nucleus of the spermatozoon.
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5.7. Intrauterine de6elopment: parasitism. Brood care: an act of ensla6ing? In mammals with placenta, the oocyte is devoid of yolk. Their ancestors, the reptiles had yolky eggs. Apparently, some mutation that occurred, perhaps in the starting codon of the vitellogenin gene, ‘wiped out’ vitellogenin biosynthesis. An embryo that develops from such an egg can survive only if it is ‘hooked’ to a constant food supply. This was achieved by invading the epithelial lining of the uterus and developing in the uterus. Nutrition-wise these embryos are parasites of their mother. The developing embryo contains many proteins, coded for by paternal genes, which should be recognized as foreign by the immunological system of the mother. However, the developing embryo has mechanisms which prevent the mother from making antibodies against it. At least in humans, nobody will question that parents who foster their offspring display ‘love’. But is this love a deliberate strategy from the side of the parents themselves, or is this behavior imposed by the offspring in which the germ cell anlage of the next generation is already at work? To a large extent, the offspring enslaves one or both of the parents who would have had an easier life without the offspring. Yet, having the offspring means transmitting 50% of the genes of each parent to the next generation, thereby ensuring genetic success.
6. Conclusion Humans find that the opposite of aggression is reproduction in all its aspects: courtship, copulation, pregnancy, love, care for partner and offspring etc. Therefore, the view we presented here may elicit rejection at first reading. After a careful analysis of the interactions between the different cell types involved in animal reproduction, we came to the apparent conclusion that the somatoplasm treats the germ cells as if they were cancer- or foreign cells that have to be destroyed with all means. From our approach, it follows that the origin of ‘love’ in the course of evolution, might have been actually due to the ‘nasty’ behavior of germ cells which elicited a whole array of hostile defense mechanisms by somatic cells. Thus, our story turns the ‘romance of reproduction’ into a story of cellular aggression and hostility. Should one be shocked? Not at all. The oocytes, these ‘obese’ beauties with their ‘dominant’ and ‘nasty’ character succeed in making both the male and female somatoplasm believe that caring for them is a joy, which cleverly ensures their survival and development. The final outcome of this story of hostility is genetic/evolutionary success. Reproduction at the or-
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ganismal level is a mechanism that ensures that genes survive longer than the organisms themselves; it tries to attain immortality. At the level of the population, reproduction can be considered as regeneration of the population [11]. We are evidently aware that in describing physiological/biochemical phenomena it is not common practice to use terms referring to human feelings, such as hostility, pleasure and love. No doubt, some readers will find that cell biologists and physiologists should keep far away from such an antropomorphic approach. Our view is that this is a too narrow approach for biology in general. Communication is essential to life. In fact, what we call ‘life’ is the communication activity of a communicating compartment [11]. In communication, feelings play a very important role, which is ignored in contemporary biology. Finally, we would not be surprised if some of the cell’s strategies that are used to inhibit oocyte growth might one day lead to practical applications in treating cancer. Acknowledgements The authors thank the Fonds voor Wetenschappelijk Onderzoek, the Onderzoeksraad of the K.U.Leuven and NATO for supporting their research, Dr Anne´ for providing data on bacterial reproduction and Julie Puttemans for the drawings. References [1] Borovsky D, Carlson DA, Griffin PR, Shabanowitz J, Hunt DF. Mosquito factor: a novel decapeptide modulating trypsin enzyme like biosynthesis in the midgut. FASEB J 1990;4:3015 –20. [2] Briers T, De Loof A. The molting hormone activity in Sarcophaga bullata in relation to metamorphosis and reproduction. Int J Invertebrate Reprod 1980;2:362 – 72. [3] Bylemans D, Borovsky D, Hunt DF, Shabanowitz J, Grauwels L, De Loof A. Sequencing and characterization of trypsin like oostatic (TMOF) from the ovaries of the grey fleshfly, Neobellieria (Sarcophaga) bullata. Regul Pept 1994;50:61 – 72. [4] Bylemans D, Proost P, Samyn B, Borovsky D, Grauwels R, Huybrechts R, Van Damme J, Van Beeumen J, De Loof A. Neb-colloostatin, a second folliculostatin of the grey fleshfly, Neobellieria bullata. Eur J Biochem 1995;228:45 – 9. [5] Callaerts P. Lectin binding sites during Drosophila embryogenesis. PhD thesis. Belgium: K.U. Leuven, 1992. [6] Callaerts P, Vulsteke V, Peumans W, De Loof A. Lectin binding sites during Drosophila embryogenesis. Roux’s Arch Dev Biol 1995;204:229 – 43. [7] Campbell NA, Mitchell LG, Reece JB. Biology. Concepts and Connections. Redwood City, CA: Benjamin-Cummings, 1994. [8] De Loof A. All animals develop from a blastula: consequences of an undervalued definition for thinking on development. Bioessays 1992;14:573 – 5. [9] De Loof A, Bylemans D, Schoofs L, Janssen I, Huybrechts R. The folliculostatins of two dipteran insect species, their relation to matrix proteins and prospects for practical applications. Entomol Exp Appl 1995a;77:1 – 9.
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