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Perspective |
a 2441 Kenilworth Road, Cleveland Heights, Ohio 44106
One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity. (Willard Gibbs, 1881)
Omne vivum ex ovo. (Wm. Harvey, 1651)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONBACKGROUND: REPRODUCTIONBACKGROUND: HOW THINGS EVOLVEPUTTING IT ALL TOGETHERNOTES TO THE READERREFERENCES |
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corpus luteum, developmental biology, evolution, follicular development, oocyte development, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONBACKGROUND: REPRODUCTIONBACKGROUND: HOW THINGS EVOLVEPUTTING IT ALL TOGETHERNOTES TO THE READERREFERENCES |
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Viviparity is a form of reproduction in which the embryo either begins or begins and also completes its development within or on a part of the parent's body. In the eutherians ("placental mammals"), it differs from all other forms of viviparity in at least two essential respects. First, progesterone (P) is indispensable for both its initiation and its maintenance. Second, the blastocyst's trophoblast cells have properties lacking in all other forms of vertebrate embryogenesis.
The vertebrate ovarian follicle—the basic unit of ovarian hormonal activity and the means by which the oocyte reaches maturity—in eutherians is also unique. All known vertebrate follicles have the same basic form, but the mammalian follicle has four unique characteristics: 1) in marsupials and eutherians, the mature follicle is vesicular; 2) its granulosa cells are more highly differentiated at follicular maturity than are those of other vertebrates; 3) it contains proportionately more of these cells than in other vertebrates; and 4) in eutherians, its oocyte is the smallest of all vertebrate oocytes and contains no yolk.
The common ancestor of all vertebrates almost certainly reproduced by spawning. Both in the transformation of the spawner's follicle into that of the eutherians and in the accompanying changes leading to the unique character of eutherian viviparity, two features of the vertebrate follicle stand out above all others: atresia and yolk.
Among the eutherians, the vast majority of all the gametes a female has are lost through atresia before ovulation. Among spawning vertebrates, most of the oogonia produced during a female's life are ovulated as mature oocytes. Yolk is the principal—and sometimes only—source of nutrition through which the zygotes of all nonmammalian vertebrates start embryogenesis and through which most complete it. It is still used during the early stages of embryogenesis in monotremes and marsupials. The eutherian zygote, however, depends directly on maternal nutrients from the very beginning of embryogenesis.
Since the structure of all vertebrate follicles is the same and all follicles function in a way that brings the oocyte to maturity, the fact that atresia is as widespread among the eutherians as it is rare among spawners tells us there must be a connection of fundamental importance between the ability of the oocyte and the follicle to survive and the particular process through which the animal reproduces (*see Notes to the Reader, p. 353). I think the source of the connection between the eutherian follicle and viviparity will be found in genetic changes that affected the viability of oogonia, the stability of the follicle, and the liver's ability to produce vitellogenin (VTG), the principal lipoprotein constituent of yolk. I will try to spell out the basis for this idea in as much detail as it requires and our store of information allows (**see Notes to the Reader, p. 353).
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BACKGROUND: REPRODUCTION |
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TOPABSTRACTINTRODUCTIONBACKGROUND: REPRODUCTIONBACKGROUND: HOW THINGS EVOLVEPUTTING IT ALL TOGETHERNOTES TO THE READERREFERENCES |
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In all known vertebrates, the follicle consists of the primary oocyte surrounded by one or more layers of epithelial cells, called granulosa cells (GCs), enclosed in a spherical envelope, called the basement membrane. The ovarian blood vessels do not penetrate this membrane until ovulation occurs—and even then not in all species. At some point during development of the follicle, a well-vascularized layer of epithelial cells derived from the ovary's interstitial tissue cells surrounds the follicle. This layer is called the theca interna, and its cells are called theca interna cells (TICs). In some, but not all, species, still another layer of smooth muscle-like connective tissue cells, called the theca externa, may surround the TICs [3–6].
The ovary is usually, but not always, paired. In birds, for example, only a single ovary is active; the other remains undeveloped. The ovaries are suspended in a mesovarium from the dorsal wall of the abdominal cavity on either side of the vertebral column in the region of the kidneys; in the embryo, this area is called the germinal ridge. Between the cystic ovary of most fishes and amphibians and the solid ovary of eutherian and marsupial mammals there is an enormous variation in size and appearance of the ovary. The ripe follicles may lie just beneath or bulging from the ovarian surface (eutherians), hang pendulously (birds and most reptiles), or lie in ramified branches of the ovarian stroma, facing the interior of the ovarian cavity (bony fishes and amphibians). Both the amount and kind of stroma also vary enormously [3, 5]. Nevertheless, the common organization of the follicle in all the vertebrates must mean that it was a specific trait of the common ancestor of all vertebrates—and probably of all chordates.
Stages of Oocyte and Follicle Growth and Development
From Oogonia to Oocytes Oogonia, the female descendants of the primordial germ cells, proliferate by mitosis in either a discontinuous or a continuous pattern. The discontinuous pattern, which is practiced by cyclostomes, elasmobranchs, birds, and mammals, results in a single, large population of oogonia, the only source of all the oocytes a female will have during her lifetime. In the continuous pattern, which is practiced by almost all bony fishes, amphibians, and most (or all?) reptiles, oogonial stem cells persist throughout the female's life and produce a batch of new oogonia during each breeding season from which the oocytes come. Each oogonium becomes an oocyte when it begins the first of the two meiotic divisions [7–12].
The Primordial Follicle The GCs arise from a population of stromal cells only when an oogonium becomes an oocyte and in response to the latter [13]. They then form a single layer of small, flat, relatively undifferentiated epithelial cells around the surface of the brand new oocyte; the basement membrane then forms around them and completes the structure of the primordial follicle. The primordial follicle is very small (diameter, 10–20 µm) and does not grow until it is transformed into a primary follicle. Once this happens, the follicle is committed to one of three fates: 1) death through atresia, 2) ovulation, or 3) death through luteolysis if the postovulatory follicle (POF) becomes a corpus luteum (CL).
Once meiosis begins, the oocyte's chromosomes proceed through the leptotene, zygotene, and pachytene to the diplotene stage of prophase, where they remain (meiotic arrest) until ovulation occurs [7, 9, 10, 13, 14].
From Primordial to Primary or Previtellogenic Follicle In response to still unknown intrinsic controls [15–17], growth of the follicle begins with the metamorphosis of the GCs into cuboidal-columnar cells with a reorganized internal structure connected to the oocyte through microvilli [18, 19]. The GCs then begin to proliferate and the oocyte and follicle to grow while the name changes to primary in marsupials and eutherians and to previtellogenic in monotremes and nonmammalian vertebrates. Follicle growth proceeds in two main stages: previtellogenic and vitellogenic in the latter and primary (or preantral) and vesicular (or antral) in the former. Previtellogenic and primary growth are more or less homologous, but vitellogenic and vesicular growth are quite different processes.
Previtellogenic and Vitellogenic Follicle Growth During previtellogenesis, as the result of GC proliferation and the increase in cytoplasmic elements in the oocyte, the follicle and oocyte grow in direct proportion to one another. During vitellogenesis, however, the follicle grows mainly through the oocyte's accumulation of yolk and other cytoplasmic constituents. The GCs continue to proliferate. However, the rate of increase in their number falls behind that of the oocyte volume, and at maturity, they form only a single layer of somewhat flattened cells between the oocyte and the basement membrane [12, 20–24].
Primary and Vesicular Follicle Growth in Mammals As in previtellogenic follicles, and for the same reasons, the oocyte and primary follicle grow in direct proportion to one another until the very first sign of follicular fluid (FF) appears and vesicular follicle growth begins [7, 15, 19, 25]. At this point, the eutherian oocyte is almost mature and virtually full size [17], while the marsupial oocyte continues to grow through yolk accumulation—although at a slower rate than before—until ovulation occurs [26, 27]. In both, the vesicular follicle grows as the result of GC proliferation [26–28] and accumulation of FF in the antrum (the cavity of the follicle). The vesicular, or antral, follicles are usually referred to as small, medium, large, and preovulatory; the largest are also called Graafian follicles.
Differences among Vertebrates In the nonmammalian vertebrates and monotremes, yolk is the main determinant of oocyte size; a few examples will illustrate its importance. The smallest nonmammalian oocyte (in a viviparous fish) has a volume more than 4-fold that of an average marsupial oocyte, and the latter's volume is 4-fold that of an average eutherian oocyte [26, 29].
The growth stages vary enormously, because they depend on the pattern of the ovulation cycle (OC) in each species. However, they bear the same general relation to each other in all. Primordial follicles do not grow; they also live the longest. Primary and previtellogenic growth may last for weeks or months, whereas vesicular follicle growth is limited to the duration of the follicular phase (maximum, 4 weeks) of a marsupial or eutherian OC [26, 30]. Vitellogenesis consists of several stages and may even extend over more than one breeding episode, as in some spawners [12, 23, 31]. In birds and reptiles, it may consist of a slow phase lasting several weeks and a rapid phase lasting no longer than the time limits set by the ovulation and oviposition of a clutch [22, 24, 32]. Growth of the vesicular follicle and at least the rapid phase of vitellogenesis are strictly dependent on the effects of the pituitary gonadotrophic hormones.
Vesicular follicles vary between 1 millimeter and several centimeters in diameter, depending on body size, but a whale's oocyte differs from a shrew's by only a few micrometers in diameter [29]. Marsupial oocytes also tend to be unrelated to body size [26]. In addition, the follicles and oocytes of birds and most oviparous reptiles tend to be proportional to body size. In viviparous nonmammalian vertebrates, little or no relation is found between body and oocyte size, with the primary determinant being the egg's yolk content. Among spawners, outside rather narrow limits, the size of the oocyte tends to be inversely related to the number of eggs that are shed [23, 33].
The Oocyte and Its Follicular Environment
The oocyte becomes fully mature only after a series of developmental changes. The first is breakdown of the germinal vesicle (GVB) (dissolution of the nuclear membrane) and resumption and completion of meiosis I; the next is progression to metaphase of meiosis II. These primarily nuclear events are followed by cytoplasmic changes that lead to the ability to be ovulated, to be fertilized, and to form a viable zygote [17, 25, 34]. In the nonmammalian vertebrates, full maturity also includes the accumulation of yolk and maternal mRNAs, proteins, and other substances on which at least the early stages of embryogenesis depend [35]. In the eutherians, however, the fully mature oocyte is devoid of yolk and almost devoid of maternal mRNAs, proteins, and associated substances [16, 35–37]. By the end of primary follicle growth, the oocyte can complete meiosis I independently of the follicle, but its ability to reach this stage—as well as the remaining ones to full maturity—can be fully expressed only in the environment of the follicle [15, 17, 25, 34, 35, 38].
The follicle is thus an essential element of the processes that allow the oocyte to become mature. Until the beginning of growth of the vesicular follicle in mammals and of vitellogenesis in nonmammalians, the steps to maturity are largely—or entirely—independent of the internal environment of the organism [17, 20, 39]. However, the latter environment is indispensable for completion of the later stages of maturation, because these depend not only on intrinsic follicular factors but also on the effects of the pituitary gonadotrophic hormones and other extrinsic factors.
Oocyte/GC Symbiosis When Nalbandov and his colleagues [40] claimed that the oocyte prevented luteinization of rabbit follicles, these findings were greeted more with skepticism than with excitement, because during the early 1970s, the oocyte was still seen only as a passive occupant of the follicle. Things have changed since then. It is easy to understand that the oocyte's ability to reach full maturity would depend crucially on its GCs [17, 19, 25, 31, 34, 35, 38, 41]. Other investigators, however, including those led by Eppig, have helped us also understand that the oocyte is not the passive recipient of GC attention but, instead, plays an equally crucial role in its own development by regulating the growth, activity, and differentiation of its GCs [16, 25, 42–50]. The detailed evidence will be found in the references included with this statement. It is unnecessary to repeat it, for its significance is clear: the oocyte and its GCs are in constant communication with each other from the time of formation of the follicle. In other words, the oocyte reaches maturity through its ability to induce the formation of GCs and to regulate their activity, but in a way that causes both the oocyte and the GCs to depend on one another for their continued existence.
The oocyte's relation to its GCs is thus a mutualistic (obligatory) symbiosis. With the addition of TICs to the follicle and the beginning of vesicular follicle growth, the oocyte's ability to reach final maturity depends on its control of two conflicting drives in the GCs: proliferation and differentiation. The follicle's development to ovulability depends on how effective the oocyte is in controlling its GCs' behavior (see also [42]); I will discuss these aspects of the follicle in connection with atresia.
A very significant difference between nonmammalian follicles and those of marsupials and eutherians is the morphologic differentiation of GCs, especially during the phase of rapid growth. In mammals, the accumulating FF separates the GCs surrounding the oocyte from those lining the antrum; these anatomical differences lead to physiologic ones, depending on how close a GC is to the oocyte (the GCs of the cumulus oophorus) or to the basement membrane or antrum (the mural GCs). The absence of yolk in the eutherian oocyte may also underlie a further difference between its GCs and those of marsupial follicles (see p. 351, Loss of the genes for VTG changed the character of the vertebrate CL). In birds (if the chicken is typical), the GCs lying directly over the germinal disc (the part of the oocyte containing the nucleus and almost all the cytoplasm) are connected by microvilli to the oocyte. Throughout the rapid phase of vitellogenesis, these GCs continue to proliferate, spreading over the surface of the expanding oocyte. They stop dividing as they do so and dedifferentiate morphologically into a single layer of flattened cells as they differentiate physiologically into P secretors [47]. Except for this instance in birds, no other example of spatial differentiation of GCs is known among the nonmammalian vertebrates, since the yolk-filled oocyte occupies the entire volume of the follicle. Specialized GCs may develop during previtellogenesis or the slow phase of vitellogenesis, such as in reptiles, but as in birds, they become dedifferentiated during the final stages of vitellogenesis into a single layer of flattened cells around the oocyte [22, 24, 32].
Vertebrate oocytes thus do not mature independently of GCs, and GCs do not form follicles independently of oocytes. In the absence of oocytes, as, for example, in Turner's syndrome or after busulfan treatment, "GCs" also do not produce steroids, or if they do, in amounts much smaller than, and in patterns totally different from those of normal ovaries [51–53].
The active role of the oocyte in its own development should not surprise us, because it is both the least and the most specialized cell in the body. After all, the follicle is only the oocyte's way of getting to where it can become a zygote. Within the borders of this symbiosis, however, each symbiont retains a small but distinct amount of autonomy, which can be affected—but never abolished—by the other. For example, the oocyte can actually be fertilized at the end of primary follicle growth, even if not very well [17]. Under a variety of in vitro conditions, GCs can produce steroids for much longer durations than normal [54, 55]. Within the confines of the intact follicle, in the intact organism in most known vertebrates, however, the symbiotic arrangement affects the oocyte and its GCs in a way that allows only a small proportion of the ovary's vast stock of gametes to express their potential for maturity by becoming zygotes or the GCs to express their potential for differentiation by becoming a CL. The vertebrate follicle is, in a word, unstable. With the possible exception of losses among oogonia, this instability is almost certainly the basis for most of the gametes lost through atresia. Although factors outside the environment of the follicle (e.g., the pituitary gonadotropins) may be considered as causes of atresia, they probably act ultimately by contributing to the follicle's basic instability.
Atresia of Gametes and Follicles
Like many biologic terms, atresia, which literally means a narrowing or closure of a body orifice, has acquired many wider meanings. I will use it here to mean female gamete losses at all stages of development.
Atresia among the Eutheria Gamete losses in the human female may be somewhat above average for all eutherian species, but they emphasize the great difference between mammalian reproduction and that of spawning vertebrates. In round figures, oogonial proliferation peaks during midgestation at a population of 6 million. During the next 4.5 months, as each oogonium enters meiosis I, the female fetus loses gametes at the rate of 1 million per month, and the baby girl is born with only approximately 2 million primordial follicles. During the next 12 years, she loses follicles at the rate of approximately 12 000 per month and enters puberty with no more than 300 000 follicles. Between puberty and climacteric, a span of approximately 38 years, she can ovulate no more than 400–500 eggs and so loses all the others at the rate of approximately 700 per month (calculated from the data of Baker [56]).
Atresia among other mammals differs from the human pattern only in minor details [57, 58] and, indeed, is not much different from that among other vertebrates in which the parental energy used for reproduction is invested in a relatively small number of zygotes. A peculiar aspect of atresia is that most of what we know about its causes comes from the study of follicles in which the smallest number of gamete losses occurs! As a step at least toward a better understanding of all its causes and its significance in vertebrate reproduction, let's examine five major transitions or nodal points in gamete development (see also [35, 58, 59]).
Nodal Points in Development of the Oocyte and Follicle The five nodal points in development of the oocyte and follicle are: 1) transition from mitosis to meiosis, 2) formation of the primordial follicle, 3) metamorphosis of the GCs, 4) initiation of growth of the oocyte and follicle, and 5) transition from primary to vesicular follicle growth in mammals or from previtellogenesis to vitellogenesis in nonmammalians. The transition of the ripe follicle to ovulability can also be thought of as a nodal point, but I will discuss this in connection with instability of the antral follicle. Each of these nodal points distinguishes between gametes and follicles that can pass to the next stage of development and those that, for the lack of whatever quality, fall into the discard pile of atresia [58].
Transition from mitosis to meiosis No one knows how this switch occurs, except that it must be intrinsically controlled [59, 60]. Mitosis is a critical event in the life of any cell. However, meiosis must bring even more hazards, because it includes fundamental changes in how the chromosomes are duplicated and segregated [14, 15, 60, 61]. For example, one-third of all losses at this transition occur during the pachytene stage [60].
Formation of the primordial follicle The first step is the formation of GCs among the neighboring somatic cells, a form of embryonic induction with the oocyte being its specific agent [13]. Even if an oocyte reaches the diplotene stage but stumbles along the way, it may not acquire adequate competence to induce GCs or to participate with them in formation of the primordial follicle. Losses could be caused by failure to form a follicle or by formation of a defective one [59].
Metamorphosis of GCs and initiation of growth of the oocyte and follicle The lack of any sign of atresia among primordial follicles, in spite of a steady drop in their number, as among the eutherians, may be because they do not undergo atresia. Their number falls almost certainly because of losses during the transition from quiescence to activity and may be traced to defects in GC metamorphosis and/or in the signals arising in both the oocyte and the GCs as the changes occur that allow the oocyte to start growing and the GCs to resume proliferation. The survival of the symbiotic arrangement that follows these changes depends on how effectively the oocyte's and the GC's intrinsic signals are coordinated. When we consider the many steps in converting GCs to proliferating cells and in the processes that permit the oocyte to grow [19, 42, 59, 62, 63], it is no wonder that this nodal point is the second most frequent site of gamete loss.
Transition to rapid growth of the follicle A major difference exists between the eutherian and marsupial mammals as a group and all the other vertebrates in what happens at this point. It will be easier, perhaps, to explain this difference if I consider the mammals first.
In contrast to the first four nodal points, in which the danger of atresia arises almost entirely from the transition itself, here the danger also includes all the phases of rapid growth that follow. Let's look at the hazards of becoming a rapidly growing vesicular follicle and then at the hazards of being one.
The basement membrane separates the GCs from their stem cells. As each cell divides, therefore, the daughter cells form a population of increasingly more differentiated GCs, which also become spatially differentiated as the antrum enlarges (see above). This process ultimately leads to ovulation and luteinization [28].
As the GCs begin to increase in number and differentiation, the interstitial cells form a layer of nascent TICs around the basement membrane, and soon after this, the intrinsic controls of the follicle's activity are supplemented and then largely replaced by extrinsic controls as the TICs and, through them, the GCs become responsive to the pituitary's gonadotrophic hormones [13, 30, 59, 62, 63]. All further development toward ovulability depends on the follicle's ability to maintain this responsiveness. Thus, if communication between the primary follicle's GCs and oocyte is defective, if GC differentiation is less than ideal, if the number of GCs is less than a critical level, induction of the TIC layer may be defective. Because all further maturation of the oocyte and of proliferation and differentiation of the GCs depends crucially on the rich blood supply of the TICs, through which they receive all their nourishment and extrinsic controls, any faltering of the TICs to join the follicle will make atresia inevitable.
Instability of the Vesicular Follicle Even if an oogonium had survived all the hazards of these transitions, it is still faced with those of being an oocyte in a vesicular follicle, for it is now developing to full maturity as its environment is changing from moment to moment. In contrast to the preantral follicle, the vesicular follicle now also responds to much of its external environment, and this is loaded with potential danger. More than any other characteristics, meiotic arrest, GC proliferation and differentiation, estrogen and P production, and the feedback arrangement between the follicle and the gonadotrophins contribute to the antral follicle's instability.
Meiotic arrest Because the oocyte is already partially mature at the end of primary follicle growth, it has at least the potential ability to complete meiosis I spontaneously and prematurely [17, 35, 64]. Should this occur, its life expectancy would be shortened, because it cannot be fertilized inside the follicle. The GCs will also die as a result, even if they show some signs of luteinization in the process [64].
Proliferation and differentiation of GCs The rapid growth of the vesicular follicle is the result mainly of FF production and a great increase in GC proliferation. The FF is itself a product of GC activity as well as a serum exudate passed through them to the antrum [30]. Both activities are stimulated by estrogens made by the GCs. With the acquisition of TICs, the GCs also acquire FSH receptors and make the oocyte maturation inhibitor (OMI) in response to FSH; estrogens and the OMI help to maintain meiotic arrest [35, 64]. However, FSH also promotes the GCs' innate capacity for P production, and large-scale P production is the ultimate expression of GC differentiation (see p. 350, The follicular GC is the larval form of luteal GC). The GCs' ability to proliferate thus falls as their state of differentiation rises [28, 62, 64, 65]. The oocyte also encourages GC proliferation and suppresses GC differentiation [46].
Obviously, then, the precarious balances between GC proliferation and differentiation in general, between the effects of FSH on estrogen production and on P production, between the ability of OMI and estrogen to maintain meiotic arrest and of P to end it, and between all these and the oocyte's effect on proliferation and differentiation of GCs can be easily upset and lead to atresia. The number of GCs in mitosis at any one time may also affect these balances, because secretory activity stops during mitosis [37, 66]. The lack of stem cells also may affect their number, because losses cannot be replaced. Both the total number of GCs and their state of differentiation [64] are critical factors in regulating ovulability and the oocyte's attainment of full maturity [17, 42]. The wonder is, on the one hand, that any vesicular follicle in any mammal ever reaches ovulation and, on the other, that all of them do in a few (see below)!
Estrogen production by the vesicular follicle More than 60 years ago, P.C. Williams [67] found that treating rats with high doses of estrogens prevented posthypophysectomy atresia. Since then, many others have confirmed Williams' finding and extended it to the current concept of estrogen as a powerful GC mitogen [62, 63, 68, 69], probably a reflection of the effect of a small amount of autonomously produced estrogens limited to the GC itself (autocrines). Autocrine estrogens are also made by the GCs in response to FSH in cooperation with growth factors and other intracellular substances; the mysteries of their production and interactions are the subject of very active current research [16, 59, 70, 71], most of which is outside the scope of this paper.
How the ovary makes large amounts of estrogen in response to both gonadotrophins, with effects almost entirely outside the ovary (hormonal estrogens), is much more well known. Its most popular explanation is the so-called "2-cell theory," the essence of which is that TICs can make androgens but cannot convert them to estrogens, whereas GCs cannot make androgens but can convert them to estrogens [72, 73]. The theory fits neatly with two sets of facts: First, FSH acts primarily on GCs and LH on TICs, and second, the amount of estrogen made by the ovary in response to both gonadotropins is much greater than the sum of the amounts made in response to either one alone [30]. Nevertheless, the theory is misleading, because both GCs and TICs can—and do—make estrogens independently of one another [74]. However, because the synergistic effect of the two gonadotropins is also indisputable, we must conclude that whatever the reasons may be for GCs and TICs to cooperate in making estrogens, an idiosyncratic deficiency of specific steroidogenic enzymes in either cell is not among them. In other words, the two cells are not obliged to make estrogens together; they just do.
TICs are not really an integral part of the follicle, but like the hermit crab's empty cockle shell's relation to the hermit crab, their addition to the follicle's immediate environment had a profound influence on what it could do. About the time this change in the follicle's economy was evolving, FSH and LH were probably evolving from the pituitary's original single gonadotrophin into separate but related protein hormones [75–77]. These two sets of changes also allowed estrogens to evolve from their autocrine state (as one of several intracellular regulators of GC mitosis) into a hormone with effects on growth and development of the body as a whole, on behavior, on the pituitary, on the female reproductive tract, and through some or all of these, on the stability of the vesicular follicle.
Hormonal estrogens, however, are not necessary for either maturation of the oocyte or ovulability of the follicle, for pure FSH in the virtual absence of LH can induce both effects without more than a barely detectable increase (or none at all) in estrogen production (e.g., [78, 79]). The importance of hormonal estrogens in vertebrate reproduction is in the coordination of their effects on the central nervous system (making the female interested in coitus) [80], on the pituitary (facilitating the surge of LH that induces ovulation) [81], and on the female reproductive tract (making it a zygote-friendly place), with the intrafollicular events leading to oocyte maturation and ovulability. This coordination increased enormously the chances for successful fertilization of the ovum and survival of the zygote. Accompanying these selective advantages was a continuation of gamete losses into the phase of rapid follicle growth itself, a phenomenon that may be unique to mammals, because it does not seem to occur in birds or reptiles or, probably, in amphibians and fishes (see below, Gamete Losses among All Vertebrates).
Aside from the negative feedback of estrogens and several nonsteroidal GC products on gonadotropin secretion, the main reason why hormonal estrogens make the follicle unstable comes from the way that GCs and TICs make them. TICs respond to LH by becoming the main source of androgen production, and androgens are the principal—or sometimes only—precursor of estrogen production. Regardless of what proportion of these androgens is converted to estrogens by TICs, by GCs, or by the combined activity of both cell groups, their moment-to-moment conversion must affect the androgen : estrogen (A:E) ratio in the GCs, and atresia is associated more with high A:E than otherwise [58]. The ability of any follicle's GCs to convert whatever androgens it is exposed to into an optimal amount of estrogens therefore becomes a measure of its ability to survive because atresia, in essence, involves only the oocyte and its GCs [58]. (TICs, unlike GCs, remain in contact with their stem cells [the interstitial cells] and can survive even when the rest of the follicle dies.) If the rate of conversion to estrogens falls below a critical level or the rate of androgen production rises above one, atresia will become almost inevitable [64, 82].
Production of P by GCs A capacity for autonomous P production by the mammalian CL, and perhaps even of all vertebrate CL [83–85], may be an attribute of follicular GCs in a nascent state, but it may also be dependent on FSH for its expression. This small amount of P may fit into the follicle's economy only as a precursor for estrogens made in the GCs, but the potential capacity for large-scale P production may lead to atresia. Only proliferating GCs will maintain meiotic arrest and the oocyte's progression to full maturity [17, 42, 64], but GCs will luteinize spontaneously if removed from contact with the oocyte [44, 46]. Normal luteinization during the periovulatory period may even depend on P itself [86]. If, for example, the GCs escape from the influence of the oocyte and begin to luteinize prematurely within the intact follicle, meiotic arrest will be broken, and atresia will become inevitable [64]. It is conceivable, in fact, that one of the connections between androgens and atresia may come from their stimulation of P production by GCs [64, 82].
Negative-feedback control of gonadotropin secretion During the normal OC, the vesicular follicle, in response to both FSH and LH, increases its secretion of hormonal estrogens in direct proportion to its increase in size [63] until just before ovulation, when estrogen and androgen production switch to P production [58, 87]. Less consistent changes in production of the nonsteroidal hormones inhibin, activin, and folistatin by the follicle [59, 71] accompany the increase in hormonal estrogens. At their peak, these estrogens facilitate the sudden surge of LH and FSH secretion that leads to ovulation [81]. The ability of the follicle to become ovulable, however, depends on GC proliferation (probably to an optimal number), and in turn, this depends on autocrine estrogens, which in turn depend on FSH. The complex of hormonal estrogens and nonsteroidal hormones depresses FSH secretion and, to a lesser extent, LH secretion. Only those follicles, therefore, that can remain responsive to the steadily falling level of circulating FSH will continue growing to ovulability. Most of the total cohort of vesicular follicles will be unable to do so, and the resulting imbalance between oocyte and GCs will drive them into atresia.
These losses, although only a tiny proportion of all gamete losses among mammals, probably are only the fine-tuning of a process that ends with the production of a very small number of viable zygotes (see below).
Gamete Losses among All Vertebrates
I have restricted the examination of atresia until now almost entirely to its manifestations in the eutherians. However, among all vertebrate species in which the amount of energy expended in reproduction is distributed among only a very small number of zygotes, a high incidence of gamete losses always precedes ovulation. We can only speculate about how these losses, in most cases, are connected to the way in which a particular species reproduces.
Take, for example, the process usually referred to as selection of the dominant follicle. Among the eutherians, this takes place during the rapid growth of a cohort of vesicular follicles, as just described, with 1 oocyte left to be ovulated in primates and approximately 5 in dogs, 10 in rats, etc. [2]. In birds and reptiles, however, in which the ovulated eggs form a clutch (homologous to a eutherian litter), selection through atresia is completed before, not during, the phase of rapid vitellogenesis that precedes ovulation [22, 32, 57]. When atresia does occur among these follicles, it is only after ovulation of the entire clutch or in response to random environmental events, such as poor nutrition, stress, disease, etc., regardless of the pattern of oviposition [22].
Another especially pertinent example occurs among mammals. In the elephant shrew, an insectivore [88], and the plains viscacha, a rodent [89], among eutherians and in the American opossum and the dunnart among marsupials [29], almost all of the cohort of rapidly growing vesicular follicles—which may number 50–100—are ovulated, and their eggs are fertilized! Only those blastocysts that can find room in the uterus or neonates that can find a free teat have a chance to survive, and these are never more than about a dozen [90]. Thus, selection in these species occurs neither before nor during rapid growth but only after fertilization!
Among spawners, selection is preovulatory and is similar in principle, but with one very significant difference. Losses at each of the nodal points in the oocyte's development are minimal, so the "cohort" or "clutch" of ovulable follicles comes from almost all the primordial oocytes with which the animal started an OC [12, 21, 23, 57].
The distribution of degrees of instability among a group of follicles could be the basis of the selection process—that is, a reflection of the number of the group's least unstable follicles, genetically determined for each species. In all the departures from the spawning pattern, and in association with the change in the oocyte/GC relationship that was the basis of the new form of reproduction, the hazards in the oocyte's transition from one developmental stage to the next were undoubtedly intensified, but where most gamete losses occurred seems to have depended more on which nodal point proved to be the most susceptible to breakdown rather than on any taxonomic relation between species or rule applicable to all taxa.
The difference between the shrew and most mammals illustrates one example of this guess. Another may be the transition from mitosis to meiosis, which in mammals is the site of greatest loss, but is this true of other vertebrates? Still another is the difference in the incidence of atresia between previtellogenic follicles in birds and reptiles and their homologue, primary follicles in mammals. More than 90% of these follicles in birds and reptiles are lost during their growth, while in mammals most losses occur in the transition to and from rather than during primary follicle growth [58, 59, 64, 87, 91].
What is more interesting than all the attempts to understand the specific causes of atresia of vesicular follicles in all their biochemical, physiologic, and genetic variety is how such enormous losses of female gametes became such a dominant theme in most forms of vertebrate reproduction. The next step in our search for the origins of the eutherian follicle and viviparity, of which this question is an essential element, is to examine how parental energy is invested in the production of offspring.
How Parental Energy Is Distributed among Offspring
Of all the energy living things expend in maintaining themselves, the portion invested in reproduction varies enormously in amount and character, for it can be affected by even the smallest details of an organism's existence. This is a subject of absorbing interest, complexity, and importance (e.g., [92, 93]), but my concern is limited exclusively to how the amount invested in reproduction (regardless of its proportion of the total energy expenditure) becomes distributed among an organism's potential offspring. This is a critical factor in the origin of any system of reproduction. In particular, I will focus on a problem that does not seem to have interested most students of the subject: Faced with the compromise between size and number of offspring, how did those vertebrates that opted for a small number of large offspring (K-selection to ecologists) reduce the number of oocytes available for fertilization?
I will use parental energy invested per zygote (PEI/Z) to describe how this distribution is related to atresia and to evolution of the mammalian ovary and form of viviparity. The term zygote is used rather than gamete, because the postfertilization part of reproduction can be as important as what went before. In addition, the term parental is used rather than maternal because of the importance of the male in some forms.
Spawning as a System of Low PEI/Z In typical spawning vertebrates (most bony fishes and most anurans), the majority of the oogonia a female produces during a breeding episode become oocytes, and almost all of these reach maturity, are ovulated and fertilized, and become viable zygotes [21, 94]. Nearly all the energy a mating pair expends in reproduction is invested in producing sperm, producing and yolking eggs, and migrating to the breeding and/or nesting site, where the eggs and sperm are shed in a freshwater or marine environment. The investment in most cases ends at this point, but in a few species, a relatively small amount of energy may also be invested in parental care or other variants of the spawning pattern [21, 94–96].
Since the number of zygotes, especially in broadcast spawners, typically runs into the thousands or even millions, each one gets only a very small part of its parents' total investment. The success of spawning rests on the likelihood that at least one pair of breeding adults will be produced by each pair of parents. Cod fishing off the Grand Banks—before human greed turned it into a disaster area—tells us that it is a very good way to reproduce if you are a fish [97].
High PEI/Z Alternatives to Spawning In high PEI/Z reproduction, all of a mating pair's reproductively applied energy is invested in very few zygotes. The male's production of sperm is not different from that of spawners, but in some species, the male's participation in postfertilization reproduction is as important as the female's participation.
Although one cannot make an absolute distinction between low and high PEI/Z or among the many variants of either, it is convenient to fit systems of high PEI/Z into three very broad categories: 1) cleidoism, 2) viviparity, and 3) brooding (i.e., parental care).
Cleidoism Joseph Needham [98] coined the term cleidoic egg (from the Greek cleid, meaning key and implying locked or enclosed) to describe an egg containing all the ingredients necessary for completion of embryogenesis outside the mother's body, enclosed in a protective shell (e.g., the hen's egg). He originally described the shell as being firm and permeable to gases but not to water; the ovum itself was always megalecithal. Cleidoism applies the principle of the cleidoic egg to eggs with a protective shell that may also be permeable to water, as in some reptilian species; whether the water is included among the egg's ingredients or is available from its environment is only of secondary importance. In spawners, the egg is mio- or mediolecithal, and the shell is gelatinous [21, 94, 95]. The spawners' hatchling is usually a free-swimming, independently feeding larva; the cleidoic egg hatchling may be either precocial (i.e., advanced enough in development to feed itself, such as chickens or sea turtles) or altricial (i.e., almost fully developed but dependent on parental care for full development, such as robins or gulls). Although the cleidoic egg is clearly a change that made terrestrial life for vertebrates possible, it also occurs in somewhat different form in elasmobranchs (sharks, rays, and skates) [99] and in principle but totally different form in parasitic wasps. Parental energy is thus invested mainly in producing a few heavily yolked eggs and mating behavior, including migration to the breeding and/or nesting site. It may end with oviposition (e.g., elasmobranchs and most oviparous reptiles). It may also include brooding eggs until they hatch and feeding of altricial young (e.g., birds). Cleidoism is typical of all birds and oviparous reptiles. It does not occur among bony fishes, amphibia, or mammals.
Viviparity Oviparity, ovoviviparity, and viviparity are terms used to describe offspring at the moment of separation from a parent, but even the worthwhile effort by Wourms [96] to distinguish between different forms of oviparity by use of the terms ovuloparity, zygoparity, and embryoparity is of limited value. Isn't it more interesting to know how a pair of gametes became an offspring rather than whether it left its parent inside a shell or wrapped only in its own skin? Cleidoism includes oviparity in its meaning, but it tells us much more about how an organism reproduces. Viviparity has so many meanings that biologists do not agree on precisely how to define it [100–103], probably because its very variety obscures the fact that it is only the flip side of cleidoism. It differs from cleidoism mainly in the fact that an embryo develops inside or on a parent's body instead of inside a shell and, in addition to or in place of yolk, it feeds on parental nutrients that reach it through a variety of delivery systems. Aside from several fascinating exceptions, like the rockfish Sebastes or the cichlid Tilapia [96], the essence of viviparity is that each zygote receives a very high proportion of the parental energy invested in reproduction, because the limited amount of available space determines how many can be accommodated.
It is of only minor importance, therefore, whether the conceptus develops in a shark's oviduct and feeds on yolk, its siblings, or through trophonemata [99, 100, 104, 105]; inside its shell in a lizard's oviduct [103, 106, 107]; within a bony fish's ovarian follicle, where it started life as an oocyte [108]; in the marsupium of a male sea horse [109]; in the stomach of a frog [95]; or in a rabbit's uterus—all are versions of viviparity (see also [110]). We can better appreciate the unique quality of eutherian viviparity in the context of this broader definition than in that of the classical definition (i.e., "bearing of live young").
Brooding and other forms of parental care Parental care refers to behavior of either or both parents that protects and, in many cases, also feeds the young during their development. It may be expressed only as nest building, which can also be part of mating behavior, or also as brooding, which can vary in extent and complexity from simple guarding (as in several families of bony fishes [33]) to lactation (as in mammals). Brooding may also be associated with spawning, cleidoism, and viviparity. In some rockfish species, it is even associated with both spawning and viviparity, for here an entire brood, numbering in the thousands, may be spawned as free-living larvae [96]. Viviparity in some reptiles is little more than the brooding of an embryo in the mother's oviduct [103, 107, 111]. Mouth brooding, even of fairly large clutches, is common among cichlids and a few other families of bony fishes [33, 94, 112], and skin pits or pouches in either sex are used as brooding sites in several species of frogs and toads [95, 113]. Parental care, however, even in the rockfishes may add little to the low PEI/Z of typical spawners when compared to the cost of yolking thousands of eggs, but in birds and, most of all, in mammals, brooding is a very large item in the total cost of reproduction. In the marsupials and monotremes, as lactation, it is the main site of the investment of reproductive energy.
The Unique Character of Eutherian Viviparity
Diversity of Its Expression Eutherian pregnancy occurs in so many patterns that it does not seem possible for a single thread of uniformity to connect them. For example, it can last as little as 16 days (hamster) or as long as 2 years (elephant). Litters can consist of one (human) or a dozen offspring (pig). The interval from fertilization to implantation is usually approximately 4 days (rat and monkey) or up to 2 weeks (pig), but it can also be 10 months (European badger). (The long interval is the result of delayed implantation [i.e., embryonic diapause], a condition in which the zygote, on reaching the blastocyst stage, remains free in the uterine lumen; it is usually associated with a lack of estrogen and a long period of CL quiescence [2].) Decidualization of the endometrium is an essential part of implantation (rat and human) but is absent in ungulates; where it does occur, it may involve only the epithelium (monkey) or only the stroma (rat). The blastocyst, when it implants, may be only slightly larger than it was as a zygote (rat and monkey) or large enough to almost fill the lumen of a uterine horn (sheep and horse). It can implant anywhere on the endometrial surface (human), only on the antimesometrium (rat), or only on the mesometrium (some bats). The placenta may be diffuse (sheep), discoid (human), or bracelet-shaped (dog). Its trophoblast can be in direct contact with maternal blood (human) or separated from it by one to three layers of maternal tissue (sheep, pig, and dog). The ratio of zygotes to implanting blastocysts can range from 1:1 (human) to 100:1 (elephant shrew). The uterus can be two completely separate tubes opening through individual cervices into the vagina (rat) or a single body, the lumen of which is connected to the vagina through a single cervix (primates). Progesterone secreted by the CL may be necessary throughout pregnancy (pig) or only during the first one-sixth of pregnancy (human). The placenta may secrete only trivial amounts of P (rat) or enough to maintain the last five-sixths of pregnancy in the absence of the CL (human). Pregnancy can last as long as the luteal phase of the OC (dog) or 10-fold longer or more (human and elephant). Newborns can be altricial (mouse and dog) or precocial (sheep and guinea pig) [2, 29, 90, 105, 114].
Six Common Characteristics of Eutherian Viviparity As a group, six common characteristics define the uniqueness of eutherian viviparity. 1) From the blastocyst stage to term, all of the conceptus' nutrition, respiration, and disposal of metabolic waste occur chiefly through a chorioallantoic placenta supplemented to a varying but minor extent by a choriovitelline (i.e., yolk sac) placenta, depending on the species. 2) The eutherian trophoblast is unique in its derivation from the blastomeres of the morula, its duration and growth, and its function as a medium of exchange and protection of the embryo against immune rejection by the mother. 3) The conceptus develops at a higher and more constant temperature than does that of any other vertebrate. 4) P secreted by the CL is essential for the establishment of pregnancy, and P secreted by the CL and/or the placenta is essential for its maintenance to term. 5) Except for disease, the uterus is the only site of pregnancy. 6) In the most altricial eutherian neonate, the major organ systems are much more advanced in development than they are in either the least altricial monotreme hatchling or marsupial neonate.
A Key to Eutherian Viviparity? It is by now a truism that viviparity has appeared in many forms among many members of every vertebrate class, except birds, and that virtually every case of similarity is probably more an instance of convergence or parallelism than of an evolutionary trend [107, 110, 115]. Even among reptiles, evidence for a progression from oviparity to viviparity is questionable [103]. The eutherian style is only another such instance, distinct in its own way even in the quality of those characteristics it shares with other viviparous vertebrates, such as the association with P secretion, the uterus as the site of gestation, or trophoblast (which it shares with marsupials). Nevertheless, a definite trace of evolution from reptilian to eutherian viviparity is not only likely but is the key to its uniqueness.
Two great leaps separate the eutherian from a reptilian form of viviparity The first great leap was from a reptilian to a marsupial-like form, and the second great leap was from the latter to the eutherian form. The uniqueness of the eutherian form of viviparity is in the extent to which it still resembles that of the reptile but differs from that of the marsupial.
In all known marsupial species, pregnancy, from implantation to parturition, lasts no longer than between 4 and 10 days, and in none does its maintenance depend on P [26, 116–118]. These differences from the eutherian pattern stem from the nature of the marsupial CL and uterus [83].
The relative uniformity of pregnancy length, the similarity between the CL of the OC and of pregnancy in life span and pattern of P secretion, and the lack of effect of hysterectomy in either pregnant or nonpregnant animals or of hypophysectomy on CL activity make it safe to assume that with two exceptions, the marsupial CL secretes P autonomously, is relatively insensitive to extrinsic controls, and has a life span limited roughly to approximately 2–4 weeks [26, 83, 118]. One exception is a form of embryonic diapause in kangaroos and wallabies [26] that occurs with seasonal anestrus or lactation. The blastocyst lies free in the uterine lumen for weeks or months, but resumption of the OC, or removal of the pituitary (anestrus) or of the nursing young (in lactation), activates the CL and induces implantation [26]. The pituitary thus seems to maintain the CL in a viable but dormant state. The other exception occurs in bandicoots; their CL do not regress at parturition, as in other known marsupials, but instead secrete P for several weeks afterward, especially during lactation. In fact, parturition occurs at the peak of P secretion, 11 days after fertilization [26, 119]. These exceptions, however, do not affect the basic differences between marsupial and either reptilian or eutherian CL and pregnancy. In five species, ovariectomy or luteectomy after Days 8–10, as the unilaminar blastocyst is transformed into the trilaminar blastocyst, does not prevent pregnancy from proceeding to term [118, p. 706]—that is, presumably in the absence of P. Luteectomy after implantation may even delay parturition by a day [118].
Treatment with P also does not prolong pregnancy [118]. In both marsupials and monotremes, the interval from fertilization to parturition (discounting diapause, if it occurs) or to hatching, respectively, is approximately 2–4 weeks—only long enough to produce a tiny (<1 g), extremely altricial neonate [26, 116, 120]. The inability of the marsupial uterus to retain an embryo beyond a brief period of development and of its CL to secrete P for longer than approximately 2–4 weeks differs strikingly from reproduction in modern reptiles and, probably, also in their Permian and Triassic ancestors. Even in oviparous reptiles, the CL's life span and egg retention in the oviduct tend to coincide, and in viviparous reptiles, the duration of gestation and of P secretion are also similar [110, 111, 121, 122], even though P is probably unnecessary for its maintenance [110]. The first two pivotal events, therefore, in the switch from reptilian to eutherian reproduction were the loss of the uterus' ability to retain the embryo, except only briefly, and the loss of the CL's ability to secrete P beyond approximately 2–4 weeks. Such a drastically truncated viviparous reproduction of the first mammals succeeded only because it happened in endothermic brooders, the skin of which responded to prolactin (PRL) by producing nutrients on which an embryo could feed and develop [123, 124].
In the switch from marsupial to eutherian viviparity, the pivotal events resemble, but are not the exact reverse of, the previous ones: The uterus regained, or now definitively acquired, responsiveness to P long enough to retain an embryo until it had completed most of its development, with only the finishing touches being left for lactation. At the same time, the CL recovered its ability to respond to extrinsic stimuli, including a greater variety than before, and thus the ability to secrete P well beyond the short period of autonomous production.
Key to eutherian viviparity The eutherians seem to have returned to a reptilian mode of viviparity through a marsupial detour, but the resemblance is one of parallelism, not of homology. Behind it lie fundamental changes from reptilian reproduction, the most critical of which are in the nature of the CL and of placentation. Let's look first at placentation.
Placentation in eutherian viviparity Among viviparous fishes and amphibia, placentation of any kind is either absent if the site of gestation is in the oviduct, as in the toad Nectophrynoids occidentalis [125] and among the caecillians [21, 39, 95], or is rare, usually being only a minor source of nutrients, and the organ systems used are, in most cases, not at all homologous with the eutherian placenta [21, 39, 95, 105, 108, 113, 126]. In some elasmobranchs, placentation occurs only during the later stages of gestation. However, it can be as complex as in any eutherian arrangement, although the organs used as trophotaenia and trophonemata bear no homology to a eutherian placenta [99, 100, 104]. Among reptiles, only the squamates (snakes and lizards) contain viviparous species. They also use the oviduct as the only site of gestation, and their placentas are somewhat homologous to those of the eutherians. Yolk, however, continues to be the principal form of embryonic nutrition for most species. Placentation is not usually a part of viviparity, and in the vast majority of cases, it serves primarily for respiration, excretion, and as a supplement to yolk [103, 107, 111]. Only among the Scincidae (skinks), a family of lizards, does placentation occur in any form at all resembling that of the eutherians [127, 128]. For example, Chalcides and Mabuya have very small oocytes, and in Chalcides, placentation accounts for 99% of the embryo's nutrition [128]. However, these oocytes are, respectively, 200- and approximately 800-fold greater in volume than the eutherian oocytes; the difference is due to yolk, on which the skink embryo's earliest stages of development depend [128]. Both skinks form chorioallantoic placentas with an intimate connection to maternal tissue. In Chalcides, however, it is the last of a series of five placentas that form during development, and it does not appear until the embryo has reached the limb bud stage. It also has no trophoblast. In a sense, skink placentation is as unique among reptiles as eutherian placentation is among all vertebrates.
The marsupial embryo does not implant until it is past the pharyngula stage, at 20 somites or more; it uses a yolk sac placenta and, in bandicoots, a chorioallantoic one as well, although mainly for excretion [26]. In eutherians, the blastocyst implants even before the primary germ layers have formed (a unique eutherian trait), and the chorioallantoic placenta is the principal one used, with the yolk sac placenta being used as a supplementary one in some species. The eutherian trophoblast also differs from the marsupial trophoblast in how it is formed, intimacy of contact with the endometrium, growth and differentiation, duration of function, and amount and kind of hormonal activity [26, 29, 105, 118, 129, 130]. It probably protects the fetus against immune rejection, but whether this is also true of marsupials is still unknown, except that the very short period of placentation is not an expression of immunity-based rejection [26]. Eutherian trophoblast and placentation, in short, may be improvisations on the marsupial form in a way that made prolonged intrauterine gestation possible (see below).
The Unique Quality of the Eutherian CL
How the CL Is Formed Four specific, coordinated changes in the eutherian follicle cause it to become a CL. The first is luteinization of the GCs (and, to a lesser extent, of the TICs), which begins just before ovulation and is completed several hours to days later, depending on the species [83]. Then, beginning with or just after ovulation, the basement membrane disappears, the gap junctions [18, 19] between the GCs close [131], and endothelial cells invade the mass of luteinizing GCs (angiogenesis) and quickly invest each cell in a dense network of capillaries [83, 132–134]. To my knowledge, these four steps are typical of postovulatory CL formation in all eutherian species in which it has been studied to date.
Luteinization is the second metamorphosis in the life of a GC. Mainly through an increase in cytoplasm, each cell becomes several times larger than before, and its more or less spherical shape changes to a polyhedron with distinct borders. As the GCs hypertrophy and become vascularized, profound changes in the cytoskeleton, endoplasmic reticulum, mitochondria, Golgi, nucleoli, and nucleus complete the metamorphosis. Luteinized GCs are the primary functional element of the mature CL and the main reason why it is larger than the follicle it came from, although TICs, blood vessels, and connective tissue also contribute significantly to its enlargement. The eutherian CL goes through a typical pattern of growth and regression; P secretion [83] and angiogenesis of the GCs [135–137] keep pace with this pattern. At the peak of its activity, the CL's blood circulation is among the highest of any organ in the body [132, 133].
CL Are Not Peculiar to Eutheria or to POFs
Postovulatory follicles, containing enough luteinized GCs to look like CL, occur among several species of elasmobranchs, a few species of bony fishes and amphibia, all reptiles [85, 115, 138], and according to some scientists, even in birds [85]. Atretic follicles with varying stages of GC and/or TIC luteinization may also look enough like CL to be labeled as CL atretica [58, 87, 139]. The resemblance to the eutherian CL, especially among those formed from POFs, in the characteristics of luteinization itself, in GCs as the predominant cell type, in secretion of P or of 
4–53-one pregnenes related to P, and in a tendency to be associated with some form of viviparity [85, 125, 138] blurs the distinction between eutherian and noneutherian CL. Nevertheless, the differences are important.
Examples of crucial differences between eutherian and other CL In no case, even including marsupials, do the CL of other vertebrates form exactly as they do in eutherians. Marsupials ovulate naked oocytes; the cumulus is not only included in the CL, it does not form a corona and does not mucify [140]. In almost all known eutherians, the oocyte is ovulated while still embedded in its mucified cumulus [42], and the CL is formed only from mural GCs [139], which are physiologically different from cumulus GCs [17, 25, 28, 46]. A few insectivores ovulate naked oocytes, but their follicles become filled with GCs rather than FF [139], and no differentiation into cumulus and mural GCs is possible, although they all acquire the characteristics of both cell types. The CL of nonmammalians is formed from GCs that are not spatially differentiated and, in general, less physiologically differentiated at ovulation than eutherian GCs [22, 32, 35].
Although luteinization of nonmammalian GCs is more or less like that of eutherian GCs, they do not hypertrophy as much, and they tend to form a luteal cell mass (LCM) surrounded by a distinct TIC and, in some species, theca externa layers [125, 138]. In most species, the LCM remains avascular, and angiogenesis, when it occurs, is quite different from the eutherian pattern. For example, in several reptilian species, finger-like wedges of thecal tissue with intact capillaries penetrate the LCM, isolating and vascularizing it into units [138] rather than investing each GC with a capillary network of its own as in eutherians. The endothelial investiture of individual cells can significantly stimulate their potential for development [141]. More important still is that vascularization occurs not as the LCM is forming but well after it has been established, and the intrusion of blood vessels looks more like a response to a moribund tissue than an accompaniment of healthy growth. This fits with the persistence of the basement membrane, at least during the early stages of CL development, and its fragmentation rather than complete dissolution when the thecal capillaries invade the LCM [125, 138]. The nonmammalian CL also often tends to be smaller than its follicle, especially if it is megalecithal, but also because the follicle has fewer GCs than the eutherian follicle when it ovulates.
Aside from a brief secretion of P or of related pregnenes around the time of ovulation and an equally brief and only partial luteinization of the GCs after ovulation, the POFs of birds and of broadcast-spawning fish and amphibians do not resemble the CL of eutherians or even of other vertebrates. Even their contribution to reproduction is different from that of the CL, because they are physiologically related to the comparatively short interval between ovulation and oviposition rather than to keeping an offspring in a parent's body during its development. Their short life and their importance as agents of oviposition, as in birds [83], sets them apart from the CL, except in relation to the evolution of CL in general and of eutherian CL in particular.
Uniqueness of the eutherian CL Like the uniqueness of eutherian viviparity, that of the CL lies not so much in its particulars—except for angiogenesis of the GCs, which is uniquely mammalian—as in all of them as a group. The most important of all the CL's aspects that emerges from this point of view is the relation between the CL itself and of P secretion to the regulation of viviparity. In eutherians, the CL's secretion of P became an indispensable factor in the establishment of pregnancy, and P secreted by the CL, the placenta, or both became an integral component of the complex of factors that maintained pregnancy to term. In the nonmammalian vertebrates, and even in marsupials (except for the establishment of pregnancy), this essentiality is either lacking or questionable [110, 125]; their CL and P secretion seem to be only another expression of the viviparous process itself rather than one of its essential causes or, at most, only a nonessential accessory in its maintenance. In many cases, the CL is dispensable [125, 142]; (see also p. 350, The follicular GC is the larval form of luteal GC).
It is the essentiality itself of the eutherian CL and P that gets in the way of seeing noneutherian CL other than as playing the same role but not doing it as well. If, instead, we could look on noneutherian CLs simply as POFs, no matter how much any of them may resemble the eutherian CL, we may see that their relation to viviparity can be altogether different from that of a eutherian CL. Perhaps then the concept of the latter as a mutant form of the vertebrate POF will emerge and its uniqueness become clear.
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BACKGROUND: HOW THINGS EVOLVE |
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TOPABSTRACTINTRODUCTIONBACKGROUND: REPRODUCTIONBACKGROUND: HOW THINGS EVOLVEPUTTING IT ALL TOGETHERNOTES TO THE READERREFERENCES |
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Adaptations
An adaptation is a trait that because of its particular form and function is useful to its possessor in its way of life, including the ability to reproduce [146]. For example, a streamlined body adapts fish to life in water, lungs adapt terrestrial vertebrates to life in air, and sticky tentacles adapt the sea anemone to a sessile life among moving nutrients. Although all parts of an organism are not adaptations, they are what give an organism selective value. Biologists seem to have two conflicting views about how they arise. One is that usefulness makes an adaptation appear and that uselessness makes it go away. I'll call this the use/disuse viewpoint. The other is that an already existing trait becomes an adaptation when a change in its possessor's way of life happens to make that trait useful. I'll call this the improvisation viewpoint. The difference between them rests on where and how change occurs.
According to the use/disuse viewpoint, natural selection very slowly transforms a minutely useful trait into an adaptation. According to the improvisation viewpoint, a relatively rapid change in a trait's environment can transform it into an adaptation almost immediately; natural selection may then lead to further modifications.
Although the use/disuse principle differs from Lamarck's, its adherents too often see it as if usefulness itself was the cause rather than the result of each small change. Even Darwin believed (among several other such Lamarckianisms) that the teats of dairy cattle were larger than those of wild cattle because of more frequent use [143, pp. 10–11]. He also attributed the blindness of animals living in darkness "wholly to disuse" [143, p. 113]. The much more reasonable reason, of course, is that whatever caused the animal's blindness made darkness a more favorable habitat than a well-lit one. Yamamoto and Jefferey [147], for example, found in a species of teleost that the lens of embryos of blind fish living in darkness was unable to induce normal eye development, whereas members of the same species living at the surface had normal eyes. When they transplanted the lens of sighted embryos into the optic cups of embryos of the blind fish, the latter developed into normally sighted fish.
Distortions of the relation between use/disuse and the origin of adaptations are frequent even today—see any issue of Science!—and they make excellent material for parody, with Dr. Pangloss' opinion that the nose was designed to support spectacles as the high point [148]. Adaptationism, however, does not mean that natural selection cannot lead to adaptations. The fish's streamlining is a typical example, and many others can be cited [146]. However, it is the aleatoric quality of improvisation that is the much richer source of adaptations.
Improvisations
I improvise when I use a dinner knife as a screwdriver. A pianist improvises when he or she plays a dozen ad lib variations of Yankee Doodle. Improvisation (from Latin roots meaning "not seen before") is an unplanned, usually sudden change that makes a thing become or do something it could not be or did not do before while still retaining enough of its original character to be recognizable, even if only by an expert. For example, some viviparous sharks, by continuing to ovulate well-yolked eggs while gestating a few embryos in the oviduct, are using these eggs as embryonic nutrient [99, 100] instead of as gametes. The first vertebrate to switch its habitat from water to land already had a bony infrastructure in its fins and an organ with properties similar enough to a lung's to function as one in air [149]. However these attributes may have arisen and contributed to their possessor's economy during its life in a watery environment, even if they included locomotion and air breathing, they had nothing to do with how they were eventually used on land. What is of first importance is that these attributes could be improvised into being used for something that could not have been foreseen [150].
The biologic improvisation is analogous to the technical change in design that, for example, led to the airplane. Here, the "already existing trait" was the internal combustion engine, the change in its environment was the new form of vehicle, and the combination of these two elements produced a vehicle that could fly. The analogy of the diversification of flying machines to that of terrestrial vertebrates that followed is too obvious for comment.
Darwin certainly recognized the principle of improvisation, for he wrote: "Moreover ...a modification of structure ...may at first have been of no advantage to the species, but may subsequently have been taken advantage of by descendents of the species under new conditions of life and with newly acquired habits" [143, p. 160]. He also knew about mutants ("sports") and admitted not knowing how they or a trait "of no advantage" could arise. (If only he had known about genetics!) Improvisations arise most often from mutations in a trait's environment but also from mutations in the trait itself, and the essence of a mutation, no matter how small, is a sudden change—in plain words, a leap or saltation. What else but a leap was the switch from an exchange of genes between haploid unicellular organisms, to the union of haploid gametes and the formation of a diploid zygote (i.e., sexual reproduction)? How else did sex itself or multicellularity originate? Natura non facit saltum may have the flavor of authority, but it does not keep Dame Nature from taking leaps.
Saltations
Saltation is a dirty word in evolutionary biology largely because of its misuse by pre-Darwinian naturalists and the strong saltationist flavor of Goldschmidt's idea of the "hopeful monster" starting a new species (see Schwartz [151]). Nevertheless, the most important form of biologic change is through saltations. Natural selection would be practically useless if it acted solely on what Darwin called a trait's "infinitesimally small variations." It may have had little difficulty with a trait's extremes of normal variation and none with small mutations, but it could not at all account for the emergence of neutral—or even harmful—traits.
Saltations are coming back into polite biologic society disguised as punctuated equilibrium, the history of which (1972–1999) has been well described [151, 152]. Its basic premise is that rapid, major changes are a valid alternative to Darwinian gradualism. This premise, in one form or another, is being accepted by many biologists [107, 150–156]; mutations are the richest source of saltations.
Mutations are the result of the flexible, versatile, but inherently uncertain system through which genetic information is converted into the working parts of an organism during embryogenesis or is passed on to the organism's offspring during gametogenesis [36, 37, 66, 144, 150]. The most remarkable thing about the system is that with so many possibilities for error, mistakes are so rare. Mutations can be caused by substitution of one base pair by another or by deletion of a base pair (point mutations); gene duplication; deletion of a nucleotide sequence or an entire gene; change in the position of a gene or group of genes in relation to other genes or the DNA strand as a whole; change in the order of expression or repression of a series of genes controlling development or operation of a trait; errors in transcription, in translation of the transcript, in molding of the amino acid sequence into a working protein, in how a chromosome break occurs and how it is repaired, in inversions, deletions, translocations, or insertions of chromosome parts or of entire chromosomes; change in the amount, complexity, or pattern of the nongenomic DNA; etc. [144, 157–159]. Many, and especially large mutations are lethal, but many are neutral (i.e., harmless but useless). Some are even useful. The neutral ones can lead through improvisation—and the useful ones directly—to major changes in evolution.
Improvised Adaptations as Saltations
The term preadaptation is often applied to traits that became adaptations, but this implication of prepared uniqueness draws our attention away from the most outstanding feature of all adaptations: any trait, especially during its development, or any part of a trait, down to any of the base pairs of any of the genes encoding its formation, has the potential ability to be improvised into an adaptation, depending on its structure and on whether and how its environment changes [36, 144, 150, 151, 154]. These changes are usually, if not always, associated with or expressed as mutations.
The mutation connection may not always be easy to see if the primary change is in the organism's external environment, as in the first vertebrate's transition from water to land (unless its bony fins and "lung" were themselves mutations, which is likely). The increased incidence of the four-winged fruit fly mutant in the presence of ether (the "Waddington fly"), however, shows clearly that ether affected the genes controlling appendage development [36, p. 592]. If the primary change is in the trait's internal environment, the improvisation is almost always connected to a readily identifiable mutation in either the trait itself or, more commonly, some of the constituents of its environment in a way that affects their interactions with the trait. Any of these changes may also uncover hidden properties of a trait or its parts that can themselves be or can lead to an adaptation [160].
For example, a mutation that caused vitellogenesis and ovulation to continue during pregnancy probably lies behind the shark's use of yolky eggs as embryonic nutrient. Almost all instances of bacterial strains resistant to antibiotics or phage are due to spontaneous neutral mutations that function as adaptations to, but only in, a hostile environment [144, 158]. In several poecilid teleosts, the follicle in which the oocyte develops also serves as the site of fertilization and viviparity [108], probably as the result of a mutation that by blocking ovulation at the normal time allowed the follicle to express a hidden ability to nourish a zygote as well as the oocyte it started as. The skin as a site of embryogenesis in a variety of amphibian species [95, 105, 115] is a further example of improvisation, in some cases the result of the uncovering of hidden properties. Gerhart and Kirschner [36] describe many such examples at the level of proteins, transcription factors, genes, cells, and tissues. The lens of the vertebrate eye, for example, could have begun as a mutation that caused certain epidermal cells to accumulate large amounts of crystallins, a very ancient protein that in a solution of high concentration happens to have a very high light refractive index.
These authors especially emphasize the basic principle that novelty (at any level and of whatever kind) usually occurs not so much through a change in the trait itself as through the variety of circumstances in which a trait may find itself. The essence of these changes, however, whether in the form of a small mutation or any of the ways of improvisation, like my use of a dinner knife as a screwdriver, is in the size of the difference from the original; it is a leap. Very gradual changes do occur, of course, but only between leaps. If life forms had evolved from the beginning only through such minute changes, they would still be crawling near the Cambrian, somewhere in the foothills of Mt. Improbable [161].
It has been said that Dame Nature is like a frugal housekeeper who never throws a thing away if a chance exists that it could be used for another purpose [77]. She can also be pretty sloppy and may, like a pack rat, be collecting and hiding bits of useless but harmless mutations, genes, traits, and properties. After all, who knows when one of them may come in handy?
How Things Evolve
Most of the major changes in evolution, I think, took place either as the direct result of mutations or as adaptations improvised from already existing traits because of changes in the trait's internal or external environment, which in turn were due to mutations among their constituents. I am also fairly sure that Darwin's original theory, its modifications after the new synthesis, punctuated equilibrium, and other schemes also describe a part of the story—but not all of it. Most evolutionary biologists, however, seem to be divided into two vehemently contentious groups regarding the validity of macroevolution itself. The neo-Darwinians concede that mutations can lead to major change, but only as micromutations and only then through the gradual accumulation of minute differences [144, 145, 161] analogous to a slow climb up a moderate incline on a virtually unbroken surface. The others believe that major changes can also occur, chiefly as saltations—that is, in mutational steps that are large and frequent enough to produce a phenotype sufficiently different from the wild type to become the basis of a new species [150, 151, 153–155]. Mutations with these qualities are possible as long as they occur only in the recessive state.
Mutations in recessive alleles Almost all mutations in a dominant allele are harmful or lethal, but even if a viable, useful one did appear, the individual carrying it would be unable to produce viable or fertile offspring even if it could persuade another individual to mate with it [144, 151]. But most mutations, even major ones, occur in recessive alleles, and their fate is altogether different.
A recessive allele is never (or is rarely) expressed as long as its possessor remains heterozygous for the gene. A normally breeding population, therefore, will "soak up heterozygotes like a sponge" [162], thus allowing a useful, major mutation in a recessive allele to silently increase in frequency inversely to the population's size. Eventually, the number of such heterozygotes would become large enough to produce enough homozygotes (through Mendelian segregation) for them to appear suddenly, even in sufficient numbers to form the nucleus of a new breeding population. The recessive allele could also, with time, become dominant, although no one knows how (see Schwartz [151, p. 357] for R.A. Fisher's, J.B.S. Haldane's, and S. Wright's contributions to this explanation and Mayr [145, p. 537] for Chetverikof's contributions).
Homeobox and other selector genes Schwartz [151] has theorized that a good deal, if not most, of macroevolution can be accounted for if such useful mutations were to occur in homeobox and related regulatory genes, for these control almost all the principal stages of embryonic development [36, 37, 150, 163, 164].
When a metazoan embryo reaches the phylotypic stage, it has acquired the basic features of its phylum's body plan, and the organizer, the main source of induction signals, begins to activate a group of special genes, called selector genes. At this stage, the embryo contains the three germ layers arranged spatially, and gastrulation, neurulation, and the main anteroposterior and dorsoventral (or radial) axes are in place. For example, the vertebrate phylotypic stage is the pharyngula, which has a notochord, a dorsal hollow nerve cord, pharyngeal gill slits and branchial clefts, and a postanal tail [36, 150, 163]. This stage can also be described as the spatial organization of phylotypic processes, and these in turn as "...the selector genes and (their) signaling agents, organized spatially, (that) provide the means for activating and ordering subsequent, more local development" [36, p. 297]. The selector genes, in other words, through their transcription factors, regulate the laying-down of the various regions of the body, the main organ systems within them, the rate of proliferation and differentiation of the cells specific to each region and organ, and the order of their appearance. They are thus responsible for how the body's parts will be related to one another in space, time, form, and function [36, 37].
The genetic basis of each phylum's body plan seems to have evolved and become fixed some time during the Cambrian (
500 million years ago) [36] but with qualities in the organizer and selector genes that endowed each compartment of the post-phylotypic-stage embryo with a degree of "flexibility, versatility, and robustness" that made extensive diversification of form and function within each compartment not only possible but very probable [36, chapters 11 and 12; 150]. It is this potential for change at the level of ontogeny that led Schwartz [151] to propose that mutations among the homeobox genes—also known as Hox or homeotic genes, the largest and most important of the selector as well as other regulatory genes and their proteins—could be the primary source of major evolutionary change [151].
This is a very reasonable idea, but it, too, is probably not the last word. If it is indeed true that novelty, in the majority of cases, is the result of the variety of combinations of circumstances in which a trait may find itself [36, 150, 165], it will be in the capacity of any of a trait's hidden properties to be uncovered by its environment, in the plasticity of the genetic system [144], in the influence of compartmentalization on the responsiveness of body parts to induction signals, and in the buffering processes that permit large-scale change without loss of viability, acting in highly conserved systems of development, that we may learn how major mutations among the regulatory genes become the source of macroevolution [36, 150, 165]. Evolution, however, is far too complex a process for a single, all-embracing, basic principle of change to explain all of its manifestations, and at any rate, to my knowledge no one has so far succeeded in finding one.
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PUTTING IT ALL TOGETHER |
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TOPABSTRACTINTRODUCTIONBACKGROUND: REPRODUCTIONBACKGROUND: HOW THINGS EVOLVEPUTTING IT ALL TOGETHERNOTES TO THE READERREFERENCES |
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Einstein, in response to Heisenberg's complaint that he had to abandon several good theories because he lacked "directly observable magnitudes," said "...in principle, it is quite wrong to try founding a theory on observable magnitudes alone. In reality the very opposite happens. It is the theory which decides what we can observe" (cited in Root-Bernstein [166, p. 419]). Of the many ways to paraphrase this pithy statement, my favorite is "Facts don't make a theory; it's the theory that makes facts visible" (source unknown). In other words, how we look at a fact can be more important than the fact itself.
A New Look at the Ovary: Loss of the Genes for VTG Changed the Vertebrate Follicle and Made Eutherian Viviparity Inevitable
I will lay out the basis for this viewpoint in a series of statements and expansions on them. What facts lie behind each one are in the background sections or will accompany each statement as necessary. In the end, they are a more or less idiosyncratic look at how things could have happened—and maybe even did.
Yolk is the oldest and most common means of maternal investment in reproduction The yolked gamete probably took the first great step toward anisogamy. With only a few exceptions among invertebrates and only the eutherians among vertebrates, all known metazoans reproduce through systems based on a yolked oocyte, regardless of how much yolk the zygote inherits or how much of embryonic development depends on yolk. Among vertebrates, the intrafollicular processes through which the oocyte reaches maturity are thus inextricably bound with those through which yolk is made, gets to, is incorporated into, and is stored in the oocyte. The absence of yolk in the eutherian oocyte, therefore, indicates a profound shift in the direction of reproduction.
VTG, the principal constituent of yolk in vertebrates, is made in the liver in response to ovarian estrogens VTG is a very large (150–600 kDa), complex glycophospholipoprotein belonging to the family of lipoprotein complexes that range in size and density from chylomicrons to high-density lipoproteins and through which lipids are transported and metabolized in vertebrates [37, 167, 168]. A few threads of homology in amino acid or nucleotide sequences connect VTG to some of the yolk proteins found in invertebrates, where they are also made mainly outside the ovary, indicating a very old evolutionary history [169–171].
Vitellogenin passes from the TIC capillaries through the basement membrane and the GC layer to the perivitelline space, where it binds to specific receptors on the oocyte's surface. It is then taken into the cytoplasm through endocytosis, cleaved into two or more smaller molecules, which are stored as droplets or platelets, depending on the species [23, 169, 171–173]. Estrogens specifically activate the four or more genes involved in VTG production [172], and their secretion by the follicles matches that of the liver's production of VTG, but estrogens probably do not act alone, because they are not always effective. In Rana esculenta, for example, the liver can make VTG in response to PRL or growth hormone under conditions in which estrogens are ineffective [174]. Androgens stimulate VTG production in some fish species, probably through conversion to estrogens [172]: P tends to inhibit VTG production [121].
Estrogens act directly on the liver, and they also activate the genes responsible for the formation of the oocyte's surface VTG receptor [169, 171, 172]. This may be one of the ways in which GCs process VTG into the oocyte.
Vitellogenin is made in species-specific varieties; although the eutherian liver does not make it, it does make similar lipoproteins. At least two of these—apolipoprotein B-100, which is involved in cholesterol metabolism, and the von Willebrand factor, which is involved in clotting—have portions homologous to the VTG apolipoprotein [169–172], but to this day, no one seems to know the genetic basis for the eutherian liver's inability to make VTG (W. Wahlli, personal communication).
Involvement of GCs in processing VTG into the oocyte limits their ability to proliferate Once anisogamy became fixed in the metazoan genome, the female gamete became the only site of an organism's early development—and, in many cases, the site of most of its development. It should be obvious, then, that in all but the most primitive metazoans, the follicle arrangement survived as the system through which the oocyte matured because the oocyte's chances of surviving were much greater as part of a follicle than as a naked cell.
Vitellogenin and other yolk proteins were probably improvised from the lipoprotein constituents of cell growth long before the invention of follicles. Their success as embryotroph, therefore, must have depended crucially on, among other things, the extent to which the follicle's somatic cells facilitated the transfer of yolk proteins from where they were made to the interior of the oocyte [115]. The vertebrate oocyte induced two such essential properties in its GCs. One, discussed above, was part of the mutualistic symbiosis that controlled the oocyte's maturation. The other helped to yolk the oocyte.
Through a ping-pong-like exchange of signals with the oocyte, the GCs may be necessary for the oocyte's formation of its surface membrane VTG receptors. These appear during previtellogenesis, long before yolking begins [23].
The GCs may also be involved in the storage of VTG platelets [175], and they may be responsible for the formation of patent intercellular channels that allow VTG to bypass most of the cytoplasm and cell membranes in the GC layer on its way to the oocyte [23, 24, 171]. Facilitated diffusion through all of the GC layer is also possible and may be helped by a gonadtropin-induced increase in GC permeability [172], although it is hard to see how this could continue after the VTG concentration in the oocyte equaled or exceeded that in the circulation. This may leave open the possibility of a more active role for GCs in vitellogenesis. Nevertheless, even if only facilitated diffusion turned out to be how VTG gets to the oocyte, the negative relation between yolking the oocyte and GC proliferation [23, 32, 47, 110] tells us that vitellogenesis, in some way, interferes with GC proliferation. This, too, may be an adaptation to yolking, because thinning of the GC layer would facilitate passage of VTG to the oocyte.
The follicle in spawning vertebrates is stable In various sections above (see The Oocyte and Its Follicular Environment, Atresia of Gametes and Follicles, Gamete Losses among All Vertebrates, and How Parental Energy Is Distributed among Offspring), I drew together evidence that the vertebrate follicle's instability was the ultimate cause of atresia, no matter what the proximate cause. This conclusion leads to a critically important question related to evolution of the eutherian follicle: what accounts for the low incidence of atresia [21, 23, 39, 57] in spawning vertebrates?
It is unlikely that a very high level of gonadotropin secretion, for example, could explain the survival of most of a broadcast spawner's follicles, because most gamete losses in nonspawners take place before the follicle responds to gonadotropins (see p. 340, Nodal Points in Development of the Oocyte and Follicle). A connection between yolking itself and the difference in the incidence of atresia between spawning and nonspawning vertebrates is also highly unlikely for much the same reason: Most gamete losses in nonspawners occur before yolking begins.
Atresia in spawners is not only much less frequent than in other vertebrates, it occurs mainly in response to random environmental changes and not, as in the other vertebrates, as a built-in element of the follicle's development. Strictly as an exercise in logic, therefore, it makes sense to conclude that spawners have the least unstable follicles of all vertebrates (see also p. 342, Gamete Losses among All Vertebrates). When vertebrates moved from the ancestral spawning style of reproduction to high PEI/Z forms, they may have lost whatever quality it was that kept the follicles stable. The most likely reason for that loss was the genetic basis of the transition and its effect on the oocyte/GC relationship.
The follicular GC is the larval form of luteal GC The GC is not like any of the other ovarian somatic cells. The cell line it comes from is the only one competent to respond to the oocyte's induction signals; it is avascular, is separated from its stem cells, and has the potential ability, through metamorphosis, to luteinize and secrete large amounts of P [83]. The TICs may also luteinize. In them, however, this is primarily an enlargement of already existing traits, whereas in GCs, it is an almost complete replacement of old ones by new ones.
The success, in a spawning style of reproduction, of the follicles of the ancestral vertebrates, in addition to the formation by GCs of a mutualistic symbiosis with the oocyte and their help in yolking it, depended on their ability to proliferate with an advance in differentiation during each successive cell division [28]. Each of these traits was either induced de novo by the oocyte or existed as a latent trait in the GC cell line, with a capacity for improvisation (e.g., in response to an oocyte signal).
The drive toward differentiation was probably included in the induction by accident, but it proved to be a happy one, for in this case, differentiation meant the switch from secretion primarily of nonsterodial substances and only a modest amount of estrogens to the potential ability to secrete very large amounts of P or P-related pregnenes [76]. These had qualities that proved useful in reproduction; the oldest probably facilitated the final steps to oocyte maturation and ovulation [35, 76].
Progesterone also could induce luteinization [86, 176, 177], but from the beginning of vertebrate evolution, two things kept this trait from being fully expressed. One was vitellogenesis, which hindered GC proliferation. The other was the oocyte's ability to suppress GC differentiation, even though it encouraged their proliferation [46]. In the spawners (even today), the balance between these forces allows a moderate but adequate amount of GC proliferation. When the oocyte is fully yolked and almost mature, however, the suppression of P production by GCs falls, and the GCs secrete enough P-related pregnenes (fishes) or P itself (amphibia) to complete oocyte maturation and to induce ovulation, but not enough to luteinize the POF or to delay oviposition by very much [23, 35].
Among the other nonmammalian vertebrates, vitellogenesis still limited the GCs to less than their maximal potential for proliferation, but the oocyte must have lost some of its ability to suppress their differentiation. Partial forms of luteinization in elasmobranchs and in some teleost and amphibian species as well as, among the reptiles, a universal but limited kind of CL formation reflect this change.
What the tadpole is to the frog, in other words, is something like what the follicular GCs are to the mammalian CL. Their metamorphoses are a manifestation of the way that particular genes behave in particular situations. Each of the instances of GC luteinization among the vertebrates should be seen only as the result of chance combinations of genes and circumstances, not as parts of a progression. The CL of early mammals was, in essence, a mutant improvisation on the theme of the vertebrate POF, and the eutherian CL was a further improvisation on that (see below).
Genetic changes that led to an increase in PEI/Z also made the oocyte more fragile and the follicle unstable How can a single cause of atresia be common to all the varieties of high PEI/Z reproduction, i.e., viviparity, brooding, cleidoism, or even of both cleidoism and viviparity?
One clue is that atresia includes gamete losses before as well as after follicle formation, so the nature of even the oogonia must be considered as a source of gamete losses. Another important clue is that every switch from spawning to a variant of viviparity, brooding, or cleidoism was based mainly on a redistribution of parental energy, not necessarily—or even usually—on a marked change in its amount. This means that major changes must have occurred in both the development and physiology of the systems through which this energy was distributed among the potential offspring; such a change must have happened through mutations.
The common cause of the atresia associated with high PEI/Z reproduction, therefore, must be the change itself in the organism's genome, regardless of the kind of change it induced in the style of reproduction. The pleiotropic nature of genes [144, 145] would then have caused these mutations to change the character of the entire genome. The oogonia would differ enough from those of the spawner to drastically affect the transitions to and the steps in meiosis, the oocyte's induction of GCs, and the formation and symbiotic basis of the follicle. All the hazards of oocyte development and the causes of follicle instability leading to the high incidence of atresia can be traced to this genomic change. In the end, as Harvey said (in somewhat different words): The egg is the ultimate source of everything. This view of the high incidence of atresia in nonspawning vertebrates is not quite pure speculation. Translocations and inversions in female mice are associated with an increase in early genetic losses without, however, affecting fertility in the survivors; disorders of meiosis in humans, which are very common, may account for many of the losses in early gamete development (discussed in [60]).
Atresia in itself has no adaptive value; it is only the accidental result of the retention of the ancestral practice of producing huge numbers of gametes in animals that no longer reproduce by broadcast spawning. This ancient trait has been conserved, probably because it is very highly selective. The only way any oocyte could ever have survived the hazards of development in high PEI/Z reproduction was because so many were there at the start. In short, atresia is to high PEI/Z reproduction what losing a hand is to high stakes poker: You have to start with a very large stake if you have any hope of winning.
Loss of the genes for VTG changed the character of the vertebrate CL The GCs, freed from the restraints yolking the oocyte had placed on their ability to proliferate, were able to realize their full potential for differentiation. A CL emerged with unique characteristics (see The Unique Quality of the Eutherian CL) that made it one of the three pivotal events in the transition to eutherian viviparity.
This conclusion does not seem to fit with the nature of the marsupial CL. In spite of enough yolk in their oocytes to make them 4-fold larger in volume than eutherian oocytes, marsupials also produce vesicular follicles, yet their CLs differ markedly in physiology from eutherian CL. However, the formation of a vesicular follicle does not necessarily mean that marsupial GCs do exactly what eutherian GCs do. Their cumulus GCs differ from eutherian cumulus and are included in their CL. The nature itself of the yolk lipoproteins (which may differ from VTG [118, 140]) could also be among the causes for the difference.
My view of the origin of the eutherian CL disagrees with that of Callard et al. [121], who see secretion of P by the CL as the cause of the loss of VTG rather than as its result, since P tends to oppose the effects of estrogens and specifically inhibits VTG production under certain conditions. The combination of yolking the oocyte and long-term secretion of P, however, is not rare [178]. Furthermore, the liver's response to stimuli or inhibitors of VTG production depends on season and stage of the reproductive cycle [171, 172, 174], and P can act in both ways (even though it usually acts as an inhibitor). A very large difference, therefore, exists between P's ability to suppress expression of the VTG genes under some conditions and its hypothetical ability to make them disappear altogether—or even to suppress them under all conditions, including the absence of P itself! The general inverse relation between viviparity and yolking of the oocyte among some vertebrates does not have to mean that P is the only cause of reduced VTG production, because reduced yolking may just as well be genetically connected to the origin of viviparity (see below). If P had anything to do with the loss of VTG in eutherians, it must have been only in association with more fundamental causes, probably mutations.
The yolkless oocyte became a new kind of zygote The connection between the absence of yolk in the zygote and the formation and implantation of a unique layer of trophoblast cells before the three embryonic germ layers had been laid down [129, 130] was not a coincidence, but it raises many questions. For example, how could the loss of VTG genes be connected to the pattern of cleavage and blastomere differentiation in a yolkless zygote? Was the oocyte's paucity of maternal mRNAs and proteins also involved in the connection? Could the absence of yolk have been only a microenvironmental change that permitted expression of a hidden trait for a unique trophoblast in the vertebrate genome? I cannot answer any of these questions, but there is good reason to believe that the two events are causally connected.
Except that it occurs much later than development of any other organ system, development of the reproductive system is also regulated by selector genes, and yolking the oocyte is as unique to this development as, for example, production of nerve growth factor by a limb bud's striated muscle is to that of the peripheral nervous system [37] or as the lens is to optic cup development [147]. The effects of mutations in the genes controlling the liver's capacity for VTG production, therefore, can spread to all parts of the reproductive system, for selector genes operate almost exclusively as a network [36]. My guess, then, is that whatever the immediate genetic basis for the loss of VTG production may have been, it allowed the absence of yolk in the oocyte to be connected, through related genetic changes [36, 154, 155, 159, 165], to the formation of a new kind of zygote and trophoblast—the second of the pivotal events in the transition to eutherian viviparity.
The maintenance of pregnancy in the uterus became crucially dependent on P In many nonmammalian vertebrates and in all pre-eutherian early mammals, the zygote had enough yolk, maternal mRNAs, proteins, and access to nutrients in the oviductal or uterine lumen to develop on its own almost to—and, in some cases, beyond—the pharyngula stage. At this point, the difference between survival and extinction must have also depended on a placental connection to an oviduct or a uterus with the capacity to be a source of nutrition during the embryo's further development. It was probably at this point that P, in addition to delaying the zygote's passage through the reproductive tract in all vertebrates, became an accessory, but not essential, factor in the success of viviparous systems in the nonmammalian vertebrates. In the monotremes and early marsupials, lactation replaced the uterus as a source of nutrient for development beyond this stage. If not for the somewhat accidental events that changed the uterus into a friendly place for a yolkless zygote, we would not be wondering now about how P became an essential element in its success. If we knew how P maintains eutherian pregnancy, we might be able to answer such questions, but we do not [179], so I offer a speculation.
From its beginning, the Müllerian duct may have had a latent trait that could be improvised into making the uterus responsive to effects of P that would both prolong pregnancy and make it crucially dependent on P to maintain this responsiveness. Such a trait could have been uncovered by the mutations that led to emergence of the eutherian follicle, CL, and zygote as part of a developmental process regulated by selector genes. It is even conceivable that the environmental element that induced the expression of this trait was the peculiar nature of the eutherian trophoblast and that P acted as an essential synergist in the process. Perhaps this is how P became indispensable in the maintenance of eutherian pregnancy (see also [176, 180]).
Evolution of the Eutheria
From Reptiles to Early Mammals In the transition from reptilian reproduction to avian cleidoism and eutherian viviparity, the most striking feature is that the many variants of reptilian oviparity were reduced to only one in birds and of reptilian viviparity [103] to only one in eutherians. Endothermy was the deciding factor in both transitions, exemplifying the way complexity can lead to major changes in evolution [154]. Much of what follows below is speculation, but the transition, through endothermy and lactation, from reptilian to eutherian viviparity is definitely not.
It is generally assumed that the mammals descended from a megalecithal, egg-laying, mammal-like reptile through a moderate (monotreme), then much greater (marsupial), and then finally complete (eutherian) reduction in yolking of the egg [149, 181–183]. This is more or less the basis of my earlier interpretation [83], but since I said very little then about yolk in vertebrate reproduction—and nothing about the pivotal role of its absence in the evolution of eutherians—this interpretation needs to be revised. I now think that the transition from reptiles to early mammals could have followed quite a different path.
Mitochondrial DNA evidence suggests that monotremes did not arise long before and separate from a lineage that united marsupials and eutherians but from one in which they and marsupials were sister groups that separated from eutherians approximately 150 million years ago and from each other approximately 15 million years later [184]. Although genomically based phylogenies must be interpreted with caution [155], this information fits with the fact that marsupial reproduction is more a variant of than truly different from the monotreme style. For example, the intervals from fertilization to oviposition in monotremes or to implantation in marsupials are not only roughly similar, they are homologous forms of aplacental viviparity; the subsequent intervals and degrees of organogenesis at hatching or birth, respectively, are also virtually the same (see [26, p. 407] and also [118, 185]). Paleontological evidence also brings monotremes and marsupials closer to when eutherians appeared [186]. The small amount of FF in the monotreme follicle [7], the signs of preovulatory luteinization of the follicular GCs, and the intense vascularization of their luteal GCs [187] also support this interpretation. The monotreme egg is thought to be a leftover reptilian trait [149, 181], but reptilian follicles never form FF, their GCs dedifferentiate before ovulation, and the vascularization of their CL is different from—and much poorer than—that of mammalian CL (see also [26, p. 407]). These features of early mammalian biology hint at a different line of evolution from the standard one, as described below.
Fossil amphibian-to-reptile transitional forms are still undiscovered [149], and the earliest cleidoic egg fossil is millions of years younger than the oldest fossil reptiles [149, 181]. Also unknown is whether the amnion evolved in embryos gestated in the oviduct or in externally laid eggs [188, p. 15]. The first reptiles, therefore, may have survived as terrestrial vertebrates, not only through the eventual appearance of the amnion, but also by evolving from a reptilian-like amphibian ancestor that, like most caecilians or anurans like Nectophrynoides, practiced an aplacental form of viviparity. The gestation, based on a moderately small egg, yolk, and oviductal nutrients in the lumen, would have been long enough to produce an independently feeding juvenile.
All the branches from this ancestral line, except for the mammal-like reptiles, led to reptilian lineages in which cleidoism eventually replaced aplacental viviparity and remained the main style of reproduction in all modern reptiles (and, through the dinosaurs, in birds). Squamates were the only exception; cleidoism became somewhat modified in some and replaced in a few others by many varieties of viviparity, none of which was phylogenetically related to either the ancestral or mammalian form [122].
Meanwhile, the original ancestral lineage eventually became the line of mammal-like reptiles (synapsids 
therapsids cynodonts tritylodonts) [149], and these continued to reproduce viviparously, without placentation, until they became either extinct or early mammals (pantotheres, etc.) [181, 182]. The monotremes and marsupials may have become sister groups through a marsupial branching from a monotreme-like lineage when they switched to lactation and truncated viviparity.
The leap to lactation Postnatal/hatching parental care among modern reptiles is rare; even egg brooding is relatively uncommon, except in crocodilians [103, 188, 189]. This is not because of exothermy; both pre- and posthatching parental care occur in many species of fish and amphibia [33, 95]. Since PRL is the most important single factor responsible for parental care, the rarity of such behavior among reptiles may mean that PRL lost contact with the physiology of parental care during the evolution of reptiles. When the switch to the truncated viviparity of the earliest mammals occurred, however, postnatal/hatching parental care must have already been in place, because the ultra-altricial neonates could not otherwise have survived. This could mean that the first small leap toward mammalian reproduction was a mutation that restored the connection between PRL and parental care. It probably took place as hair and feathers were evolving and was included in the changes that made endothermy possible. Endothermy had almost certainly evolved in the late mammal-like reptiles (cynodonts and tritylodonts) by the early Triassic, because fossil evidence suggests that they digested well-chewed food and had rapid, sustained locomotion [149, 182]. Endothermy became an essential part of egg brooding in birds and of nesting/nursing behavior in the first mammals, in which skin glands that secreted material their embryos could grow on had already evolved [123, 124].
From Marsupials to Eutherians In 1981, I saw this transition as a gradual change, primarily of the CL and uterus, and I reasoned that if bandicoots could evolve long-lived CL responsive to PRL, the other marsupials could also eventually acquire such CL. However, the questions of how this change came about as well as how the uterus became responsive to the pregnancy-maintaining effect of P were not considered. They remained unanswered until recently, when the position of yolk in vertebrate reproduction struck me—not quite as his bath struck Archimedes but more like eureka? rather than EUREKA! Somewhat as follows.
With marsupial reproduction in place, the loss of the genes for VTG results in a yolkless oocyte; hidden traits in the oocyte, GC, and uterus are then uncovered either by the physical absence of yolk, the physiologic effects of its absence, the change in genome caused by mutation in the VTG genes, or any combination of these and other factors. The result is a follicle with more differentiated and numerous GCs and, therefore, a more versatile and long-lived CL and P secretion, a zygote with a special kind of trophoblast, and a uterus with new properties.
Each of these features of eutherian viviparity could have existed as a potential improvisation in the vertebrate genome and remained hidden forever. But once uncovered—given the epigenetic nature of ontogeny and the inherent mutability of genes [36, 150, 151, 154–156, 159, 165]—the ability of these features to be converted into adaptations that allowed a yolkless oocyte to survive in the female reproductive tract was not only possible, it was virtually inevitable. The transition itself from marsupial to eutherian viviparity, however, was not inevitable. It was an enormous improbability—but only in the events that led to the loss of VTG, not in what followed that loss. If they had not occurred, we would still be reproducing like kangaroos.
Could the transition have occurred gradually? The Insectivores (e.g., shrews and moles) are generally assumed to be the earliest of the eutherians, and indeed, some aspects of cleavage, trophoblast formation, and early embryogenesis still have a few marsupial-like features [26, p. 406]. Nevertheless, they also switched from an essentially lactation-based to a placental-based system of reproduction without any apparent intermediates. It is this great gap, in fact, more strongly than anything else that points toward a saltation.
None of these ideas should even suggest that the eutherians were to be expected or that theirs is an improvement over other forms of vertebrate reproduction, especially the marsupial form. A great deal has been written about their respective merits and drawbacks [26, 182], but in the end the only certainty is that the arguments will continue without resolution. Eutherian reproduction is not better than other forms; in fact, it does not hold a candle to the way the insects or bacteria do it. It is only different.
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NOTES TO THE READER |
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TOPABSTRACTINTRODUCTIONBACKGROUND: REPRODUCTIONBACKGROUND: HOW THINGS EVOLVEPUTTING IT ALL TOGETHERNOTES TO THE READERREFERENCES |
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** This paper is oriented toward the reader who is familiar with at least the basic language of biology and who has some familiarity with that of reproductive biology. Although most of the less commonly used terms are defined, the glossary of Knobil and Neill's Encyclopedia of Reproduction [2] is a rich source of answers to any remaining questions.
List of Abbreviations
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ACKNOWLEDGMENTS |
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My grateful thanks go also to Kathleen Blazar, Joanna Maniglia, and Kathleen Meneely, the reference librarians at the Cleveland Health Center Library, and to Virginia Saha, its director, for their uncomplaining assistance in tracking down, checking, and obtaining hard-to-find references and related information. To Elaine Iannicelli and Rosa Garnett go many thanks for their skillful preparation of the manuscript. Most of all, however, my warmest thanks go gladly to my dear friend Patricia Cliffe, without whose help as my reader, proofreader, copier of needed articles, study-to-library-go-between, and general all-around indispensable amanuensis, this paper could never have been completed!
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FOOTNOTES |
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2 Emeritus, Department of Reproductive Biology, Case Western Reserve University, Cleveland, OH 44106
Received: 13 February 2002.
First decision: 22 April 2002.
Accepted: 12 August 2002.
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TOPABSTRACTINTRODUCTIONBACKGROUND: REPRODUCTIONBACKGROUND: HOW THINGS EVOLVEPUTTING IT ALL TOGETHERNOTES TO THE READERREFERENCES |
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- and ß-subunits of glycoprotein hormone gene family. J Endocrinol 1998 156:529-542[Abstract]-subunit from human chorionic gonadotropin, which can synergize with progesterone to induce decidualization. J Clin Endocrinol Metab 1998 83:575-581This article has been cited by other articles:
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  Y. Katsu, R. Ichikawa, T. Ikeuchi, S. Kohno, L. J. Guillette Jr., and T. Iguchi Molecular Cloning and Characterization of Estrogen, Androgen, and Progesterone Nuclear Receptors from a Freshwater Turtle (Pseudemys nelsoni) Endocrinology, January 1, 2008; 149(1): 161 - 173. [Abstract] [Full Text] [PDF]
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 V. D. Castracane and G. Gibori In Memoriam: Irving Rothchild, Ph.D., M.D. 1913-2006 Biol Reprod, April 1, 2007; 76(4): 736 - 736. [Abstract] [Full Text] [PDF]
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 P. Parham NK Cells and Trophoblasts: Partners in Pregnancy J. Exp. Med., October 18, 2004; 200(8): 951 - 955. [Abstract] [Full Text] [PDF]
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