Embryology and Teratology

Embryology and Teratology

C H A P T E R 23 Embryology and Teratology Manu M. Sebastian1,3, Tiffany Marie Borjeson2 1 Epigenetics & Molecular Carcinogenesis, MD Anderson Cance...

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C H A P T E R

23 Embryology and Teratology Manu M. Sebastian1,3, Tiffany Marie Borjeson2 1

Epigenetics & Molecular Carcinogenesis, MD Anderson Cancer Center, Smithville, TX, United States; 2Animal Care, Brown University, Providence, RI, United States; 3Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX, United States

I. INTRODUCTION The rat (Rattus norvegicus) is the rodent of choice to extensively study the development of the mammalian embryo. It is one of the species of choice for the regulatory assessment of developmental toxicology. The purposes of this chapter are to present the normal embryology of the rat and the methods used in experimental teratology.

II. EMBRYOLOGY A. Preimplantation Embryo 1. Fertilization Fertilization is a process by which the haploid male gamete, the sperm, and the haploid female gamete, the oocyte or egg, fuse together to create a diploid zygote. Fertilization functions to allow genes to be transferred from parent to offspring and the process of development to begin. The rat is an excellent model for the study of fertilization because the timing of events has been well established. Similar to other rodents, rats have estrous cycles that last approximately 4e5 days. Proestrus, characterized by nucleated epithelial cells on a vaginal smear, is approximately 12 h. Estrus, or the receptive phase, has a duration of 9e15 h, and is characterized by anucleated, cornified epithelial cells on vaginal smears (Long and Evans, 1922, Blandau et al., 1941). Ovulation takes place approximately 11e13 h after estrus, but may occur as early as 6.5 h after (Boling et al., 1941; Odor and Blandau, 1951). Early diestrus (also referred to as metestrus) demonstrates a mixed population of cell types, including cornified and nucleated epithelial cells with The Laboratory Rat, Third Edition https://doi.org/10.1016/B978-0-12-814338-4.00023-4

leukocytes, on vaginal smears and is approximately 14e18 h in duration (Freeman, 1988; Borjeson et al., 2014). An abundance of leukocytes seen on vaginal smears characterizes late diestrus, which lasts 60e70 h (Long and Evans, 1922). Within the ovarian follicle, oogonia become oocytes as they enter meiosis I in the primordial follicle. As the oocyte matures within the primary follicle, meiosis I resumes, and the oocyte forms the first polar body. At the end of meiosis I, the first polar body is lost and the second maturation division, or meiosis II, begins immediately after the first meiosis within the secondary follicle. The oocyte is arrested in the metaphase of meiosis II until recruitment and growth of the egg in response to fertilization occur. Granulosa cells affiliate with both the follicle and the oocyte: mural granulosa cells that surround the antral, or tertiary, follicle and cumulus granulosa cells that surround the oocyte and are expelled at ovulation. The zona pellucida is a glycoprotein layer that surrounds the oocyte and appears to be a secretory product of the Golgi complexes from granulosa cells (Kang, 1974). Cumulus granulosa cells directly surround the oocyte, forming the cumulus oophorus, or cumulus mass (Khamsi, 2001). The innermost cell type of the cumulus oophorus, the corona radiata, is held closely in approximation to the zona pellucida. Once the oocyte is expelled out of the follicle, the cumulus cells and the oocyte pass through the ciliated, fimbriated infundibulum of the oviduct into the ampullary portion. Oocytes are held in the ampulla for fertilization and then move down the oviduct to the isthmus, which contains secretory cells and eventually opens into the cavity of the uterus. In the male, spermatogonia, the earliest cells of spermatogenesis, are formed within the base of the seminiferous tubule epithelium. These spermatogonia mature

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into spermatocytes (via meiosis) and later become round spermatids (Jan et al., 2012). Round spermatids undergo dramatic morphological and cytological changes with support from the Sertoli cells of the seminiferous tubule, termed spermiation, which result in spermatid elongation and development of a distinct headpiece, midpiece, and tail (O’Donnell et al., 2011; Jan et al., 2012). This process of acquiring both motility and fertility takes place mostly within the caput and corpus of the epididymis. Mature spermatozoa in the male rat are stored in the cauda epididymis. At the time of ejaculation, sperm still lack the capacity to bind to and fertilize an egg. Capacitation, or the functional maturation of sperm, occurs after the sperm are inside the female reproductive tract. Capacitation also allows the sperm to produce hydrolytic enzymes, including hyaluronidase, that aid in the process of dispersion of the cumulus mass that surrounds the oocyte. When the fertilizing spermatozoon comes into contact with the zona pellucida, the cell membranes of both the spermatozoon and the oocyte rupture in the area of contact and fuse with each other as the spermatozoon passes into the cytoplasm of the egg (Austin, 1961). The zona pellucida undergoes a change (zona reaction) after the entry of the first spermatozoon that tends to exclude other spermatozoa. The zona reaction is thus a mechanism that helps to prevent the occurrence of polyspermic fertilization. In the rat, the mean time to complete the zona reaction has been estimated to be not less than 10 min or more than 1.5e2 h (Austin, 1961). Spermatozoa have been found disseminated throughout the uterine horns and already migrating into the uterine segments of the oviducts within 15 min after ejaculation. At 30 min after ejaculation, spermatozoa were found in 88% of the oviducts examined, and 100% of the examined oviducts had spermatozoa present 1 h after ejaculation. Blandau and Money (1944) and Huber (1915b) found that the number of spermatozoa entering the oviduct is exceedingly small compared with the number present in the uterine horn. The ampulla of the female is thought to aid in transporting sperm to the site of fertilization by both cilia and muscular activity in the mouse, and this is extrapolated to be the same in the rat (Kaufman and Bard, 1999). It is estimated that the life span of spermatozoa in the female reproductive tract is only about 10 h in the albino rat (Huber, 1915a). After sperm penetration of the oocyte occurs, the second maturation division of the egg continues in the oviduct. The sequence of events that lead to the formation of the two-cell embryo are discussed in detail by Odor and Blandau (1951). The second polar body is formed and extruded by the ova, and the sperm head undergoes enlargement during the first 4 h after sperm penetration in the oviduct. From 5 to 8 h after sperm

penetration, the female pronucleus forms but remains close to the extruded second polar body. The typical hook-shaped sperm head is transformed into an elongated slipper-shaped pronucleus that remains in the vicinity of the proximal end of the middle piece of the sperm flagellum. There is a rapid increase in the size of the pronuclei and in the number of their nucleoli and a gradual migration of the pronuclei toward the center of the embryo during the interval of 9e19 h after sperm entry. Fertilization may be considered complete with the condensation of the chromosomes in the male and female pronuclei and the coming together of the two groups of chromosomes to form a single chromosome group, which constitutes the prophase of the first cleavage mitosis (Austin, 1961). By the end of the first day, the fertilized eggs have traveled about one-fourth the length of the oviduct and are found lying free in its lumen (Huber, 1915a,b). 2. Cleavage/Morula Formation Cleavage is the initial phase of cell division after fertilization and is similar to mitotic division except that the individual blastomeres do not undergo a period of growth between successive cleavages, as with other cells. As a result, the blastomeres become progressively smaller as cleavage continues (Gulyas, 1975). After fertilization, the chromosomes proceed immediately thereafter to become arranged at the metaphase plate of the first cleavage spindle (Austin, 1961). The first cleavage occurs during the early part of the second day after insemination, and the resulting two-cell stage lasts for a period of about 24 h. By the end of the second day after insemination, the two-cell embryo has traversed a little over one-half the length of the oviduct. One of the cells usually divides before the other, resulting in a three-cell stage. By the end of the third day, only four-cell embryos are found in the oviduct. Compaction of the embryo starts at approximately the four-cell stage, and is characterized by cellular flattening (Pampfer and Donnay, 1999). During the early cleavage of the embryo, up to the eight-cell stage, all blastomeres appear morphologically identical, and each is competent to contribute to descendants of both trophectoderm and inner cell mass (ICM) cells (Fleming, 1987). At the 16-cell stage, the term morula(e) is used and cells become further tightly bound and are no longer readily distinguishable as individual cells. Initially, both turnover of energy substrates and synthetic activity are low, but they increase significantly over the first 3 or 4 days when glycolysis and tricarboxylic acid cycle activity increase significantly. Increasing macromolecular synthesis leads to the appearance of new RNA, DNA, protein, and polysaccharides (Wales, 1975). In the mouse and rabbit, RNA synthesis is low

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during the early cleavage stages and increases rapidly at the early morula stage, while the rate of protein synthesis increases rapidly at the eight-cell stage in the mouse (Biggers and Borland, 1976). Both phenomena are thought to be similar in the rat. During the preimplantation period, development follows a program determined partly by the transcription of the maternal genome during oogenesis and partly by reading of the new genome after fertilization. Biggers and Borland (1976) concluded that the mammalian embryo is not completely selfcontained but undergoes important exchanges with the dam. These exchanges are of three types: exchanges concerned with homeostatic functions that preserve the internal environment of the embryo (e.g., pH and osmotic pressure), exchanges involving the uptake of substances that can be metabolized (e.g., glucose and amino acids), and specific signals that pass between dam and embryo. Early on the fifth day, the 24- to 32-cell morulae are found lying free in the lumen of the uterus, spaced in a manner similar to how they will implant. Krehbiel and Plagge (1962) concluded that the random scattering of rat preimplantation embryos at the time of implantation determines the spacing of the implanted embryos. No predetermined sites for blastocyst attachment have been found (Abrahamsohn and Zorn, 1993). 3. Blastocyst The blastocyst (Fig. 23.1) is a roughly spherical epithelial structure that ranges in size from 60 to 85 mm and is surrounded by the 2.5- to 3-mm-thick zona pellucida. Formation of the blastocoel by cavitation of the morula, which is now composed of 30e32 cells, begins at approximately day 5 (Schlafke and Enders, 1967). This initially appears as small, irregularly shaped cavities beginning

FIGURE 23.1 Implantation of the rat blastocyst. Histological section showing a rat blastocyst engaged in the process of adhesion to the uterus. ICM, inner cell mass, TE, trophoectoderm, EP, uterine epithelium; ST, decidualizing uterine stroma. Adapted from Pampfer, S., Donnay, I., 1999. Apoptosis at the time of embryo implantation in mouse and rat. Cell Death Differ. 6 (6), 533e545.

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between two or three cells, which slowly increase in size (Schlafke and Enders, 1967). It is the ion- and fluidtransporting ability of the single layer of cuboidal trophectoderm cells, with their apical surface facing the zona pellucida, that enables blastocoel formation (Schlafke and Enders, 1967; MacPhee et al., 2000). This event prepares the blastocyst for implantation. The ICM of the blastocyst is made up of inner eccentrically placed large (7e12 mm), roughly spherical, and loosely associated cells from the polarized, compacted morulae, which occupy about onequarter of the volume of the sphere and have lineage fates to make the embryo proper The trophoblast cells (also referred to as the trophectoderm epithelium) are continuous and surrounded by the zona pellucida and will eventually form the extraembryonic tissues (amnion, yolk sac, allantois, and mesodermal components of the placenta). The blastocysts shed, or hatch, from their zona pellucidae during the afternoon of day 5 in the uterus before implantation, when the pregnant rat is under the influence of both progesterone and estrogen (Dickmann and Noyes, 1961; Dickmann, 1969; Enders, 1971; Lundkvist and Nilsson, 1984). The shedding of the zona pellucida depends on the stage of development of the embryo (Dickmann and Noyes, 1961), and this is important because it marks the beginning of the first direct contact between the embryonic and the maternal tissues (Davies, 1971). In the rat, the loss of the zona pellucida is probably the last event that precedes the beginning of implantation. Orientation of the blastocyst during initial attachment to the mouse and the rat uterine wall has the ICM facing the mesometrial aspect of the uterus (Fig. 23.2) (Pampfer and Donnay, 1999). During the sixth day, the size of the blastodermic vesicles increases, partly as a result of flattening of the roof cells and partly as a result of rearranging and flattening of the cells constituting the floor of the vesicle. By the end of day 6, the blastodermic vesicle consists of a discoidal area, the germinal disc, and the remainder of the vesicle wall, a single layer of very flattened cells (Huber, 1915a,b). During the early hours of day 7, cell proliferation, rearrangement, and enlargement of cells take place in the region of the germinal disc, initiating the phenomena known as inversion of the germ layers or entypy of the germ layers. The anlage of the ectoplacental cone, or tra¨ger, can be recognized (Huber, 1915a,b). The preimplantation period is usually regarded as a period during which toxic insult generally results in embryonic death or the absence of an effect because of the regenerative powers of the pluripotent cells of the embryo at this stage. However, preimplantation exposure to actinomycin D and methotrexate has been shown to affect growth and development of the embryo (Christian, 2001), whereas dichlorodiphenyltrichloroethane, nicotine, or methyl methanesulfonate results in body and/or brain weight deficits or death (Fabro, 1973, Fabro et al., 1974). Malformations have been caused by administration of

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FIGURE 23.2 Diagrams of an implanting rat conceptus illustrating the yolk sac in myomorphic laboratory rodents. (A) At 7 days postcoitum (dpc) the hypoblast has delaminated from the inner cell mass (icm). At the periphery of the ICM, the hypoblast proliferates to line the blastocoel, converting the chamber into a yolk-sac cavity. (B) At 8 dpc, an amniotic cavity has separated the endoplacental cone from the icm; the cells of the icm have arranged to form a bilaminar embryonic disc. The adembryonal (i.e., visceral) portion of the yolk sac has become trilaminar by an interposition of extraembryonic mesoderm between epiblast (ectoderm) and hypoblast (endoderm); the parietal yolk-sac remains two layered. (C) by 10 dpc, the visceral yolk-sac mesoderm has split to form an extraembryonic coelom and undergoes angiogenesis to vascularize this portion of the yolk sac. Abbreviations: ac, amnionic cavity, ec, ectoplacental cone, eec, extraembryonic coelom, ed, embryonic disk, pys, parietal wall of the yolk sac, vys, visceral wall of the yolk sac, ysc, yolk sac cavity. Adapted from Jollie, W.P., 1990. Development, morphology, and function of the yolk-sac placenta of laboratory rodents. Teratology 41(4), 361e381.

methylnitrosourea, cyproterone acetate, and medroxyprogesterone acetate during the preimplantation period (Eibs et al., 1982; Takeuchi, 1984). 4. Yolk Sac Placenta Development Brunschwig (1927) and Everett (1933) suggested that the yolk sac epithelium of the rat is physiologically a placenta that functions as an organ for maternal and embryo exchange. Over much of its surface, Reichert’s membrane, a thick basement membrane in the parietal wall of the yolk sac, is directly bathed by circulating maternal blood that allows for diffusion of materials into the yolk sac cavity, from which they are absorbed into the embryo (Everett, 1935, Inoue et al., 1983). Therefore, in the rat there are two placentas that serve as organs for maternaleembryo exchange and are present together throughout most of gestation. The first to develop is a villous highly vascularized yolk sac placenta, which will be followed on days 11.5e12.5 by the chorioallantoic placenta (Beck et al., 1967a,b; Jollie, 1990; Abdulrazzaq et al., 2001). The formation of the yolk sac placenta begins concurrently on day 7 with the proliferation at the periphery of the ICM of the hypoblast, which will line the blastocoel cavity, converting this chamber into a primitive yolk sac cavity lined with a bilaminar structure of trophoblast and a single layer of hypoblast (endodermal cells) (Fig. 23.2A) (Jollie, 1990). Subsequent development consists of two separate processes: an inversion of the embryonic disc and an inversion of the yolk sac that lies peripheral to it. Entypy of the yolk sac is a fundamental phenomenon observed in

rodents; it is associated with a precocious development of the ICM that proliferates rapidly and becomes invaginated into the yolk sac cavity. The inversion of the germ layers involves a process that temporarily brings the embryonic hypoblast (endoderm) external to the embryonic epiblast (ectoderm). As a result of the inversion, the cavitation forms the amniotic cavity. By day 8, the amniotic cavity has separated the ectoplacental cone, the thickened portion of the embryo that eventually will become the fetal portion of the placenta, from the ICM (Fig.23.2B). Three cavities eventually form within the ectoderm of the egg cylinder: the amniotic cavity (most ventral cavity), the extraembryonic cavity (middle), and the ectoplacental cavity, which is just under the ectoplacental cone and is transitory. The membrane separating the extraembryonic cavity from the ectoplacental cavity is the chorion, and the membrane between the extraembryonic coelom and the amniotic cavity is the amnion (Beaudoin, 1980). The visceral (adembryonal) portion of the yolk sac becomes trilaminar by an interposition of extraembryonic mesoderm between epiblast and hypoblast and will undergo angiogenesis. The vascular adembryonal portion or visceral wall of the yolk sac membrane, with its endodermal epithelium directed outward, completely surrounds the embryo external to the amnion. The more peripheral portion or the parietal wall of the yolk sac membrane remains bilaminar, but the ectoderm (trophoblast) is separated from the yolk sac endodermal epithelium by Reichert’s membrane. The space between the two walls of the yolk sac membrane constitutes the yolk sac cavity. By day 10, the visceral yolk sac mesoderm has split to form an extraembryonic coelom (Fig. 23.3C),

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FIGURE 23.3 Diagram of implanting rat conceptus. By 11.5 dpc, anatomic relationships of rat fetal membranes are definitive. The distal allantoic diverticulum is invading the ectoplacental cone to form the definitive chorioallantoic placenta. Note that the allantoic stalk (umbilical cord) attaches to the placenta. As a consequence, the embryo and the amniotic cavity are enclosed by both yolk-sac membranes. Abbreviations: ac, amnionic cavity, eec, extraembryonic coelom, ys, yolk stalk, ysc, yolk sac cavity. Adapted from Jollie, W.P., 1990. Development, morphology, and function of the yolk-sac placenta of laboratory rodents. Teratology 41(4), 361e381.

undergoes angiogenesis, and becomes vascularized by the peripheral vitelline circulation. The yolk sac is thus the first hematopoietic organ. Division of the yolk sac into visceral and parietal walls is complete in the rat by gestation days 11e12 (Fig. 23.3). Meanwhile, the visceral wall of the yolk sac cavity also differentiates into the villous portion, a more highly vascular and more absorptive area, and a smooth portion that is less vascular and presumably a less absorptive area. In rodents, the visceral yolk sac villi are generally found close to the fetal surface of the chorioallantoic placenta. At this stage of development, the yolk sac is the only placental exchange site (Jollie, 1990). Inversion (Fig. 23.3C) occurs as a result of the apposition of the inner (visceral) wall of the yolk sac, which is vascular, to the parietal wall of the yolk sac. The parietal wall of the yolk sac with its associated trophoblast then disappears about two-thirds of the way through gestation in rodents. This brings the absorptive cells of the visceral yolk sac into contact with the uterine epithelium and its secretion.

B. Implantation and Chorioallantoic Placenta Development Implantation is a complex sequence of events that requires proper synchrony between embryonic and endometrial development. It has been shown that implantation

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requires strictly timed hormonal conditioning, consisting of continuous preparation of the endometrium by progesterone for at least 48 h and then a brief intervention of minute amounts of estrogen on the fourth day after fertilization. A delay in the presence of estrogen or a lack of estrogen, attributed to lactation or ovariectomy, results in the postponement of implantation and will induce a state of diapause, or delayed implantation, for 4e10 days in the rat (Psychoyos, 1966; Abrahamsohn and Zorn, 1993; Renfree and Shaw, 2000). During the period of diapause, the hatched blastocyst experiences no growth (Psychoyos, 1966; Abrahamsohn and Zorn, 1993; Renfree and Shaw, 2000). Between days 5 and 7 of gestation, the implantation chamber, which is epithelial degeneration on the mesometrial side, develops from a flattened shallow crypt to an elongated tube (Schlafke et al., 1985; Pampfer and Donnay, 1999). On day 5, the blastocyst is still free in the lumen but is pressed between the enlarged cells of the superficial epithelium of the antimesometrial wall (Psychoyos, 1966). This first association between the blastocyst and the uterus establishes a definitive position for the blastocyst in relation to the uterus and is frequently referred to as attachment (Schlafke and Enders, 1975). This attachment stage can be further divided into an appositional stage and a subsequent adhesion stage. It is during the appositional stage of implantation that the first structural interaction occurs between embryonic and maternal cells. Apposition is characterized by increasing contact between the microvilli on the surface of the trophoblastic and uterine epithelial cells, leading to their interdigitation (Abrahamsohn and Zorn, 1993). Contact between the embryo and the surface of the uterine epithelium increases gradually at the beginning of implantation and is probably caused by a combination of blastocyst swelling and closure of the uterine lumen (Abrahamsohn and Zorn, 1993). The attachment reaction that obliterates the uterine lumen occurs when the uterine surfaces approximate each other (Schlafke and Enders, 1975). By the seventh day, the decidual growth that has occluded the uterus has isolated each segment of the uterus that contains an embryo (Bridgman, 1948). This encapsulating uterine tissue underlying the ectoplacental cone becomes the maternal part of the definitive placenta, the decidual basalis. Implantation proceeds into the adhesion stage when the surfaces of trophoblastic and luminal epithelial cells have lost their microvilli and run parallel to one another (Pampfer and Donnay, 1999). The rat blastocyst is in contact with the uterine surface for nearly a day because of the initial uterine vascular response and the first invasion of the luminal epithelium of the uterus by the trophoblast (Enders et al., 1980). According to Schlafke et al. (1985), the decidual cells are principally responsible for the formation of the implantation chamber and may assist in the expansion of the blastocyst by initiating disruption of the residual uterine

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luminal epithelial basal lamina. During the seventh day, the blastodermic vesicles become definitely oriented in the decidual crypts, which are lined by slightly flatted cubic epithelium and directed toward the antimesometrial border (Huber, 1915a; Abrahamsohn and Zorn, 1993; de Rijk et al., 2002). In the rat, embryo implantation normally occurs at the antimesometrial side of the uterus. The ICM always faces toward the mesometrial border, and the final placenta is formed at the mesometrial side (Abrahamsohn and Zorn, 1993; de Rijk et al., 2002). Uterine luminal epithelium is present both mesometrial and antimesometrial to the blastocyst, but it is discontinuous in a central band on the walls of the implantation chamber, indicating that these are the initial areas of cellular interaction between embryonic and maternal tissues. In this area, trophoblastic giant cells, which are considered invasive and phagocytic, penetrate between the decidual cells, extending mesometrially between the basal lamina and the overlying uterine cells, forming a narrow avascular layer of transitory fibroblasts around the embryo or the primary decidual zone proper (Tung et al., 1986; Simmons et al., 2007). Maternal blood now begins to circulate through spaces in the network of giant cells. All of these events happen in the antimesometrial endometrium at the site of initial embryonic attachment (Schlafke and Enders, 1975; Abrahamsohn and Zorn, 1993). In the rat, the chorioallantoic placental development is a strictly timed and synchronized process defined by the appearance of five different layers, based on the origin of the placental membranes, and is further defined as hemotrichorial due to its three trophoblastic layers, both of which are discussed in detail below (de Rijk et al., 2002). The chorioallantoic placenta results from the growth of the ectoplacental cone, differentiation of its cells, and formation of an association with cells of allantoic origin. The placenta will form on the mesometrial side of the uterus. An angiogenic allantoic diverticulum differentiates and invaginates into the extraembryonic coelom during day 9 through 10. By day 9.5 the rat allantois first appears as a small cluster of cells attached to the caudal region of the embryo in the angle between the amnion and the yolk sac (Ellington, 1985). Beginning about day 10, the allantois will come into contact and fuse with the chorionic mesoderm (Ellington, 1985). The allantois differentiates into fetal mesenchyme and vascular channels lined by endothelium, which will become confluent at its proximal end with the visceral yolk sac vessels. The vasculature of the allantois extends deeply into the ectoplacental trophoblast between the preexisting maternal blood channels. The allantois and chorionic mesoderm, together with the ectoplacental cone, will develop to form the fetal component of the chorioallantoic placenta beginning at days 11.5e12.5 (Beck et al., 1967a,b; Jollie, 1990; Abdulrazzaq et al., 2001). This is the labyrinth, or zone, of combined fetal and maternal vascularization

(Davies and Glasser, 1968). The allantoic stalk gives rise to the umbilical cord with its root centered on the fetal surface of the chorioallantoic placenta. In the rat, a portion of the avascular parietal wall of the yolk sac membrane covers this organ. As a result, in the rat, all embryonic tissues (intraembryonic and extraembryonic), except the chorioallantoic placenta, are enclosed within the bilaminar yolk sac. From day 12 and throughout pregnancy, five distinct morphologic layers are present in the placenta. Starting from the myometrium, there is a strongly decidualized myometrium with many large metrial cells. A stromal layer consisting of moderately large decidual cells is followed by a layer of giant trophoblasts that separates the tissue of maternal origin from that of embryonic origin. A narrow layer follows, which is called the trophospongium (also called the basal zone, reticular zone, or junctional zone), that consists of highly proliferative cells with rather large syncytiotrophoblasts and lacunae with mature blood cells. Last is a layer called labyrinth, which contains many lacunae, some filled with maternal blood and some with embryonic blood. The last two layers comprise the fetal placenta and occupy about one-third of the thickness of the uterine mesometrial wall (Davies and Glasser, 1968). The labyrinthine trophoblast of the rat is trilaminar (hemotrichorial); the layer in direct contact with the maternal blood is cellular and fenestrated, and the intermediate and basal layers are syncytial, with the third layer in immediate contact with the trophoblastic basement membrane and fetal mesenchyme (Davies and Glasser, 1968). These lacunae will develop later into capillaries with separate vascular channels for maternal and fetal blood. Fetal red blood cells are 100% nucleated on days 12 through 15 of pregnancy, but the number of nucleated red blood cells decreases to 50% by day 16 and then to 5%e10% as the end of pregnancy nears (de Rijk et al., 2002). On about day 14, the uterine cavity is reestablished ventral (antimesometrial) to the embryo, creating a capsule of decidua over the implantation site, the decidua capsularis. The remaining uterine lining is the decidua parietalis (Beaudoin, 1980).

C. Gastrulation The period of gastrulation (days 8.5e9.5 in the rat) covers the period of development of the conceptus from a bilaminar germ disc of epiblast and hypoblast that arises from the ICM through the formation of the primitive streak to the trilaminar ectoderm/mesoderm/endoderm of the embryo. Only in two areas is the original bilaminar condition retained: the prochordal plate, or buccopharyngeal membrane, and the cloacal membrane (Van Mierop, 1979). Structures formed during gastrulation include the

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notochord and neural plate (gestation day 9.5). The notochord appears as a cranial outgrowth of the primitive streak, in a location termed Hensen’s node, which is situated in an axial position between the ectoderm and the endoderm (Gajovic et al., 1989). Hensen’s node is considered the organizer for embryonic gastrulation. The first segregation of cells within the ICM involves the formation of the hypoblast (primitive endoderm) layer at the blastocoel side and will also line the blastocoel cavity, where they will form the yolk sac endoderm. No derivatives of these cells form the embryo. The remaining ICM cells are referred to as the epiblast. Derivatives of these cells will form all embryonic tissues and the amniotic ectoderm. During the middle and latter part of day 9, active cell proliferation in the embryonic ectoderm in the region of the future caudal end of the embryo leads to a distinct thickening of the embryonic ectoderm of this region. This thickening constitutes the primitive streak region and defines unequivocally the anteroposterior axis of the embryo. Within the primitive streak, a short axial groove called the primitive groove develops. During gastrulation, first the definitive endodermal cells, which will replace the primitive endodermal or hypoblast cells, and then the mesodermal cells leave the primitive ectoderm and take their positions in the definitive germ layers (Svajger et al., 1986). Cells remaining in the topographic position of the primitive ectoderm become the definitive ectoderm and will, at the head-fold stage, undergo neurulation. Mac Auley et al. (1993) showed that during gastrulation, most cells of the ectoderm and mesoderm had a cell cycle time of 7e7.5 h, but the cells of the primitive streak divided every 3e3.5 h. By the end of day 9 or at the beginning of day 10, the mesoderm extends so as to form a distinct layer situated between the two primary germ layers (Huber, 1915a,b). With gastrulation and the displacement of cell groups, new spatial relationships, interactive events, and cell commitments occur that lead to regional restrictions of developmental potencies. Grafting of isolated rat embryo germ layers under the kidney capsule has shown that the primitive embryonic ectoderm of the preprimitive streak stage gives rise to tissue derivatives of all three definitive germ layers. At the head-fold stage, embryonic ectoderm differentiates into only ectodermal and mesodermal tissues, whereas the definitive endoderm, as an isograft, shows no capacity for differentiation. Definitive endoderm, together with mesoderm, differentiates into a variety of derivatives of the primitive gut. Isolated mesoderm at the head-fold stage differentiates into only brown adipose tissue. However, in combination with the definitive endoderm, it develops into  adipose tissue, cartilage, bone, and muscle (Levak-Svajger,  1974) (Levak-Svajger and Svajger et al., 1986). Smith (1985) noted in the mouse that the anteriore posterior axis of the blastocyst was determined before

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streak formation and before implantation. In mice, the hypoblast forms on the side of the ICM that is exposed to the blastocyst fluid. As development proceeds, the notochord maintains dorsaleventral polarity by inducing specific dorsaleventral patterns of gene expression in the neural tube (Goulding et al., 1993). Somites, or paired segmental masses of mesoderm in the vertebrate embryo, run along the neural tube and will eventually give rise to tissues of the body. The sidedness of asymmetric body structures is determined during the early neural plate stage, which is before the 5- to 8-somite stage when the bulboventricular loop is formed from a single heart tube and when morphological signs of body asymmetry first appear. The embryonic axis starts to rotate from a concave to a convex dorsal curvature, where the tail takes up a position on the right side of the anterioreposterior body axis at the 9- to 10-somite stage. This rotation is completed at the 17- to 18-somite stage (Fujinaga and Baden, 1993). Most defects of gastrulation are probably incompatible with survival. It is important to stage embryos, as there is considerable variation in embryonic development by gestation day 9, ranging from embryos at early primitive streak stage to those at late neural plate stage with a foregut pocket (Fujinaga and Baden, 1992; Fujinaga et al., 1992), and by day 11, embryos may be up to 12 h apart in development (Fujinaga and Baden, 1991). Causes for this difference in development include length of mating period, time required for the sperm to reach the ova, actual time of ovulation and implantation, and nutritional access of implants owing to differences in uterine site (Holson et al., 1976; Fujinaga and Baden, 1992). Two staging systems for gastrulating rodent embryos are in use: Theiler’s system (Theiler, 1972) for mice and Witschi’s system (Witschi, 1962) for rats. Both systems use consecutively numbered stages and cover all of the gestation period; however, neither system precisely defines the stages during the early postimplantation period. Fujinaga et al. (1992) proposed modifying Theiler’s system by making subdivisions of some of the stages to permit a more precise staging of early postimplantation embryos of the rat. For the somite period, embryo staging is usually based on the number of somite pairs, but this can be unsatisfactory because the number of somites varies by as much as six pairs at any one stage of development and because the total number cannot be counted throughout development, as the somites gradually become obliterated (Edwards, 1968).

D. Organogenesis 1. Neurulation and Early Central Nervous System Development Neurulation is the process of neural tube formation that forms the future brain and much of the length of

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the spinal cord. The precursor to the neural tube is the neural plate, which is found as a thick ridge of ectoderm on the dorsal surface of the embryo (Greene and Copp, 2009). The caudal portion of the spinal cord forms during secondary neurulation. The formation of the nervous system depends on cell movements and cellecell interactions that begin during gastrulation. It has also been shown that many of the Pax genes are expressed during early neurogenesis, where they exhibit distinct regional patterns of transcriptional activity (Goulding et al., 1993). The induction of the neural plate has been studied principally in amphibian embryos. Although the fundamental mechanisms of neurulation and neural tube formation have been long studied, the molecular mechanisms behind neural tube closure are poorly understood (Copp et al., 2003). Based on presumed parallels with amphibian embryos, it is believed that this migration of cells brings the mesodermal cell population into a close relationship with the overlying ectoderm and sets up the initial embryonic inductive interaction between the medial portion of the mesoderm and the overlying prospective neural plate. Numerous microsurgical and transfilter experiments have shown that the subjacent notochord is a requisite for neural induction to occur. The development of the notochord in the rat takes place between days 8.5 and 9 (Florez-Cossio, 1975). Induction is followed by elevation of the lateral margins of the neural plate to form neural folds. On elevation, the neural folds first become biconvex, then become concave, and finally meet and fuse in the midline. During this time, neuroepithelial cells change in shape from low columnar to high columnar and then to wedge shaped. The initial neural tube fusion occurs in the cervical region of the second and third somites at the 7-somite stage, extending upward into the hindbrain. At the early 10-somite stage, a separate region of fusion begins at the midbrain/forebrain junction immediately above the developing cranial flexure. By the 14-somite stage, final closure of the spindle-shaped opening between these two areas of fusion is complete (Morriss-Kay, 1981). The last portion of the cephalic neural fold to close, the anterior neuropore, is located in the future forebrain and closes by the 16-somite stage. This is followed by progressive fusion from the cervical region to the caudal region (Morriss and New, 1979; Campbell et al., 1986; Morriss-Kay and Tucket, 1991). The posterior neuropore is found at the caudal end of the embryo in the future lumbosacral region and remains open until about the 21-somite stage. In the rat, the neuropores close between days 10.5 and 11, with the anterior neuropore closing first (Long, 1938; Hoar, 1981). The final processes of neural tube closure are migration of the neural crest cells and individual fusion of the surface ectoderm and neural ectoderm (Campbell et al., 1986).

In the rat, as well as mouse and other tailed mammals, secondary neurulation, or the process of the secondary neural tube formation, starts from a process within the tail bud just after the posterior neuropore closure has been completed. The tail bud is a mass of multipotential undifferentiated mesenchyme, which consists of cells derived from the remnants of the primitive streak and Hensen’s node, the posterior end of the neural groove, and the posterior coelomic mesodermal epithelium. During the elongation of the tail, new cells are added through proliferation of those at the proximal end of the tail bud. The neural tube within the tail originates through a process of secondary neurulation by simultaneous processes of extension of the central canal of the primary neural tube and recruitment or mitosis of cells that become radially arranged around the extending lumen. This lumen formed by cavitation is always directly continuous cranially with the lumen of the fully formed neural tube (Hughes and Freeman, 1974; Schoenwolf and Nichols, 1984; Campbell et al., 1986; Gajovic et al., 1989). The notochord in the tail of the rat develops on days 12 and 13 from a common mass of condensed mesenchymal cells located ventral to the secondary neural tube, which subsequently splits to form a thin cord (which becomes the notochord) and a thick portion (which gives rise to the tail gut) (Gajovic et al., 1989). Neural crest cells differentiate at the lateral margins of the neural plate and are derived solely from the neural epithelium from the 6-somite stage onward (Tan and Morriss-Kay, 1985). They will migrate away from the neural fold, or roof of the incipient neural tube, and are induced by structures with which they come in contact to form many cell types, including neurons and glial cells of the sensory, sympathetic, and parasympathetic nervous systems; the medulla cells of the adrenal gland; adrenergic paraganglia; calcitonin-producing cells; melanocytes of the epidermis; endothelium of the aortic arch arteries; the septum between the aorta and the pulmonary artery; and skeletal and connective tissues of the face and anterior neck region (Adelman, 1925; Le Douarin and Ziller, 1993). Neural crest migration occurs in the following sequence: midbrain, rostral hindbrain, caudal otic hindbrain, postotic, and finally rostral otic hindbrain, with a general shift in migratory intensity from rostral to caudal regions (Tan and Morriss-Kay, 1985). Fukiishi and Morriss-Kay (1992) showed that in the rat, neural crest cells from the midbrain levels migrate to the maxillary process and the mandibular arch and contribute to the development of the face, and neural crest cells from the rostral hindbrain migrate to the first and second pharyngeal arches (Fukiishi and Morriss-Kay, 1992). Occipital neural crest cells migrate to the third, fourth, and sixth pharyngeal arches and are essential for the division of the outflow tract of the heart into the aorta and

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pulmonary trunk. Clonal analysis of the developmental potentialities of individual neural crest cells has shown that most of the migrating cells exhibit various levels of pluripotency, but some are fully committed at the time of migration (Le Douarin and Ziller, 1993). Inhibition or errors in neural crest migration produce anomalies of facial development, defects in the aorticopulmonary septum in the heart, small or missing dorsal root ganglia, and errors in pigmentation. In the anterior region, the neural tube balloons into three primary vesicles as the anterior neuropore closes at gestation day 10.5: forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon). The optic vesicles extend laterally from each side of the developing forebrain. On gestation day 12, the forebrain becomes subdivided into the anterior telencephalon (cerebral hemispheres) and the more caudal diencephalons (thalami, posterior lobe of the pituitary gland, pineal body and optic stalk, and optic cup, which later generate the optic nerve and retina). The rhombencephalon becomes subdivided into a posterior myelencephalon (medulla oblongata) and a more anterior metencephalon (pons and cerebellum). The cerebellum is formed in the rat on day 14. The remainder of the neural tube forms the spinal cord. Expression profiles of Wnt genes in the mouse suggest that Wnt signaling plays a major role in early central nervous system development in vertebrates (Parr et al., 1993). Neurogenesis in the rat spinal cord occurs in a temporal gradient, with neurons in the ventral spinal cord becoming postmitotic before those in the dorsal cord. Most motor neurons are generated between days 11 and 13 (Goulding et al., 1993). Neurons form in the fetal rat brain principally between days 13 and 20dexcept in the cerebellum, where they do not appear until about day 19dand continue to form during the early postnatal weeks (Hoar, 1981; Gilbert 1997, 2000). Naturally occurring neural tube defects have been extensively studied in several strains of mice (curly tail, splotch mutant, loop tail, and those trisomy for chromosomes 12 and 14). Campbell et al. (1986) list teratogens that have been administered to rats to interrupt the process of neurulation (Campbell et al., 1986). Defective closure of the neural folds results in the following malformations: anencephaly, exencephaly, rachischisis, encephalocele, or spina bifida. 2. Somite Formation In vertebrate development, the segmental pattern of the body axis is established as somites, masses of mesoderm distributed along the two sides of the neural tube. The anterioreposterior mechanism of somite formation depends on waves of gene expression associated with the Notch, Fgf, and Wnt pathways (Fongang and Kudlicki, 2013).

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The mesoderm is found on day 9 extending on each side of the notochordal plate as two lateral wings, and it divides peripherally into the somatic and splanchnic layers. Somite formation in the rat has been well described (Bucher, 1929). The somite, or embryonic segments generating a body plan, divides into three parts. When somites are formed, the most medial part, the sclerotome, migrates around the notochord and neural tube and will form connective tissue, cartilage, and bone (vertebrae and intervertebral discs of the vertebral column). Immediately lateral to the sclerotome are the cells that form the myotome, which will later become skeletal muscle. The most lateral part of the somite forms the dermatome that will form the dermal connective tissue. The first somite will later involute, and other somites develop in a head-to-tail sequence until there are 65 complete somites and a mesodermal remnant. The number of somites present (Table 23.1) has been used to classify the developmental stage of rat embryos (Schneider and Norton, 1979). Each successive somite is more advanced structurally than the one just anterior to it. The size of the successive somites increases from the first to the 31st, and then the somites are successively smaller. 3. Cardiovascular System The heart is the first functional embryonic organ and is required by the embryo for efficient growth (Christoffels et al., 2000; Linask, 2003). The cardiogenic mesoderm forms by anteriorelateral migration from the primitive streak and constitutes a single primordium, which will organize into an epithelium. The heart forms in the anterior margins of the embryonic disc in front of the neural plate. The pericardial cavity also originates in the cardiogenic mesoderm, first appearing as small spaces that coalesce to form larger single closed right and left cavities, which in turn unite into a single horseshoe-shaped cavity. Because of the rapid cephalad growth of the forebrain and cephalocaudal folding of the embryo, the prochordal plate (buccopharyngeal membrane) and pericardial cavity are carried farther under the rising head of the embryo. Cells that form the heart initially have a bilateral distribution, but they gradually become localized in the cardiac crescent lying in front of the prochordal plate and stretching along the future neural plate at the level of the first two paired somitomeres. Subsequently, after the lateral body folding, the two sides of the crescent meet in the midline to form the single heart tube (Baldwin et al., 1991; Suzuki et al., 1995). An epithelial myocardium is established and forms a tubular heart in the rat on day 9e9.5, and contractions can be observed on day 9.5e10 (Gross, 1938; Hebel, 1986; Clark, 1989; Baldwin et al., 1991). In a complex morphogenetic progression, the linear tube is transformed into a synchronously contracting fourchambered heart (Christoffels et al., 2000). Both myosin

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Number of Somites and Corresponding Gestational Age of the Rat.

Number of Somites

Age (Days from Mating)

1

9

5

9.5

10

9e10

20

10e11

25

11e12

30

11e11.5

35

12

40

12e12.5

45

13.5

50

13

60

14.75

and actin filaments become identifiable for the first time in the day 10 myocardium of the C-shaped tubular heart (Chacko, 1976). The splanchnopleuric mesoderm that surrounds the endothelial heart tube differentiates into three layers: an inner layer of cardiac jelly surrounding the endothelium, an early myocardium that acquires contractile elements, and an outer layer (the epicardium). The last two layers are referred to as the myoepicardial mantle. Cranially, at the arterial pole of the bulboventricular tube, the first pair of aortic arches arises and is continuous with the dorsal aortae. Caudally, the bulboventricular tube receives blood from the yolk sac by means of the paired sinus venosus and atrium. From this, the bulboventricular tube will develop into the embryonic ventricle and bulbus cordis, and later into the ventricle and their outflow tracts. As it grows, the bulboventricular tube forms a loop bending right in the shape of a C. Looping results in the correct spatial orientation for subsequent modeling of the four chambers of the heart (Linask, 2003). It has been shown by Christoffels et al. (2000) that the onset of the transcriptional program for cardiac genes specifically associated with the formation of the ventricular and atrial chamber myocardium is restricted to the ventral side of the linear heart and the outer curvature of the looped heart (Christoffels et al., 2000). The atrial portion of the heart dilates and fuses to form a large common atrium, which, as a result of growth, moves cranial from its original caudal position to lie behind the bulboventricular loop. The paired sinus venosi also partly fuse to form the right and left sinus horns that receive the vitelline, allantoic, and cardinal veins. Dilatations develop in the bulboventricular loop, whereas the atrioventricular junction and the junction

of the ventricle and proximal bulbus cordis remain relatively narrow. The embryonic ventricle becomes the primitive left ventricle, and the proximal one-third of the bulbus cordis becomes the primitive right ventricle. Alternatively, it has been proposed that the right and left ventricular chambers “balloon out” from the outer curvature of the heart tube (Christoffels et al., 2000). The conus cordis or outflow portions of both ventricles will be formed from the middle one-third of the bulbus. The remaining one-third of the bulbus after partitioning develops into the truncus arteriosus. The cardiac jelly in the atrioventricular canal area and in the conus cordis and truncus arteriosus becomes invaded by cells derived from the endothelium and is referred to as endocardial cushion tissue. The atrioventricular and outflow tract cushions function as primitive valves in the early heart tube, ensuring directional blood flow (Barnett and Desgrosellier, 2003). Growth of the right and left ventricles results in the formation of the muscular ventricular septum that begins on day 11.5e12 in the rat (Hoar, 1981; Hebel, 1986). The atrioventricular canal shifts position to the right, allowing blood to enter both ventricles. In the rat, the division of the atrium is accomplished by the formation of the septum primum, which contains the foramen ovale, and begins on day 11.5e12 (Hoar, 1981; Hebel, 1986). Opposing pairs of endocardial cushions appear in the atrioventricular canal, the conus cordis, and the truncus arteriosus and then meet and fuse. This divides the atrioventricular canal into right and left ostia and divides the right anterior half of the conus cordis into the right ventricular outflow tract, and the posteromedial half becomes absorbed into the primitive left ventricle to become its outflow portion. The truncus arteriosus is divided into pulmonary and aortic channels. Conotruncal septation involves the morphogenic movement of mesenchymal tissue from the branchial arches, neural crest cells, and endothelium and also involves the conotruncal cushions. As a result of shifts in the fourth and sixth aortic arches, the sixth arches become aligned with the pulmonary channel; the fourth arches, with the aortic channel. Endocardial cushion tissue derived from the atrioventricular canal and conal septa completes the ventricular septum. Additional pairs of opposing masses of endocardial cushion tissue in the atrioventricular canal area along with the atrioventricular canal septum act as provisional valves. Most of the atrioventricular valves (tricuspid and mitral valves) form by the increased diverticularization and undermining of the interior of the ventricular walls that later become fibrous and form the valve cusps and their chordae tendineae. Additional pairs of endocardial cushion tissue in the truncus arteriosus eventually develop into the anterior cusp of the pulmonary valve and the posterior cusp of the aortic valve. The remaining

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cusps of the pulmonary and aortic valve develop from the endocardial cushion tissue of the truncus septum (Moffat, 1959; Van Mierop, 1979; Vries, 1981; Clark, 1989; Barnett and Desgrosellier, 2003). After septation, these swellings thin and become “excavated” on the distal side to become leaflets (Rothenberg et al., 2003). The heartbeat is initiated and coordinated by a heterogeneous set of tissues that are collectively referred to as the pacemaking and conduction system (Gourdie et al., 2003). The sinus node, which determines the intrinsic rhythm of the heart, develops at the junction of the right common cardinal vein and the right sinus horn and can be observed on day 12 in the rat and is completely developed by day 15 (Domenech Mateu and Orts Llorca, 1976; Hebel, 1986, Gourdie et al., 2003). After initiation of a cardiac action potential within the node, activation is propagated through the muscular tissue of the atria, eventually focusing into the atrioventricular node. The atrioventricular node, located at the junction of the atria and the ventricles, functions as part of a mechanism for generating a momentary delay in the propagation of the cardiac action potential. This separates the activation of the atrial chambers from that of the ventricles (Gourdie et al., 2003). After exiting from the atrioventricular node, the action potential rapidly propagates along the bundle of His and its distal branches, finally activating the ventricular chambers via the Purkinje fibers. The HisePurkinje system is a network of fast conduction tissues that are responsible for coordinating ventricular contraction. The fast-conduction system of the ventricles is the last element of the pacemaking and conduction system to develop (Gourdie et al., 2003). The atrioventricular node and bundle of His develop from a specialized reticular segment of the myocardium that initially surrounds the atrioventricular canal (Van Mierop, 1979). The pulmonary veins grow as a bud from the lung buds and connect with the left atrium. Of the six pairs of aortic arches, the first and second arches, which appear by the 12-somite stage, regress, and the fifth arch disappears. In embryos with a crownerump length of between 3 and 4 mm, the first aortic arch has broken down, but the second and third arches are present and join the aortic sac to the dorsal aorta. In the rat, there is a temporary communication that forms between the fourth and the sixth arches, after the sixth arches are fully formed, as a result of the marked caudally directed convexity of the fourth arches. The third arches, which appear between the 15- and the 18-somite stages, form the proximal parts of the internal carotids, and the sixth arches form the pulmonary arteries and, on the left, the ductus arteriosus. The left fourth arch forms part of the aortic arch, which lies proximal to the origin of the left common carotid artery, and the right fourth arch forms part of the innominate artery (Moffat, 1959). The common carotid artery is formed as a result of the elongation

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of the cranial end of the aortic sac. Coronary arteries that arise as buds from the ascending aorta grow down across the heart surface and into the myocardium by day 15.5 in the rat (Hoar, 1981). The coronary veins form about the same time and connect with the left sinus venosus later to form the coronary sinus (Clark, 1989). Development of both the coronary arteries and the capillaries continues after birth (Hew and Keller, 2003). Structures peculiar to the fetal circulation include the foramen ovale, ductus arteriosus, umbilical vessels, and ductus venosus. The foramen ovale permits blood to flow directly from the right atrium to the left atrium, bypassing the lungs; the ductus arteriosus allows blood to flow from the pulmonary arteries to the aorta, again bypassing the lung. The ductus venosus allows blood in the umbilical vein to bypass the liver. The umbilical vein carries oxygen-rich blood from the placenta to the fetus, while oxygen-poor blood is returned to the placenta through the umbilical arteries. Two umbilical veins form in young rat embryos, but the right umbilical vein disappears on day 13. In the rat the umbilical arteries arise from the internal iliac arteries, but there is only a single umbilical artery within the cord. In the rat embryo, there is a communication between the vitelline and the umbilical arteries until day 11 (Hoar, 1981). The transition from prenatal to postnatal circulation involves three primary steps: (1) the cessation of umbilical circulation, (2) the transfer of gas exchange function from the placenta to the lungs, and (3) closure of the prenatal shunts. In addition, the heart function changes from acting as two parallel pumps to acting as two pumps performing in series. The foramen ovale is diminished in the first 2 days after birth and is completely closed by day 3. Functional closure of the ductus arteriosus is complete 2e5 h after birth in the rat. The ductus venosus narrows rapidly after birth and closes completely in 2 days (Hew and Keller, 2003). Hew and Keller (2003) report heart rate and blood pressure values for the early postnatal period in the rat. Heart malformations attributed to aberrant neural crest cell migration include aorticopulmonary septal defects (persistent truncus arteriosus, transposition of the great vessels, truncus arteriosus communis). Malformations attributed to extracellular matrix abnormalities include persistent atrioventricular canal and tricuspid atresia. Malformations related to abnormal blood flow include pulmonary valvular atresia, coarctation of the aorta, and aortic valvular stenosis or atresia. Aberrant cell death can cause muscular ventricular septal defects. Left-sided looping of the developing heart will result in dextrocardia (Kirby et al., 1983; DeSesso, 1993). Overriding aorta is a malalignment defect in which wedging of the aorta between the mitral valve and the tricuspid atrioventricular valves fails to occur because the outflow track does not lengthen normally during looping. This

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leads to a double-outlet right ventricle and tetralogy of Fallot (Hutson and Kirby, 2003). 4. Urogenital System The urogenital system is actually two separate systems united through inductive interactions during development. The urinary system develops within the intermediate mesodermal ridge, which is along the peritoneal aspect of the dorsal body wall. The rat, similar to other mammals, develops three different but overlapping kidney systems, which appear during days 10e12 of gestation: pronephros, mesonephros, and metanephros. In the rat, the pronephros and the mesonephros regress in utero. The excretory system of the rat begins as a nephrogenic ridge that runs from the 9th to the 12th somites. The pronephros develops at the cervical somite levels in the intermediate mesoderm and migrates caudally. The anterior region of the pronephric duct induces the adjacent mesenchyme to form the pronephric kidney tubules. This is a nonfunctional kidney. The pronephric tubules and the anterior portion of the pronephric duct degenerate, but the more caudal portion of the pronephric duct persists and is referred to as the Wolffian duct. In the rat, the Wolffian duct appears at the end of day 10 (Hebel, 1986). The mesonephric duct (formerly the caudal portion of the pronephric duct) initiates over time a new set of vesicles, beginning at the level of somite 12. These vesicles develop in a cranial-to-caudal pattern and will become the second kidney or mesonephros. One end of each elongating vesicle connects to the mesonephric duct, and the other end is invaginated by capillaries from the dorsal aorta to form the functional mesonephric glomerulus. The tubular components of the mesonephric nephron are found between the glomerulus and the mesonephric duct and consist of a secretory segment and a collecting segment. Twenty-four- to 30-somite rat embryos have mesonephric tubules in various stages of differentiation and paired Wolffian ducts that demonstrate varying degrees of canalization. On day 12, the mesonephros is at the height of its development, with 15e18 tubules between the levels of somites 12 and 18. The distal ends of the mature tubules are dilated to form Bowman’s capsule. Some of the more anterior tubules share a common basal junction instead of opening directly into the Wolffian duct. On day 12, the mesonephric tubules and the Wolffian duct develop a lumen that opens into the cloaca, an area in the early embryo that comprises the early urogenital and anal areas. The Wolffian duct bends rather sharply at the level of the 26th somite before it opens into the cloaca. It is at this bend that the ureteric bud forms (Hebel, 1986). The mesonephric nephrons regress with only the three most anterior tubules being retained. These three comprise the future epigonadal system (Torrey, 1943; Arey, 1974; Gilbert, 1997). The permanent kidney, or metanephros, forms from an evagination (the ureteric or metanephric bud) that

appears just proximal to where the mesonephric duct enters the cloaca on day 13 (Hebel, 1986). These buds eventually separate from the nephric duct to become the ureters that will take the urine from the kidney to the bladder. The ureteric bud elongates and migrates cranially and projects into the mesenchyme, which then is induced to condense around the buds and differentiate into the secretory part of the nephrons of the metanephros. This nephron-forming tissue then induces further branching of the ureteric bud to form the major and minor calyces of the metanephros. Outgrowths from the minor calyces develop and project into the condensed mesenchyme or metanephric blastema to form the collecting duct portion of the nephron. The metanephros consists of three cell lineages: the epithelial cells of the Wolffian ductederived ureter bud, the mesenchymal cells of the nephric blastema, and the endothelial cells of the capillaries. Kidney development requires a set of inductive interactions between the metanephric mesenchyme and the ureteric bud. First, the metanephric mesenchyme induces a bud to form on the nephric duct. This is followed by two reciprocal interactions: the mesenchyme causes the ureteric bud to grow and bifurcate, forming the collecting duct system, whereas the bud induces the mesenchyme to differentiate into stem cells within the growing kidney. Groups of these stem cells then aggregate, epithelialize, and undergo morphogenetic events to produce commashaped bodies, which change into S-shaped bodies. These S-shaped bodies later unwind into elongated tubular nephrons that interact with capillaries at their proximal ends to form glomeruli. These glomeruli, which convolute and differentiate, become the selective reabsorption apparatus and finally fuse to the collecting ducts at the distal end. The connecting tubules of the juxtamedullary and midcortical nephrons form arcades; however, the subcapsular nephrons directly join the collecting duct. Nephrons continue to be initiated at the periphery of the kidney over the fetal period so that the less mature nephrons are located in the kidney periphery. The first primitive loop of Henle appears at day 17 in the rat, and the last loops mature by approximately postnatal day 15. The thin ascending limb of Henle begins to differentiate, beginning in the tip of the papilla, and progresses outward, with all the loops differentiated at postnatal day 8 (Arey, 1974; Kazimierczak, 1980; Hoar, 1981; Neiss 1982a, 1982b; Saxen and Sariola, 1987; Davies and Bard, 1996; Gilbert, 2000). The primordial germ cells are identifiable in the yolk sac epithelium on days 9e10 in the rat and begin to migrate on day 10 or 11 (Beaudoin, 1980). The gonadal ridge containing primordial germ cells has developed by day 13 (Maeda et al., 2000). The mammalian gonad first develops through an indifferent or bisexual state, with the default sex being the female. During the indifferent gonadal stage, both the Wolffian and the

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Mu¨llerian ducts are present. The Wolffian (mesonephric) duct is in close association with the gonad and empties into the urogenital sinus. The Mu¨llerian (paramesonephric) ducts are also in close association with the developing gonad being induced within the intermediate mesodermal ridges by the mesonephric ducts, beginning on day 14. Depending on the sex of the embryo, one of these two duct systems completes its development and the other disappears almost completely. The gonadal primordium or genital ridge can be observed in the rat at day 11 at the level of somite 16. In day 13 embryos, the gonadal blastema blends with the mesonephric stroma and makes contact with the first three mesonephric tubules. It is in this area that the rete forms. The junction of the rete cords with the mesonephric tubules takes place in both male and female rats on day 17. The epithelium of the genital ridge proliferates into the mesenchymal tissue above it to form the sex cords that will surround the germ cells that migrate from the yolk sac to form the gonad. Two pairs of genital ducts are established and present at the indifferent-gonad stage. The Mu¨llerian ducts begin to degenerate in the male, and Wolffian ducts degenerate in the female on day 18 in the rat. The Mu¨llerian (paramesonephric) ducts, which can form the oviducts and uterus, are also in close association with the developing gonad being induced within the intermediate mesodermal ridges by the mesonephric ducts beginning on day 14. In the male, the genital tract develops primarily from two embryonic anlagen: the Wolffian ducts and the urogenital sinus. The formation of the male phenotype involves the secretion of the hormones testosterone, secreted from the fetal testicular Leydig cells that stimulate the mesonephric (Wolffian) duct and urogenital sinus to differentiate, and anti-Mu¨llerian duct hormone (Mu¨llerian-inhibiting substance) from early Sertoli cells that destroys the Mu¨llerian duct over days 18e22 in the rat. The Wolffian duct, whose epithelium is mesodermal in origin, gives rise to the epididymis, ductus deferens, seminal vesicle, and ejaculatory duct. The urogenital sinus, whose epithelium is derived from endoderm, gives rise to the prostate, bulbourethral glands, urethra, and periurethral glands. The mesonephric tubules give rise to the efferent ducts. The testes can be observed toward the end of day 13 or beginning of day 14, when primary sex cords are seen at right angles to the surface of the testis. An area of unorganized blastema found between the peripheral ends of the primary cords and the coelomic epithelium is the tunica albuginea. The mesenchyme-like cells found in the narrow angles between some of the cords are the progenitors of the interstitial tissues of the testis. Testicular secretion of testosterone begins on day 15 and is a requirement for differentiation of the reproductive organs that develop from the urogenital sinus, Wolffian ducts, and perineal

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tissue (Timms et al., 2002). By day 16, the testis cords have become elongated and tortuous, and Sertoli cells are first observed. Leydig cells are found among the stromal cells around day 17. At the anterior end of the testis, the cords are beginning to radiate toward the mesonephros and abut on the developing rete cords. After forming a junction with the first three mesonephric tubules on day 17, the rete cords in the male begin to cavitate late on day 18. By day 20, most of the volume of the cord is occupied by germ cells. By postnatal day 1, the rete tubules are broad and branching, and open into the mesonephric tubules that comprise the efferent ducts that connect to the Wolffian duct or vas deferens. On about postnatal day 15, the cavitation of the sex cords begins to form the seminiferous tubules. The seminal vesicles appear as diverticula just proximal to the ejaculatory duct, and the prostate gland develops from the pelvic portion of the urogenital sinus on day 19 (Torrey 1945, 1947; Merchant-Larios, 1976; Hebel, 1986; Cunha et al., 1992; Gilbert, 1997). If the Y chromosome is absent, the gonadal primordial develops into ovaries that secrete estrogenic hormones and enable the development of the Mu¨llerian duct into the uterus, oviduct, and upper end of the vagina. The mesonephric (Wolffian) duct disappears almost completely. Ovarian differentiation is first observed in the rat at day 16 as a pronounced clumping of cells and the beginning of cord formation within the blastema. The rete cells extend through the mesovarium into the stroma of the mesonephros. They are continuous with the primary sex cords and are in contact with the distal ends of the mesonephric tubules that will become the tubules of the epoo¨phoron. The cords become tightly packed in the interior of the ovary, and no tunica albuginea separates the cords from the coelomic epithelium. Secondary cords that originate in the epithelium form by day 17. The Wolffian duct disappears, and the rete breaks up into sharply delineated cords. Cavitation of the rete cords usually begins during postnatal day 3. At parturition, the central two-thirds of the ovary is divided into rectangular columns of sex cords by connective tissue septa. Degeneration of the medullary cords and formation of the primary follicles occur during postnatal development (Torrey 1945, 1947; Hoar, 1981; Gilbert, 1997). 5. Craniofacial Development Proper temporal and spatial patterns of growth are critical for normal development of the craniofacial region, which includes externally the face and head (excluding the caudal pharyngeal arches) and internally the brain; skull, jaws, and facial skeleton; teeth; and associated soft tissues. The face develops from the growth and fusion of five facial prominences that surround the stomodeum: the frontonasal prominence and the paired

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maxillary and mandibular prominences (Sulik and Schoenwolf, 1985). The cells that form the skeletal and connective tissues of the face, with the exception of tooth enamel, are derived from cranial neural crest cells that migrate into the first and second pharyngeal arches (Johnston, 1980). The frontonasal prominence is composed of the tissue that surrounds the forebrain. The olfactory placodes located on the lateral aspects of the frontonasal prominence will invaginate to form the widely separated nasal pits. Cells from the nasal pits will line the nasal cavities and later form the sensory epithelium for olfaction. Midbrain crest cells migrate into the frontonasal region and then form the lateral nasal prominence on the rim of the nasal placodes. The median portion of the face develops from the frontonasal processes that separate the nasal pits. Forebrain crest cells also migrate into the frontonasal region and then contribute to the formation of the medial nasal prominence that surrounds the nasal pit closer to the midline (Osumi-Yamashita et al., 1997b; Hall and Miyake, 2000). The first and second pharyngeal arches are evident on days 11e12, the time the anterior neuropore closes (Hebel, 1986; Ross and Persaud, 1990). The first pharyngeal arch has both the maxillary and the mandibular prominences that contribute to the upper and lower jaw. The maxillary process is first distinguishable at day 12 (Smith and Monie, 1969). At day 11, the mandibular arches meet in the midline but do not commence fusion (Symons and Moxham, 2002). The maxillary processes emerge laterally from the proximal portion of the mandibular processes at the lateral edges of the stomodeum and extend upward toward the nasal region and reach the lateral nasal process by day 11.5e12. The ends of the lateral and medial nasal processes come into contact below the nasal pit and begin to fuse, with the result that the nasal pit becomes deeper and narrower. Around the 31-somite stage, the medial nasal process and the maxillary process come into contact, and the groove between these two processes becomes the nasolacrimal groove. The fusion of the mandibular arches begins at their caudal margin and extends cranially and is complete around day 13 to form the floor of the mouth (Christie, 1964; Smith, 1969). By day 13, the upper jaw of the rat is forming. The maxillary processes extend around the developing mouth, and the lateral and medial nasal processes surround the developing nasal pits. These processes fuse by the merging of the mesenchyme to form the intermaxillary segment (Lejour, 1970; Symons and Moxham, 2002). The medial nasal prominences merge in the midline and extend caudally to the oral aperture, forming the columella. Lateral extensions of the tips of each nasal medial process extend cranially between the nasolateral and the maxillary processes (Smith, 1969). By day 13.5 a swelling of the medial borders of the maxillary

processes begins to form the definitive or primary palatal processes. By day 14, the lower jaw extends about halfway along the maxillary process (Smith, 1969). On day 14, fusion occurs between the ventral end of the palatal proliferation of the maxillary process and that of the frontonasal process to form the roof of the anterior portion of the primitive oral cavity, as well as the initial separation between the oral and the nasal cavities. Later, derivatives of the primary palate form portions of the upper lip, anterior maxilla, and teeth (Johnston, 1980). In the caudal portion of the oral cavity on day 14, new outgrowths from the medial edges of the maxillary processes form the shelves of the secondary palate. The anterior four-fifths of each palatine process grows downward on either side of the tongue, and on day 15 the tip of the tongue extends beneath the posterior portion of the primary palate. The posterior one-fifth of each palatal shelf (future soft palate) grows horizontally above the dorsum of the tongue from the beginning and never elevates (Ferguson 1977, 1978). The vertical palatal shelves elevate to a horizontal position between the tongue and the nasal septum by day 16, with the shelves taking an active role in their elevation (Srivastava and Rao, 1979). After the shelves elevate, the shape of the tongue changes from a highly arched structure that fills the oronasal cavity to a broad flat structure. On day 16, the tip of the tongue protrudes from the mouth, but by day 17, the mandibular growth rate has exceeded that of the tongue, and the tongue returns to the oral cavity (Wragg et al., 1970). Within 2 h of elevation, the anterior shelves begin to fuse, leaving a Y-shaped gap in the palate in front of the region of contact and a long straight gap behind it. The anterior gap is closed later by a combination of backward growth of the nasal septum and primary palate and forward growth of the palate shelves. The more posterior shelves grow rapidly toward each other, and epithelial fusion proceeds, with the major part of the future hard palate being fused within 5 h (Ferguson, 1978). The future soft palate is still widely separated and does not fuse until day 17.5 (Ferguson, 1977). Fusion of the palate is complete by day 18, and the characteristic pattern of rugae is present. Cleft palate can be induced at several developmental stages (days 9, 11, or 15) in the rat by various agents that include high doses of vitamin A, glucocorticoids, retinoids, and b-aminopropionitrile (Kochhar and Johnson, 1965; Vig et al., 1984; Granstrom et al., 1992). Cleft palate may result from disturbances at any stage of palate development by defective palatal shelf growth, delayed or failed shelf elevation, defective fusion, or postfusion rupture (Kerrigan et al., 2000). The small eye rat (rSey), owing to a mutation in the Pax6, gene lacks eyes and nose owing to an impairment in which the frontonasal

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ectoderm fails to induce lens and olfactory placodes (Fujiwara et al., 1994; Osumi-Yamashita et al., 1997) . At day 13, the rSey/rSey homozygous embryo lacks eye and nasal pits. In addition, the nasal prominence appears to be missing and the frontonasal prominence protrudes, making an appearance at the medial nasal prominence (Fujiwara et al., 1994). 6. Limb Development Limb development begins when mesenchymal cells proliferate from the somatic layer of the lateral plate mesoderm (skeletal precursors) and the somites (Gilbert, 1997). In the rat, the forelimb limb bud is apparent on day 11 as a lateral swelling of the plate mesoderm between about the sixth and the 10th somite with an epithelial ectodermal layer (Christie, 1964; Hoar, 1981; Hebel, 1986). The hind-limb buds lag in temporal development and are apparent shortly after the forelimb limb buds are seen at day 12 (34-somite stage) opposite somites 23 to 28 (Long, 1938; Christie, 1964; Hoar, 1981; Hebel, 1986). Progenitor cells of skeletal muscle migrate from the lateral edge of limb-level somites into the limb bud of the rat on day 14 and colonize in the dorsoproximal region of the limb bud. After the establishment of myogenic masses, these cells continue migrating within the limb (Hayashi and Ozawa, 1995; Rahman et al., 1997). Once limb buds are formed and initiate lateral growth, the apical ectodermal ridge (AER) appears and runs along the distal margin of the bud. The AER is considered the major limb bud signaling center and the proximaledistal growth and differentiation require interaction between bud mesenchyme and ectoderm (Gilbert, 2000; Williamson et al., 2016). The elongation of the limb bud is mediated by the proliferation of the mesenchymal cells under the AER. In the mammalian embryo, the anteriore posterior axis is defined by tissue along the posterior margin of the limb bud, or the zone of polarizing activity (ZPA), and provides positional information for the specification of the digits (Tickle et al., 1975; Fallon, 1977; Summerbell, 1979). The exact cascade of genetic determination and transcription factors of limb formation is beyond the scope of this chapter. In summary, the AER expresses fibroblast growth factor 8, which induces the Sonic Hedgehog polarizing morphogen in the posterior mesoderm (Tickle and Towers, 2017). This is localized in the region of the limb bud shown to contain the highest ZPA activity (Lopez-Martinez et al., 1995; Riddle et al., 1995). Development along the dorsoventral axis is mediated through epithelialemesenchymal interactions and does not appear to require an organizing center (Williamson et al., 2016). The Wnt7a gene is expressed selectively in the dorsal but not ventral ectoderm and is required for the establishment of a normal dorsal pattern in the limb mesoderm of mice, whereas Wnt5a is expressed in the ventral ectoderm of limb buds (Parr et al., 1993). The

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Wnt7a gene induces the Lmx1 gene, which is expressed selectively by the dorsal mesenchyme and encodes a transcription factor that appears to be essential for specifying the dorsal cell fates in the limb (Riddle et al., 1995). By day 14 in the rat, the forelimb has formed digital rays. Apoptosis of mesenchyme that does not integrate into the developing cartilage of the limb is utilized to further refine the rat limb as we know it (Williamson et al., 2016). Among the areas that undergo cell death, the most predominant are those located between the digits, which are called the “interdigital necrotic zones,” and at the peripheral margins of the limb, which are called the “anterior necrotic zone” and the “posterior necrotic zone,” and by day 16, the forelimb digits have fully separated (Christie, 1964; Hoar, 1981; Hebel, 1986; Williamson et al., 2016).

III. EXPERIMENTAL TERATOLOGY A. Historical Overview Experimental teratology in the modern sense began in the 1940s when Warkany, who is considered the father of experimental teratology, and his associates first called attention to the fact that maternal dietary deficiencies and X-rays could adversely affect the in utero development of mammals (Wilson, 1977). The major teratology events or experimental studies prior to the thalidomide catastrophe include embryonic lethality induced by X-rays in cats, disorder of limbs in pigs induced by lipid diet, microcephalia caused by X-ray irradiation of the pelvis, eye disorders in pigs due to hypovitaminosis A, masculinization of female fetuses in mice due to the action of androgens, report on maternal rubella viruse induced human malformations, decreased learning ability in rats caused by the administration of sodium bromide, experiments with alkylating agents and trypan blue that showed chemically induced teratogenicity, multiple malformations in fetuses caused by aminopterin, and disorders of the central nervous system and dentition caused by methylmercury (Ujhazy et al., 2012). Three human tragedies resulting from in utero exposure to drugs or chemicals led to the development and revision of testing guidelines. The thalidomide tragedy of 1961 resulted in the birth of infants with rare limb malformations, amelia (absence of the limbs), or various degrees of phocomelia (absence of the proximal portion of a limb) to mothers who took this sedative. Additional malformations seen with thalidomide treatment included defects of the external ears, facial hemangioma, atresia of the esophagus or duodenum, tetralogy of Fallot, and renal agenesis (Shepard, 1995a,b). This completely changed the perception regarding the vulnerability of the intrauterine embryo to outside influences.

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In the early 1970s, adenocarcinoma of the vagina began occurring in young women whose mothers had been treated with diethylstilbestrol during the first trimester of pregnancy. This raised concerns regarding adverse effects that were not evident until after puberty. The third event was the epidemic of organomercurial poisoning that resulted from the dumping of metallic mercury into Minamata Bay, Japan, and its conversion by aquatic plant life into methylmercury. The concern for the potential to affect postnatal development resulted in additional testing for postnatal behavioral changes (Christian, 2001). Wilson (Wilson, 1977) formulated six principles of teratology and they remain valid today: 1. Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with environmental factors. 2. Susceptibility to teratogenic agents varies with the developmental stage at the time of exposure. Wilson (Wilson, 1965a,b) graphically illustrated this concept, showing periods at which specific organs are more susceptible to teratogenic insult (Fig. 23.4). 3. Teratogenic agents act in specific ways (mechanisms) on developing cells and tissues to initiate abnormal embryogenesis (pathogenesis). 4. The final manifestations of abnormal development are death, malformation, growth retardation, and functional disorder. 5. The access of adverse environmental influences to developing tissues depends on the nature of the influence (agent). 6. Manifestations of deviant development increase in degree as dosage increases, from the no-effect to the totally lethal level. In addition, Wilson (1977) listed eight mechanisms of teratogenesis: mutation, chromosomal nondisjunction

A Brief Pulse of Teratogenic Treatment on the 10th Day of Gestation Would Result in the Following Incidence of Malformations 35% Brain Defects 33% Eye Defects 24% Heart Defects 18% Skeletal Defects 6% Urogenital Defects 0% Palate Defects

50 % Malformation

FIGURE 23.4 Hypothetical representation of how the syndrome of malformations produced by a given agent might be expected to change when treatment is given at different times. The percentage of animals affected as well as the incidence rank of the various types of malformations would be somewhat different from that shown for the 10th day if treatment were given instead on the 12th or the 14th day, for example. Data from Beaudoin, A.R., 1980. Embryology and Teratology. New York, Academic Press.

and breaks, mitotic interference, altered nucleic acid integrity or function, lack of precursors and substrates needed for biosynthesis, altered energy sources, enzyme inhibition, osmolar imbalance, and altered membrane characteristics. Schardein and Shepard (Shepard, 1995; Schardein, 2000) regularly update compilations of animal teratogens that provide overviews of the effects of test agents. After the thalidomide tragedy, the US Food and Drug Administration (FDA) issued more extensive testing protocols (segments I, II, and III) (FDA, 1966). In 1994, as a result of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), a new internationally accepted set of guidelines was issued that the FDA, the European Union, and Japan accept (ICH, 1994; ICH, 2005). These guidelines cite recommendations for flexible study designs, kinetics, requirements for mechanistic studies, and essentially equal emphasis on all endpoints (death, malformation, and growth). Additional guidelines for evaluating chemicals for teratogenic potential include those by the US Environmental Protection Agency (EPA) (EPA, 1998) and the Organization for Economic Co-operation and Development (OECD): Guidelines for the Testing of Chemicals (Section 4, Nos. 414, Teratogenicity, and 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) (OECD, 1981; 1996). Harmonization efforts are ongoing between the EPA and the OECD (Christian, 2001). Christian (2001) gives a listing of the majority of the regulatory guidelines in use as of this writing. Since 2000, teratology information services (TISs) have been developed in many countries throughout the world. In North America and Europe, collaborative organizations of TISs have emerged (Organization of Teratology Information Specialists/Mother to Baby and European Network of Teratology

Eye

40

Brain 30

Palate

Heart and Axial Skeleton

20

Urogenital

10

Aortic Arches 8

9

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11

12

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Information Services, respectively). See https:// mothertobaby.org/ and https://www.entis-org.eu). These services focus on advising pregnant women, women planning pregnancy, women who are breastfeeding, and their families and health care providers on the reproductive safety or risk of prenatal exposures. These include medicinal drugs, drugs of abuse, chemicals, infections, and occupational and environmental agents. The most common treatment period to evaluate teratogenicity requires exposure from implantation to closure of the hard palate or from gestation day 6 through day 17 in the rat. Studies to evaluate effects on embryoefetal development are required in two species: one rodent and one nonrodent; usually rat is used as the primary species and rabbit as the secondary species for reproductive toxicity testing. The animal species selected for testing of vaccines should demonstrate an immune response to the vaccine. The ICH guidelines (ICH, 1994; ICH, 2005) state that the rat is the most often used rodent species for reasons of practicality, general knowledge of pharmacology in this species, the extensive toxicology data usually available for interpretation of nonclinical observations from development of the pharmaceutical, and the large amount of historical background data. The rat has the following characteristics that make it a suitable species for evaluating effects on embryoefetal development: it is relatively disease resistant, withstands operative procedures well, and has a short reproductive cycle (estrus stage can be determined) and a high breeding rate with a relatively short gestation period and good litter size. The fetuses have a low spontaneous malformation rate and are of sufficient size (Beaudoin, 1980). Historical data are available in the scientific literature and in a compilation of historical control data from 1992 to 1994 (MARTA/MTA, 1995), but the most relevant data are control data generated in the specific laboratory using the same animal strains, scientific nomenclature, and technical conditions. An internationally developed glossary of terms for structural developmental abnormalities, which gives developmental findings, synonyms, and definitions, has also been published (Wise et al., 1997). The disadvantage of using the rat for developmental and reproductive toxicity testing include different placentation (e.g., timing, inverted yolk sac); dependence on prolactin as the primary hormone for establishment and maintenance of early pregnancy, which makes them sensitive to some pharmaceuticals (e.g., dopamine agonists); high sensitivity to pharmaceuticals that disrupt parturition (e.g., nonsteroidal antiinflammatory drugs, in late pregnancy); less sensitivity than humans to fertility perturbations; and limited application for humanized monoclonal antibodies due to limited or no pharmacologic activity, limited or no binding, and significant antidrug immune response (ICH, 2017).

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B. Breeding Pregnant females used for embryoefetal development studies may be obtained in one of three ways: (1) by purchasing timed pregnant females from a commercial supplier, (2) by purchasing males and females from a commercial supplier and breeding them in-house, and (3) by maintaining a breeding colony. The ICH guidelines (1994) state “that within and between studies, animals should be of comparable age, weight, and parity at the start: the easiest way to fulfill these criteria is to use animals that are young, mature adults at the time of mating with the females being virgin.” Strains with low fecundity should not be used. The amount of background data available and the experience of the laboratory with specific rat strains will determine the final choice; however, the ICH guidelines (1994) state that “it is generally desirable to use the same species and strain as in other toxicological studies.” If mating is done in-house, the stage of the estrus cycle (proestrus, estrus, metestrus, or diestrus) can be determined by evaluating vaginal smears of cells from the vaginal epithelium (Beaudoin, 1980; Taylor, 1986; Christian, 2001). Rats in the proestrus stage are placed for overnight breeding. Rats in estrus are paired with a male if bred for short periods (2e4 h) because ovulation in the rat occurs spontaneously near the end of the estrous period, the period when the female is receptive to the male. The shorter mating period is used to reduce interlitter variability. Males and females are usually cohabitated in a 1:1 ratio, but males may be cohabitated with up to five females. Insemination is confirmed by the presence of spermatozoa in a vaginal smear or the presences of a vaginal plug. The vaginal plug is a coagulated mass of semen, which may be found lying on the bottom of the cage (Beaudoin, 1980; Taylor, 1986). The day of insemination is usually considered gestation day 0, but day 1 is also used. The gestation period in the rat is 22 days (Hoar and Monie, 1981; Barrow, 2000). When evaluating the female for the estrous cycle stage or for the presence of sperm in the vagina, care should be taken to avoid excessive stimulation of tissues because this can disrupt the estrous cycle and produce a pseudopregnancy with a duration of 12e16 days before the resumption of the normal cycle (Beaudoin, 1980).

C. EmbryoeFetal Development Study Design The aim of the study is to detect adverse effects on the pregnant female and the development of the embryo and fetus consequent to exposure of the female to the drug/test material evaluated for toxicity from implantation to closure of the hard palate. The embryoefetal developmental toxicity study is designed to assess enhanced maternal toxicity relative to that in nonpregnant females, embryoefetal death, altered growth, and

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structural changes. Terms like “embryo toxicity” and “fetotoxicity” relate to the time point/period of induction of adverse effects, irrespective of the time of detection (ICH, 2005; ICH, 2017). In the rat, the ICH guidelines (ICH, 1994; ICH, 2005; ICH, 2017) for the testing of drugs require treatment from implantation (day 6) of the embryo until closure of the hard palate (day 17) and group sizes that will yield between 16 and 20 L (minimum of 16) to evaluate. Below 16 L per evaluation it may be that study results become inconsistent; above 20e24 L per group, it is believed that consistency and precision are not greatly enhanced. The EPA (1998) guidelines and OECD drafts for chemicals modify the traditional study design and require treatment to include the fetal period (gestation days 17e20). The extended duration of treatment is intended to improve the sensitivity of the test to detect toxic influences on fetal development (Barrow, 2000). Satellite animals that are not included in the fetal evaluations of the experiment are generally needed for toxicokinetic evaluations in the rat because of the volume of blood that is collected, and the possible stress induced in the animal has the potential to affect pregnancy outcomes. 1. Dose Selection Selection of the correct doses is a critical issue. Usually there are at least three test agent treatment groups and appropriate control groups, a total of four dose groups. Vehicle controls are normally included; however, in some cases an untreated or sham-treated control group and a positive control group may be included. It is recommended that control animals be dosed with the vehicle at the same rate as test group animals. When different treatment volumes are used for the different treatment groups, the control group is normally given the volume administered to the highest treated group. The selection of the high dose should be based on data from all available studies (pharmacology, acute and repeat dose toxicity, and kinetic studies), and doses should be used that are similar to those used in general toxicity studies, thus allowing for comparison. A repeated-dose toxicity study of about 2e4 weeks’ duration provides a close approximation to the duration of treatment in segmental designs of reproductive studies. Dose selection is usually based on results of a dose range-finding study in pregnant animals with about five or six animals per group. The same in-life and cesarean examinations as for the embryoefetal development study are used except that fetuses are evaluated only externally. According to the ICH guidelines, the high dose is expected to induce minimal maternal toxicity, as indicated by reductions in body weight gain, specific target organ toxicity, changes in hematology or clinical chemistry parameters, and/or an exaggerated pharmacological response. However, maternal

effects should not be overly severe so as to limit the number of fetuses (not resorbed, available intact) to be evaluated. The kinetics of the drug, both systemic exposures (area under the concentrationetime curve [AUC]) and peak plasma levels (Cmax) also should be considered, as both can influence developmental effects. Ideally, the doses selected should provide systemic exposures with an adequate margin above the estimated human therapeutic exposure. It should be noted that for some compounds, particularly cytotoxic oncolytics, a “safety” margin may not be achieved. The physicochemical properties of the test substance may impose practical limitations on the amount of drug that can be administered, and under most circumstances, 1 g/kg/day should be an adequate dose limit. The ICH guidelines (1994, 2005) state that “lower dosages should be selected in a descending sequence, the intervals depending on kinetic and other toxicity factors. While it is desirable to be able to determine a no observed adverse effect level, priority should be given to setting dosage intervals close enough to reveal any dosage-related trends that may be present.” In these kinds of studies, dose responses may be steep, and wide intervals between doses would be inadvisable. If an analysis of doseeresponse relationships for the effects observed is attempted in a single study, it is recommended to use at least three dose levels and appropriate control groups. If in doubt, a fourth dose group should be added to avoid excessive dosage intervals. Such a strategy should provide a “no observed adverse effect level” for reproductive aspects. The ICH further cautions that any dose response may be steep. 2. Routes of Administration, Frequency of Dosing, and Dose Volume For drugs, the proposed human route of administration is preferred for embryoefetal development studies, provided this route provides an adequate exposure compared with the estimated human therapeutic exposure. For chemicals, the principal route of human exposure should be used (Barrow, 2000). Gavage administration is the preferred method of oral treatment for embryoefetal development studies rather than admixture in the diet and has the advantage of allowing the exact predefined dose to be administered to each animal. The daily dose should be adjusted if possible according to the most recent body weight. Administration by admixture in the diet or by dissolution in the drinking water is the preferred method of oral administration for multigenerational studies with chemicals. Maternal food consumption and body weight increase during gestation; therefore, it is necessary to vary the concentration in the diet to maintain a constant dose level relative to body weight. Pregnant rats have

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particular nutritional requirements that can have a significant influence on pregnancy outcomes (Wilson, 1977); therefore, the percentage of drug in the diet must be considered or vitamin deficiencies may result. When the route of exposure is by inhalation, particularly with pregnant rats, whole-body exposure is preferred because of the stress associated with the restraint necessary in nose-only exposure (Astroff et al., 2000). Other acceptable methods of drug administration for the rat include intraperitoneal, intramuscular, subcutaneous, intravenous, intraarterial, or intradermal injections. Acceptable volumes for the rat are given in Table 23.2. The first value in the range is considered to be an optimal dose volume for most toxicology protocols, taking into account both animal tolerance and the characteristics of the dosing preparation. All volumes are expressed in milliliters per kilogram unless otherwise noted. Special consideration should also be given to pH, irritation potential, osmolality, and pharmacologic and toxicologic effects of the drugs to be administered. 3. In Vivo Observations The dams are monitored daily for clinical signs and mortality. Clinical observations may be increased to include a predose and a postdose time point during the treatment period. The time of expected maximum plasma concentration (tmax) is often used as the postdose time point. Body weights are measured at least twice weekly, and body weight change is determined. The ICH guidelines state “daily weighing of pregnant females during treatment can provide useful information.” Food consumption is usually measured concomitant with body weight; however, the ICH guidelines (1994, 2005) require only weekly measurements. Water consumption can be measured if there are scientific reasons to do so, but it is not normally done. It is recommended that observations that have been proved of value in other toxicity studies be recorded for evaluation. 4. Toxicokinetic Parameters Developmental effects depend on the gestational stage at the time of the exposure and the exposure level. Some effects seem to relate to the peak plasma levels achieved (Cmax), whereas other effects occur at lower levels related to AUC and sustained exposures. Nau (1985) showed in

rodents (mice) that with valproic acid, a high maternal plasma concentration at specific stages of development resulted in exencephaly in the fetuses, whereas sustained exposure to lower levels resulted in embryonic resorptions. Therefore, it is important to consider both the expected Cmax and the AUC when selecting doses for an embryoefetal development study. Many of the physiological changes that occur during pregnancy may potentially influence the toxicokinetic and pharmacodynamic properties of the test substance and its metabolites. These changes include reduced gastric secretion, increased transit time, increased plasma and extracellular fluid volume, increased body fat, decreased xenobiotic hepatic transformation, increased kidney function, and differences in protein binding (Clarke, 1993). In rats, many blood parameters change considerably during pregnancy, including drastic decreases in red blood cell counts, hemoglobin concentration, and hematocrit, which strongly suggest that hemodilution occurs in rat pregnancy. Total protein, albumin, glucose, cholesterol, high-density lipoprotein cholesterol, triglycerides, and phospholipids change significantly during pregnancy. Electrolyte values change during pregnancy in the rat as shown by significant declines in sodium and chloride levels (de Rijk et al., 2002). In summary, the physiological changes that occur during pregnancy should be considered when interpreting the data. 5. Dam Necropsy and Collection of Fetal and Placental Data In embryoefetal development studies, dams are typically euthanized on gestation day 20 or 21 by carbon dioxide inhalation and are given a detailed macroscopic necropsy to detect lesions in the maternal organs. It is recommended to preserve organs with macroscopic findings for possible histological evaluation and corresponding organs of sufficient controls for comparison. The uterus and ovaries are excised (Taylor, 1986; Barrow, 2000; Christian, 2001). The number of corpora lutea in each ovary is counted and recorded. The uterus of female rodents that do not appear to be pregnant can be examined either by pressing the uterus between glass plates and examining it for the presence of implantation sites or by staining it with ammonium sulfide (Kopf et al., 1964; Salewski, 1964). The weight of the gravid uterus may be recorded (required for chemical testing),

TABLE 23.2 Recommended Dosing Volumes. Intramuscular (mL/Injection Gavage Intravenous (Bolus)a Intraperitoneal Subcutaneous Site)

Intranasal (mL) Intradermal (mL/ Site)

10e30

0.1

5e10

5e10

5e10

0.1e0.3

a

0.05e0.1

Bolus injections are generally given over approximately 1 min. The maximum volume for slow intravenous infusions lasting up to 20 min is 4 mL/kg/min. Maximum volumes for continuous intravenous infusion are 4 mL/kg/h in rats.

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and this value can be used to calculate the corrected body weight (terminal body weight minus the gravid uterine weight), which can be useful for evaluation of maternal body weight independent of uterine effects. The uterine horns are cut open, and the chorionic and amniotic sacs are opened to expose the fetuses. The number and location of each implantation site are recorded. The status of each implant site (live and dead fetus, early or late resorption) is recorded. The number of implantations is compared with the number of corpora lutea to determine preimplantation loss. An early resorption is a conceptus in which only placental remnants (metrial gland) are visible, and a late resorption has both placental and fetal remnants visible. For apparently nonpregnant rats or mice, ammonium sulfide staining of the uterus might be useful to identify periimplantation death of embryos. A live fetus is pink in color and will respond to stimulation. A dead fetus is often pale to tan in color and is not responsive to stimulation but does not demonstrate marked autolysis. Fetuses with marked autolysis are considered to be late resorptions (Taylor, 1986; Barrow, 2000; Christian, 2001). Individually identified fetuses are separated from the placenta by cutting the umbilical cord, and any membranes are removed. The fetuses are then blotted dry and individually weighed. The individually identified placentas from live fetuses are then removed from the uterus, and after any remaining membranes are removed, they are individually examined for abnormalities in appearance and weighed. Placental weights normally correlate with fetal weights. Abnormal placentas can be evaluated microscopically. 6. Fetal Evaluations a. External Evaluations Each fetus is carefully examined for any external abnormalities, including palatine abnormalities, and the sex is determined by inspection of the analegenital distance. Taylor (1986) gives a detailed method for conducting an external evaluation. Fetal examinations are singled out in the EPA guidelines as requiring blinded examination in which the technician is unaware of the identity of the fetus with regard to treatment group or control at the time of the examination (Barrow, 2000). b. Visceral Evaluations The ICH guidelines (1994) state that a minimum of 50% of fetuses (every other fetus in the uterus) from each litter should be examined for visceral or soft-tissue alterations. Examinations can be performed by serial sectioning (Wilson, 1965a,b) or by a combination of serial sectioning of the head and fresh gross microdissection of the thorax and abdomen (Barrow and Taylor, 1967; Staples, 1974; Stuckhardt and Poppe, 1984; Barrow, 1990; Christian, 2001). In the microdissection technique, the thoracic and

abdominal cavities are carefully opened under a microscope, and the organs and major vessels of the heart are examined in situ. The heart is cut using scissors, with the first cut beginning to the right of the ventral midline surface at the apex and extending anteriorly and ventrally into the pulmonary artery. The incision is opened, and the papillary muscles, the tricuspid valve, and the three cusps of the semilunar valve of the pulmonary artery are evaluated. The interventricular septum is examined for any defects. The second cut is made starting to the left of the ventral midline surface at the apex and extending through the left ventricle into the ascending aorta. The mitral valve (bicuspid valve) and the three cusps of the semilunar valve of the aorta and papillary muscles are observed in addition to the interventricular septum. Igarashi (Igarashi, 1993) published a gelatin-embedding-slice method to evaluate cardiac malformations. The kidneys are sectioned to allow for an examination of the renal papillae. A frozen-sectioning method for the evaluation of the head permits the complete visceral examination to be completed at the time of cesarean section (Astroff et al., 2002). The fetuses are eviscerated to aid in clearing and staining of the skeleton if the skeleton is to be evaluated on the same fetus. The fetuses must be skinned if double staining of the skeleton for bone and cartilage is to be done (Young et al., 2000). Fetuses need not be skinned if only ossification sites are stained using alizarin red S (Kawamura et al., 1990). c. Skeletal Evaluations Before skeletal evaluation can begin, the fetal carcasses that were fixed in 95% ethanol must be dehydrated, macerated, stained with alizarin red S, and cleared. The stained specimens can be stored indefinitely in glycerol. This results in specimens in which all ossified parts of the skeleton are stained red and are strikingly visible through the other tissues that have become completely transparent. Alternatively, the cartilage can also be stained with Alcian blue (Inouye, 1976; Whitaker and Dix, 1979; Kimmel and Trammell, 1981; Young et al., 2000). Fetal rat specimens, previously stained only for bone, can be overstained for cartilage according to the methods of Yamada (Yamada, 1991). Automatic processors are available (Rousseaux, 1985; Conway and Erb, 1995). The EPA guidelines for chemicals suggest an examination of the fetal cartilage in addition to the ossified bones (Barrow, 2000). After staining, the ossified skeleton can be examined and checked for the presence, size, and shape of every bone (Taylor, 1986; Barrow, 1990; Christian, 2001). Also, if the maceration step with potassium hydroxide is carefully controlled and kept short, the cartilaginous areas of the skeleton remain partially visible as light shadowy regions (Barrow, 2000; Christian, 2001); this allows for an accurate distinction to be made between

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agenesis of a bone, a malformation, and developmental delay of ossification, a variation. An atlas of the fetal skeleton on gestation day 20 for the Crl:CD rat is available (Menegola et al., 2001), and Yasuda and Yuki (Yasuda and Yuki, 1996) published a comparable atlas of the skeleton of the gestation day 21 Wistar rat. Data on the sequence of fetal ossification in the Longe Evans strain from gestation day 14 to 21 are described by Wright et al (Wright et al., 1958). X-ray imaging has also been used for skeletal examination of fetuses. Counting and recording numbers of selected ossification sites in each skeleton provides a method to identify delays in ossification and analyze them statistically for significance. According to the ICH guidelines (ICH, 1994; ICH, 2005), it must be possible to relate all findings by different techniques (i.e., body weight, external inspection, visceral and/or skeletal examinations) to a single specimen to detect patterns of abnormalities. The examination of mid- and low-dose fetuses for visceral and/or skeletal abnormalities may not be necessary where the evaluation of the high-dose and the control groups did not reveal any relevant differences. It is advisable, however, to store the fixed specimen for possible later examination. If fresh dissection techniques are normally used, difficulties with later comparisons involving fixed fetuses should be anticipated. 7. Evaluation Criteria Developmental toxicity is manifested by death, structural abnormalities, and developmental delay. Malformations are structural defects that are incompatible with life or of major physiological consequence, represent gross structural changes, and are rare in occurrence. Variations are structural alterations with no significant biological effect. Ossification retardation, which represents a delay in the normal amount of ossification, or decreases in ossification sites are classified as variations. Teratogenicity is characterized by a significant increase in: (1) the percentage of malformed fetuses per litter, (2) the number and percentage of litters with malformed fetuses, and (3) the number of fetuses or litters with a particular malformation that appears to increase with dose. However, the level of concern regarding these findings is increased in the absence of causal maternal toxicity. Significant dose-related increases in the incidence of variations or reductions in fetal weights are considered an indication of developmental toxicity. A significant increase in the number of dead or resorbed embryos/fetuses is indicative of embryo/fetal lethality. Maternal toxicity is assessed by the evaluation of morbidity, body weight gain, food and water consumption, and lesions in the dam. Material toxicity may directly or indirectly influence the development of the embryos and should be considered when evaluating developmental toxicity. Maternal body weight gain

883

during the second half of gestation in the rat is influenced by the size and rate of growth of the litter. Litter influences on maternal weight can be assessed by subtracting the weight of the gravid uterus (determined at necropsy) from the terminal body weight of the dam. Treatment-related effects on gestation length have an influence on the relative state of development of the fetus at the time of examination or the pups at birth and need to be considered in the evaluation of fetus or pup weight and fetal or postnatal development. When assessing effects on embryoefetal development, one particular difficulty arises when fetal toxicity is observed at dose levels that were also toxic for the mother. It cannot be assumed that developmental toxicity was secondary to maternal toxicity unless such a relationship can be demonstrated either de novo or from published precedence. One way of doing this is to assess the degree of concordance between the severity of toxicity seen in the individual dams and the effects on their litters. Also, the consistency between studies can provide further evidence of an adverse effect of the pharmaceutical (e.g., increased fetal lethality seen in a rodent embryoefetal development study consistent with decreased live litter sizes in the pre- and postnatal development study). It is important to consider the exposure at which specific effects were seen across studies and species. Knowledge of the mechanism of reproductive or developmental effects identified in animal studies can help to explain differences in response between species and provide information on the human relevance of the effect (e.g., rodent-specific effects of prostaglandin synthetase inhibitors on cardiovascular fetal development). In the presentation of data in report form, the entire spectrum of observed effects needs to be presented. The key to good reporting is the tabulation of individual values in a clear concise manner to account for all animals that are being assessed. Because the data are derived from offspring that are often not directly treated, clear and concise tabulation that permits any individual animal from initiation to termination to be followed should be presented. Presentation of fetal abnormality findings should utilize terminology that is consistent and easily understood. Interpretation of study data should rely primarily on comparison with the concurrent control group. Historical control/reference data are most useful when an interpretation of the data relies on the knowledge of variability within the larger control population and specifically among control groups in previous studies (ICH, 2017). The conclusion should concisely state the effect of the compound on the fetuses and should include any maternal toxicity at each of the specific dose levels. Risk assessment is defined as consisting of hazard identification, doseeresponse assessment, exposure assessment, and risk characterization (Christian, 2001).

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Guidelines for developmental toxicity risk assessment were issued by the EPA in 1996 and provide guidance for interpreting, analyzing, and using the data from developmental toxicity studies. The FDA considers risk on a risk/benefit basis, and the conclusions regarding this risk/benefit consideration are published on the label (package insert) of the drug or biologic, which is given a pregnancy label category of A, B, C, D, or X (Christian, 2001). The five-letter classification system (A, B, C, D, X) was then introduced in 1979 by the FDA. This system was developed based on the amount and quality of research done on the medication, not the safety of the medication in pregnancy or lactation. This system, while relied upon to guide treatment of pregnant and lactating women, has been misinterpreted and is being misused. In June 2015, the FDA shifted from the A, B, C, D, and X categorization system to a new system for all drugs that enter the market after this time, which requires removal of the old categorization from all drug product labeling for drugs on the market over the next 3e4 years. The pregnancy subsection of the label is now presented under the following subheadings: Pregnancy Exposure Registry, Risk Summary, Clinical Considerations, and Data (Ramoz and Patel-Shori, 2014) . 8. Statistical Analysis Developmental and reproductive toxicity studies usually show a distribution of response that does not follow a normal distribution, but can vary from any continuous to any discrete distribution. So the statistical method used should be informed. When employing inferential statistics (determination of statistical significance) the basic unit of comparison should be used. The experimental unit is a concept that is oftentimes misinterpreted but refers to the units that have been randomized and treated. Therefore, cesarean and fetal data should be calculated for the litter as the unit of measure; study result inferences are made back to the mother, not to fetuses. This is because the pregnant females have been allocated to different dose groups (not the fetuses or neonates) and the development of individual offspring in a given litter is not independent. The responses of individual offspring in a given litter are expected to be more alike than responses of offspring from different litters. So in summary, the litter should be the experimental unit of comparison (Jensh et al., 1970; Haseman and Hogan, 1975; ICH, 1994; ICH, 2005; ICH, 2017). According to the ICH guidelines (1994), the use of statistics should support the interpretation of the results based on biological significance. Statistical analysis can be conducted on maternal body weight, maternal body weight change, and food and water consumption over periods of interest, that is, before dosing, during the dosing period, and after the dosing period. Fetal body weights by sex, sex ratio, and placental weights may be

analyzed by using appropriate statistical tests. Statistical analysis can be conducted on the data collected at necropsy, including number of corpora lutea, implant sites, live and dead fetuses, and resorptions (early and late may be individually analyzed). The calculated values of the percentage of preimplantation loss ([number of corpora lutea minus number of implant sites divided by number of corpora lutea]  100) and postimplantation loss ([number of implant sites minus number of viable fetuses divided by number of implant sites]  100) can also be analyzed. Numbers of specific sites of ossification can also be analyzed statistically as a measure of developmental delay. The percentage fetal incidence and litter incidence of external, visceral, or skeletal malformations and variations can also be evaluated.

D. In Vitro Methods Many different culture systemsdincluding cell culture, tissue culture, organ culture, and whole-embryo culturedhave been proposed as in vitro alternatives to existing in vivo assays for developmental toxicity. Most have been developed and tested in only a single laboratory or lack large-scale validations (Walmod et al., 2002). Teratogenesis screening systems can provide a rapid, cost-effective method of assessing potential teratogens that requires only small amounts of test material. However, in vitro assays for developmental toxicity are considered a useful method to screen structurally related compounds for teratogenic potential when at least one compound from the series has been tested in vivo. New et al. (New, 1971; New et al., 1976) described the technique for the culture of postimplantation rodent embryos from the early head-fold stage (9.5 days of gestation) for 48 h (at the approximately 25-somite stage). They found that the rates of growth and differentiation of all the embryos grown for up to 48 h in culture under optimum culture conditions were similar to those of littermates in vivo (New et al., 1976). Brown and Fabro (1981) developed and Van Maele-Fabry et al. (1990) modified an objective scoring system that provides a precise measure of morphological development, aids in the detection of retardation or dysmorphogenesis, and permits a quantitative comparison of growth and development. Studies have shown that whole-embryo cultures have a high predictive capacity for teratogens (New, 1990), and an interlaboratory evaluation of embryo toxicity that uses whole-embryo culture has been published (Piersma et al., 1995). Assay systems using artificial hydra “embryos” have been used to evaluate developmental toxicity. In this system, a differential assessment of toxicity of a compound to the adult and to regenerating artificial hydra embryos permits the determination of a developmental hazard index determined by calculating the

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REFERENCES

minimal affective concentrations in the adult hydra and developing artificial hydra embryo (A/D ratio) (Johnson et al., 1982; Johnson et al., 1988; Mayura et al., 1991). The frog embryo teratogenesis assay Xenopus (FETAX) has been used to identify the potential developmental toxicity of compounds. FETAX is a 96-h wholeembryo test that uses the embryos of the frog Xenopus laevis. The embryos are cultured with and without an exogenous metabolic activation system. The endpoints measured include mortality, malformation, and growth. The 96-h median lethal (LC50) and teratogenic (EC50) concentrations are determined. From these values, a teratogenic index (TI) is determined (TI ¼ 96-h LC50/ 96-h EC50). The TI provides a measurement of the separation between mortality and malformation concentratione response curves. The separation between the two curves is used to indicate significant teratogenic hazard. The length data are used to calculate a minimum concentration to inhibit growth (Fort et al., 1996, 2000). Embryonic development is highly sensitive to changes in cell death, growth, migration, and differentiation, and by using comparable endpoints with cell culture systems, it may be possible to predict developmental toxicity. Cell culture techniques have been developed to evaluate teratogenic potential based on the inhibition of differentiation by using limb buds, measuring chondrogenesis (Kistler, 1987; Tsuchiya et al., 1987), and midbrain cells, determining the number of neuron foci (Flint and Orton, 1984; Tsuchiya et al., 1991). Cultures of pluripotent embryonal stem cells derived from blastocysts that are able to differentiate into a variety of embryonal tissues, retain the euploid chromosome constitution, and proliferate rapidly have been developed and used to evaluate cytotoxicity; such systems appear to hold promise as an in vitro teratogenesis screen if differentiation of these pluripotent cells can be quantified (Laschinski et al., 1991). Organ cultures of explanted palates have been used to study normal palate formation and the effect of compounds/drugs on palatogenesis in vitro (Shiota et al., 1990; Mino et al., 1994). At the 17th meeting of the European Centre for the Validation of Alternative Methods Scientific Advisory Committee in 2001, three methods were endorsed as “scientifically validated” and ready for consideration for regulatory acceptance and application. These tests include, embryonic stem-cell test, in which two permanent murine cell lines are used, D3 cells representing embryonic tissue and 3T3 fibroblast cells representing adult tissue; micromass test, a test based on rat embryonic limb mesenchyme cells, when cultured in small volumes at high density from foci of differentiating chondrocytes within a background of nondifferentiating cells; and whole embryo culture test, a teratogen screening system using whole mouse, rat, and rabbit embryos cultured

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for short periods during the phase from fertilization to the end of organogenesis (Ujhazy et al., 2012). The growth in the fields of informatics and computational toxicology has led to automated, high-throughput testing methods that provide less expensive, faster, and more precise assessments of developmental risks. Computational approaches, including in silico structureeactivity relationship models as well as biologically or physiologically based pharmacokinetic models, are being developed for evaluation of compounds causing embryoefetal development risk. These models use large amounts of input data, including physicochemical properties of the test agent, as well as many parameters related to the absorption, distribution, and metabolism within pregnant animals and parameters related to placental perfusion and transplacental transport. Using the massive amount of data available through databases such as EPA’s ToxCast, these approaches have become increasingly accurate for known developmental toxicants (DeSesso, 2017).

Acknowledgments This chapter builds upon the related chapter in the second edition of this text. Portions of text from the previous edition have been retained, updated, and added to as needed. The authors of the present chapter are greatly indebted to the authors of the previous editions. The second edition author is Carol Erb.

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male urogenital tract: role of androgens, mesenchymal-epithelial interactions and growth factors. J. Androl. 13, 465e475. Davies, J., Glasser, S.R., 1968. Histological and fine structural observations on the placenta of the rat. Acta Anat. 69 (4), 542e608. Davies, J.A., Bard, J.B., 1996. Inductive interactions between the mesenchyme and the ureteric bud. Exp. Nephrol. 4 (2), 77e85. Davies, J.a.H., H., 1971. Comparative Embryology of Mammalian Blastocysts. University of Chicago Press, Chicago. de Rijk, E.P., van Esch, E., Flik, G., 2002. Pregnancy dating in the rat: placental morphology and maternal blood parameters. Toxicol. Pathol. 30 (2), 271e282. DeSesso, J., 1993. Cardiovascular Development. DeSesso, J., 2017. Future of developmental toxicity testing. Curr. Opin. Toxicol. 3, 1e5. Dickmann, Z., 1969. Shedding of the zona pellucida. Adv. Reprod. Physiol. 4, 187e206. Dickmann, Z., Noyes, R.W., 1961. The zona pellucida at the time of implantation. Fertil. Steril. 12, 310e318. Domenech Mateu, J.M., Orts Llorca, F., 1976. Arterial vascularization of the sinuatrial node in the embryonic rat heart. Acta Anat. 94 (3), 343e355. Edwards, J.A., 1968. The external development of the rabbit and rat embryo. Adv. Teratol. 3, 239e263. Eibs, H.G., Spielmann, H., Jacob-Muller, U., Klose, J., 1982. Teratogenic effects of cyproterone acetate and medroxyprogesterone treatment during the pre- and postimplantation period of mouse embryos. II. Cyproterone acetate and medroxyprogesterone acetate treatment before implantation in vivo and in vitro. Teratology 25 (3), 291e299. Ellington, S.K., 1985. A morphological study of the development of the allantois of rat embryos in vivo. J. Anat. 142, 1e11. Enders, A.C., 1971. The Fine Structure of the Blastocyst. University of Chicago Press, Chicago. Enders, A.C., Schlafke, S., Welsh, A.O., 1980. Trophoblastic and uterine luminal epithelial surfaces at the time of blastocyst adhesioni in the rat. Am. J. Anat. 159, 59e72. EPA, United States Environmental Production Agency, 1998. Health Effects Test Guidelines: Prenatal Developmental Toxicity Study. Office of Prevention. Pesticides and Toxic Substances. OPPTS. Everett, J.W., 1933. Structure and function of the yolk-sac placenta in Mus norvegicus albinus. Proc. Soc. Exp. Biol. Med. 31, 77e79. Everett, J.W., 1935. Morphological and physiological studies of the placenta in the albino rat. J. Exp. Zool. 70 (2), 243e285. Fabro, S., 1973. Passage of Drugs and Other Chemicals into the Uterine Fluids and Preimplantation Blastocyst. Raven Press, New York. Fabro, S., McLachlan, J.A., Dames, N.M., 1974. Chemical exposure of embryos during the preimplantation stages of pregnancy: mortality rate and intrauterine development. Am. J. Obstet. Gynecol. 148, 929e938. Fallon, J.F.a.C., G.M., 1977. Polarising Zone Activity in Limb Buds of Amniotes. Cambridge University Press, New York. FDA, U.S. F.a.D.A., 1966. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use. FDA, Rockville, MD. Ferguson, M.W., 1977. The mechanism of palatal shelf elevation and the pathogenesis of cleft palate. Virchows Arch. A Pathol. Anat. Histol. 375 (2), 97e113. Ferguson, M.W., 1978. Palatal shelf elevation in the Wistar rat fetus. J. Anat. 125 (Pt 3), 555e577. Fleming, T.P., 1987. A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst. Dev. Biol. 119 (2), 520e531. Flint, O.P., Orton, T.C., 1984. An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol. Appl. Pharmacol. 76 (2), 383e395. Florez-Cossio, T.J., 1975. Studies on the development of the albino-rat notochord (author’s transl). Anat. Anz. 137 (1e2), 35e55.

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