Reproductive and Developmental Toxicology Carole A Kimmel PhD Judy Buelke Sam

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2.0 Overview of Normal Reproduction and Development Relevant to Toxicology

An appreciation of normal reproductive biology and development is important for understanding and evaluating the toxic effects of chemical and physical agents. This section provides an overview of these processes and a discussion of issues that are important in toxicology. Reference texts on reproductive and developmental biology and toxicology should be consulted for more detail (11-19).

2.1 Gametogenesis

The process by which germ cells (ova and sperm) are produced in both the male and female mammal is termed gametogenesis (Fig. 3.2) (20). The germ cells originate in both sexes from cells lining the embryonic yolk sac and, during the sixth week of gestation in humans, migrate into the gonadal ridges and become spermatogonia or oogonia, residing in the testis or ovary, respectively. Although many of the processes are similar in males and females, several differences are important for reproductive and developmental toxicology.

Gametogenesis Males And Females
Figure 3.2. Gametogenesis in the male and female (used with permission of W. B. Saunders Co., and modified from Ref. 20).

Spermatogonia are dormant until after birth and up to the time of puberty when they begin to increase in number through mitosis and produce primary spermatocytes. These further divide by a process called meiosis to form two haploid (half the chromosome number) secondary spermatocytes, that further divide to form four spermatids. The process by which spermatids are transformed into mature sperm is called spermiogenesis. Mature sperm are released into the lumen of the seminiferous tubule of the testis. This transformation from spermatogonia to mature sperm, called spermatogenesis, lasts for 60 days in rats or 80 days in humans and continues throughout the life of the male, as long as undifferentiated A-type spermatogonia are present (21). Agents that affect spermatogonia are the most devastating because the effects may be permanent, whereas effects on later stages of spermatogenesis are more likely to be transient. For example, agents that affect DNA synthesis and cell division, such as the chemotherapeutic agents cyclophosphamide and cytosine arabinoside, affect spermatogonia and early spermatocytes (22, 23), whereas brief exposure to heat affects spermatocytes and early spermatids (24) and temporarily results in reduced fertility which is restored as later spermatocytes mature.

Oogenesis is the process of the development of mature ova in the female mammal. Before birth, the oogonia proliferate by mitotic division and form primary oocytes surrounded by follicular cells. The oocyte and its follicle cells are called a primary follicle. The first phase (Prophase I) of meiosis in the oocyte begins before birth, and cells are then arrested until around puberty. At the onset of each estrous cycle, a pool of primordial follicles is recruited into a growing pool of primary follicles, one or more of which go on to form the large Graafian follicle and become an ovulatory follicle. In rodents and other polytocous species, several primary follicles become ovulatory follicles. No more primary oocytes are formed after birth. There is a normal process of atresia of oocytes throughout the prenatal and postnatal periods decreasing from approximately 7 million at 5 months of gestation to approximately 2.8 million at birth, with approximately 300,000 remaining at puberty (25). These continue to decrease so that no more follicles are present by around age 50. Damage to oogonia or primary oocytes before or after birth may be permanent, and an agent that increases atresia of primary oocytes reduces the complement of total ova available for ovulation and possibly decreases the time to onset of reproductive senescence (see later). 2.2 Fertilization

Fertilization involves the penetration of the ovum, its surrounding layers of granulosa cells, and the acellular zona pellucida by the mature sperm in the upper reaches of the oviducts (Fig. 3.3) (26). Fertilization requires a mature sperm that has undergone capacitation during its traverse of the female reproductive tract. The events required in capacitation are not well understood, but require plasma membrane modifications, decreases in net negative surface charge, changes in lipid components, alterations in fluidity/mobility of membranes, increased ion permeability, and other internal modifications. In addition, the acrosome reaction, which may facilitate penetration of the granulosa cells, and activated motility, to allow penetration of the zona pellucida, occur. Factors that can alter capacitation, the acrosome reaction, or activated motility may all play a role in preventing normal fertilization (27).

Figure 3.3. Development of the human embryo in the reproductive tract from fertilization to implantation (used with permission of Raven Press from Ref. 26, p. 47).

Several genetic aspects are determined at fertilization. First, the diploid number of chromosomes is restored when the male and female pronuclei fuse and chromosomes from the two become the chromosomal complement of the embyro. Second, by gaining genetic material from two different individuals, genetic diversity is maintained. Third, sex is determined at fertilization, in that a sperm bearing a Y chromosome results in a genetically male individual (XY zygote), whereas an X-bearing sperm results in a genetic female (XX zygote). Finally, fertilization stimulates rapid cell division or cleavage to form the embryo. 2.3 Female Reproduction

Implantation Embryonic Development

Figure 3.3. Development of the human embryo in the reproductive tract from fertilization to implantation (used with permission of Raven Press from Ref. 26, p. 47).

The female reproductive system involves the ovaries, uterus, oviducts, cervix, vagina, and mammary glands. The function of these is controlled by a carefully regulated interaction between the hypothalmus, the anterior pituitary, and the ovary. The hypothalmus secretes gonadotropin-releasing hormone (GnRH) which results in follicle stimulating hormone (FSH) and luteinizing hormone (LH) release from the anterior pituitary. A preovulatory surge of FSH and LH from the anterior pituitary stimulates differentiation of the granulosa cells and further meiotic division of the primary oocytes to a pre-ovulatory state (Fig. 3.4) (25).

Figure 3.4. Endocrinology of the reproductive cycle in normal women. The dominant cycle structure, pituitary gonadotropins, gonadal steroids, and basal body temperature throughout the menstrual cycle are depicted. Note that in the follicular phase, the follicle enlarges as the serum estrogen rises. A midcycle gonadotropin surge heralds follicular rupture and release of the oocyte. Immediately after ovulation, the corpus luteum develops and secretes large amounts of progesterone with a resultant elevation in basal body temperature. In the absence of the conceptus, the luteal phase is 14 days long, and declining progesterone coincides with the onset of menses (used with permission of Raven Press from Ref. 25, p. 182).

Figure 3.4. Endocrinology of the reproductive cycle in normal women. The dominant cycle structure, pituitary gonadotropins, gonadal steroids, and basal body temperature throughout the menstrual cycle are depicted. Note that in the follicular phase, the follicle enlarges as the serum estrogen rises. A midcycle gonadotropin surge heralds follicular rupture and release of the oocyte. Immediately after ovulation, the corpus luteum develops and secretes large amounts of progesterone with a resultant elevation in basal body temperature. In the absence of the conceptus, the luteal phase is 14 days long, and declining progesterone coincides with the onset of menses (used with permission of Raven Press from Ref. 25, p. 182).

The ovary is comprised of an outer cortex, which includes the follicles, and an inner medulla. The granulosa and thecal cells of the follicle secrete estrogen in a modulated fashion during the cycle and control secretion of FSH and LH through a negative feedback mechanism. As the estradiol level rises, it stimulates the release of LH and FSH, and possibly has direct effects on the LH releasing hormone (LHRH) which is released in a pulsatile fashion from the mediobasal hypothalamus. Once ovulation occurs, the follicle forms the corpus luteum, and progesterone is secreted, which stimulates the development of a secretory endometrium in the uterus in preparation for implantation. Progesterone secretion also causes an increase in basal body temperature. If fertilization occurs, progesterone levels continue to rise. In humans, chorionic gonadotropin (hCG) secreted by the embryonic membranes is necessary to maintain the corpus luteum during early pregnancy. If fertilization does not occur, the ovum degenerates, and menses ensues.

Agents that interfere with the development of the reproductive system and the normal hormonal patterns necessary to regulate development may alter the intricate processes involved in a number of different ways. For example, the normal structure of the ovaries, uterus, oviducts, cervix, and vagina can be altered during development, resulting in interference with fertility and pregnancy. This was the case with the drug diethylstilbestrol (DES), a potent synthetic estrogen used in the 1950s and 1960s to prevent spontaneous abortion. Unfortunately, the drug was not effective in preventing labor but had profound effects on the development of the reproductive system in both boys and girls exposed before birth and produced a rare form of cancer (vaginal adenocarcinoma) in females not detected until after puberty (28). Synthetic androgens and antiandrogens also alter the structure of reproductive organs by interfering with the normal hormonal milieu during development. For example, ethinyl testosterone given to prevent spontaneous abortion resulted in masculinization (pseudohermaphrodism) of female offspring (29), and other androgenic compounds (e.g., danazole, methadriol, and methyltestosterone) prescribed for endometriosis, alopecia, hypotension, and other indications have shown similar effects. Because the endocrine activity of agents may be useful for their therapeutic value, it is sometimes difficult to separate pharmacological efficacy from toxicity. For example, raloxifene, a nonsteroidal selective estrogen receptor modulator (SERM) developed for treatment of postmenopausal osteoporosis acts as an estrogen in bone but functions in uterine tissue as a complete estrogen antagonist. Because estrogen is important in preparing for implantation, raloxifene was found, not unexpectedly, to cause delays in implantation (30); such effects have been seen with other compounds that are estrogen antagonists (reviewed in Ref. 30).

Mammary gland tissue is highly dependent on endocrine function. The mammary gland changes dramatically around the time of parturition as a result of a number of gonadal and extragonadal hormones. Milk letdown depends on suckling by the offspring, release of oxytocin from the posterior pituitary, and secretion of prolactin by the anterior pituitary. Agents that affect hormonal status, mammary gland development, and/or function may cause difficulties with milk production, milk quality, and indirectly result in adverse effects on offspring growth and development. Two neurotransmitters, dopamine and serotonin, play critical roles in the neuroendocrine modulation of prolactin secretion (31, 32), and prolactin is known to be mammotrophic and lactogenic, as well as luteotrophic and endometriotrophic (33, 34). Acute pharmacological doses of serotonin agonists stimulate prolactin release and enhance neonatal mouthing behavior, whereas acute doses of antagonists decrease these responses (35). It has long been known that bromocriptine, a preferential dopamine D2 agonist, prevents postpartum onset of lactation in humans (36), inhibits established lactation in several species including rats (37), dogs (38), and humans (36, 39), and suppresses the suckling-induced secretion of prolactin in rats (40, 41).

Reproductive senescence occurs with advancing age, depletion of oocytes, and loss of normal ovarian cycling. As indicated earlier, agents that enhance atresia of oocytes may produce early depletion and untimely reproductive senescence. The long-term consequence of early menopause is an increased risk of a number of associated diseases, including heart disease and osteoporosis. Cigarette smoking has been shown to reduce the age at onset of menopause by as much as 2 years (42). In addition, Mattison and Thorgeirsson (43) showed that benzo[a]pyrene, which occurs in tobacco smoke, can kill oocytes in mice. 2.4 Male Reproduction

The male reproductive system is comprised of the testis, accessory sex glands (seminal vesicles, prostate, and bulbourethral or Cowper's glands), and the duct system. In rodents, there are two additional accessory sex glands, the coagulating glands and the preputial glands. The duct system is comprised of the efferent ducts, epididymis (consisting of three parts: head or caput epididymis, body or corpus epididymis, and tail or cauda epididymis), ductus deferens, and ejaculatory duct. A balanced interplay among the hypothalamus, anterior pituitary, and testis regulates the function of the male reproductive system (Fig. 3.5) (44). As in the female, GnRH production by the hypothalmus permits release of FSH and LH by the anterior pituitary, which permits release of testosterone from the Leydig cells, and in turn is negatively regulated by increased levels of testosterone and dihydrotestosterone, the more active form of the androgen. Several important proteins are secreted by the Sertoli cells, including androgen-binding protein, activin which stimulates LH and FSH production by the pituitary, and inhibin and follistatin which have an inhibitory influence on the pituitary gonadotropins. Undernutrition, particularly a low protein diet (8% versus 27% in controls) started at weaning, can have a major impact on the development of the anterior pituitary-testicular axis and the feedback mechanism that controls gonadotropin secretion (45). Several antiandrogenic agents have been shown to interfere with normal development of the male reproductive system. For example, finasteride, a 5-alpha-reductase inhibitor, causes hypospadias in male offspring of rats exposed during pregnancy (46), and the most sensitive period for exposure was gestational days 16 and 17 in the rat (47). Several pesticides (e.g., vinclozolin and procymidone) are antiandrogenic and also cause hypospadias in male offspring (48-50).

Figure 3.5. Diagram summarizing the anterior pituitary-testicular axis. LH and FSH are secreted by the gonadotrophs (G) to stimulate (+) either the Sertoli cells (SC) or Leydig cells (LC). These cells subsequently produce peptides [inhibin (I), follistatin (FS), and activin (A)] or sex steroid hormones [estradiol (E2), testosterone (T), or dihydrotestosterone (DHT)], which feed back principally at the

Figure 3.5. Diagram summarizing the anterior pituitary-testicular axis. LH and FSH are secreted by the gonadotrophs (G) to stimulate (+) either the Sertoli cells (SC) or Leydig cells (LC). These cells subsequently produce peptides [inhibin (I), follistatin (FS), and activin (A)] or sex steroid hormones [estradiol (E2), testosterone (T), or dihydrotestosterone (DHT)], which feed back principally at the level of the anterior pituitary gland to regulate gonadotropin secretion. The modulatory role of these compounds is primarily inhibitory (-), although activin is known to stimulate FSH secretion. Autocrine/paracrine control over LH and FSH occurs at the level of the anterior pituitary gland via the peptides inhibin, follistatin, and activin, which are produced by the gonadotrophs and folliculostellate cells (FSC) (used with permission of Raven Press from Ref. 44, p. 6).

Approximately 90% of the testis is comprised of the seminiferous tubules, which are folded and refolded within the testis and contain the developing spermatozoa and Sertoli cells. The Sertoli cells extend from the basement membrane to the lumen of the seminiferous tubules and surround and support the developing germ cells. Tight junctions between the Sertoli cells near the basement membrane form the blood-testis barrier, which blocks access to the adluminal comparment. The interstitial tissue contains the Leydig cells (the primary source of testosterone), the vascular supply to the testis, and other cells. Testosterone is converted to dihydrotestosterone, the more active form of the androgen. Spermatogenesis takes place along the tubules in a wave form, so that cross sections of several tubules would reveal sperm in different stages of development. There are 14 distinct stages of development that have been identified in rats (51) (Fig. 3.6) (52), and six distinct stages have been described in humans (53). These stages can be used as a basis for determining the effect of an exogenous agent. A serial mating design can be used to identify site-specific lesions; this type of protocol involves mating males that have been exposed for a short time (5-7 days) with unexposed females for 4-5 day periods over several weeks. Cytosine arabinoside, a chemotherapeutic agent, caused reduced fertility 31 to 41 days post-exposure, indicating an effect on spermatogonia and early spermatocytes (22).

Figure 3.6. Cycle map of spermatogenesis for the rat. The vertical columns, designated by Roman numerals, depict cells associated with various stages. A cycle is a complete series of stages. The developmental progression of a cell is followed horizontally until the right hand border of the cycle map is reached and continues from left to right up the map, ending with spermiation (used with permission of Cache River Press from Ref. 52, p. 43).

After sperm are released from the Sertoli cells into the lumen, they pass from the seminiferous tubules through the rete testis and efferent ducts into the epididymis, then through the ductus deferens and ejaculatory duct. The sperm undergo maturation in the epididymis and are stored there until ejaculation. The accessory sex glands contribute most of the volume to the semen, and their secretions may be involved in effective transport, survival, and function of the sperm through the female reproductive tract after ejaculation. The duct system and the accessory sex glands are androgen-dependent, and can be affected by agents that alter androgen levels (54). 2.5 Development

Development can be divided into the prenatal and postnatal periods. Prenatal development includes the preimplantation andpostimplantation periods, and the latter encompasses the embryonic and fetal periods. Preimplantation is characterized by transport of the zygote from the upper ends of the oviduct into the uterine cavity during a period of 7-8 days in humans, or 5-6 days in rats or mice (Figure 3.3). Rapid cell division of the single-cell zygote (fertilized ovum) proceeds through the morula stage, a solid ball of approximately 16 cells, and further division results in a multicellular blastocyst that contains a cavity. The blastocyst implants in the uterine wall and develops into the definitive embryo. The embryo itself develops from a small group of cells in the blastocyst, the inner cell mass. The rest of the cells, the extraembryonic cells, form the placenta and surrounding membranes. Implantation occurs around gestational day (gd) 7 in humans, gd 5-6 in mice and rats, and gd 7 in rabbits. Agents that interfere with implantation result in apparent subfertility or infertility in humans. The type of effect produced on fertility can be determined in rodents by counting the number of corpora lutea (i.e., ovulated eggs) and the number of implantation sites in the uterine wall if there are enough viable implants to maintain active corpora lutea during pregnancy or if done early in gestation when there are no viable implants.

Placentation is a complex series of events that provides an intimate relationship between the embryonic and maternal tissues for the purpose of nutrition of the embryo and removal of wastes. Early on, before vascularization of the extraembryonic membranes and formation of the chorioallantoic placenta, the yolk sac placenta provides histiotrophic nutrition (breakdown and transfer of maternal macromolecules). The yolk sac placenta is the primary placenta in rodents through the early part of embryogenesis (when the early neural tube and limb buds are forming), and a gradual switchover to the chorioallantoic placenta occurs around gestation days 11-12 in the rat. In humans, the chorioallantoic placenta becomes functional around gestation day 21 when the neural tube is just beginning to form, an earlier embryonic stage than in rodents. Chorioallantoic placentation involves hemotrophic nutrition (transfer of nutrients via the circulation) and is somewhat different in different species, in that the number of maternal and embryonic layers differs. Although much has been made of these differences in terms of their role in placental transfer of toxicants, there is little evidence that these differences are important factors compared to maternal blood flow, plasma protein binding, molecular size and charge, placental metabolism, and the fetal elimination pathway. A more complete discussion of the morphology and function of the placenta can be found in embryology textbooks and excellent descriptions related to developmental toxicology are given by Beck (55), and Slikker and Miller (56).

Exposure to certain toxic agents during pregnancy may adversely affect placental function, which can in turn affect the developing embryo/fetus. Effects on the placenta may include alterations in blood flow and perfusion, metabolism, placental transfer of essential nutrients or compounds, and in extreme cases, may cause necrosis and separation from the uterine wall. Cadmium is an environmental contaminant associated with refineries, fossil fuel plants, and tobacco smoke and is a demonstrated placental toxicant in both rodents and humans (56). Although cadmium may cross the rodent yolk sac placenta very early in development and cause fetal malformations, it does not cross but accumulates in the chorioallantoic placenta. Adverse developmental effects, ranging from growth retardation to fetal death, observed following such accumulation result from placental dysfunction rather than direct actions on the embryo/fetus.

The embryonic inner cell mass forms a two-layered embryonic disc, consisting of ectoderm and endoderm. Around gestation day 17 in humans or gestation day 9 in rats, invagination of ectodermal cells occurs through a midline primitive streak to form the third intermediate layer of cells, the mesoderm. Figure 3.7 shows the major derivatives of each layer. The mesoderm cells form the viscera, blood vessels and cells, muscles, tendons, and bone. The endoderm forms the lining of the gut, respiratory system, thyroid, and pharynx. The ectoderm forms the surface layers of the body, including the skin, hair, and nails, enamel of the teeth, and the lens; a specialized portion of the ectoderm that forms along the edges of the primitive streak is the neuroepithelium consisting of the neural tube and the neural crest cells. The neural tube forms the brain and spinal cord, and the neural crest cells form the cranial and spinal ganglia and nerves, several components of the face and neck, the adrenal medulla, and contribute to the endocardial cushions that separate the chambers of the heart. Agents that interfere with the development and closure of the neural tube may result in anencephaly or spina bifida, and those that interfere with migration of neural crest cells to their ultimate site may result in craniofacial defects, cranial or spinal nerve defects, and/or cardiac septal defects. Retinoids, including excessive supplements of vitamin A or the drug Accutane (used in treating dermatologic disorders), interfere with neural crest cell migration, and both animals and children exposed during early development exhibit a number of these defects (57, 58). In addition, central nervous system malformations and a continuum of neurobehavioral disorders have been identified in animal fetuses and offspring and in children exposed early in pregnancy (59-64). Postnatal death, profound mental retardation, alterations in general learning ability, and other subtle behavioral alterations have been documented. These effects in animals and humans all depend on the dose and developmental stage at exposure, as well as the relative teratogenic potency of the individual retinoid (64).

Figure 3.7. Derivatives of the germ layers in the mammalian embryo. Cells of the inner cell mass form the embryonic disc consisting initially of ectoderm and endoderm. During gastrulation, a mesodermal layer is formed in between the ectoderm and endoderm. The primary derivatives of the three germ layers are shown.

Figure 3.7. Derivatives of the germ layers in the mammalian embryo. Cells of the inner cell mass form the embryonic disc consisting initially of ectoderm and endoderm. During gastrulation, a mesodermal layer is formed in between the ectoderm and endoderm. The primary derivatives of the three germ layers are shown.

The period during which the major conformation of organ systems occurs, called organogenesis, encompasses the period from the primitive streak stage until palate closure (gestation days 6-15 in rats and mice, gestation days 17-57 in humans). The fetal period consists of the time after major organogenesis until birth, during which organization continues at the histological level in most organ systems.

There are a number of critical periods when cells or organ systems are particularly sensitive to exposure to toxic agents. Because of this, the manifestations of developmental toxicity vary depending on the timing of exposure. As examples, exposure before conception may cause chromosomal or DNA changes in germ cells that result in heritable effects, including death, malformations, growth retardation, functional deficits, or cancer in the offspring. During very early embryogenesis when cells are multiplying at a rapid rate and are relatively undifferentiated, exposure tends to result in death or compensation and continued normal development. For several genotoxic agents (e.g., ethylene oxide, ethylnitrosourea, ethyl methanesulfonate) (65-71), exposure during this period also results in malformations and growth retardation. As organogenesis begins, cells become more and more differentiated and the major structure of organs is formed, although not all organs develop at the same time or rate. Exposure during this period may cause major structural defects, as well as death, growth retardation, or postnatal functional changes. As major organ structure is completed, organization and differentiation at the histological, physiological, and biochemical levels proceed; in most mammals, these processes occur to varying extents during pre- and postnatal development. However, there are important differences at birth among experimental animal species and humans in the staging of developmental events that must be recognized in designing studies and interpreting experimental outcome data for potential human risk. Exposure during this late gestational or fetal period may result in alterations that are detected as histopathology, growth retardation, functional changes, or cancer. Subsequent stages of development include further growth and functional maturation of organs/systems, some of which are not completed until after puberty. Exposure during this period may affect the same target organs as in adults but have different consequences because of the immaturity of the target organ itself. Additionally, other immature organ systems may be targets, and relative sensitivities may be greater or lesser due to immaturity of processes responsible for metabolism and excretion of the chemical.

Less work has been done to discern the critical timing for exposure during the postnatal period, but there are examples of neonatal exposures and effects on the developing reproductive system (72-74), and developing nervous system (75). This is an area of current interest to provide adequate guidance for pediatric use of pharmaceuticals and also for environmental exposures that may affect children where they live and play. One area that has received attention recently is the significant increase in childhood asthma (76, 77). As a result, a number of studies are being pursued to discern genetic and/or environmental factors that may be involved or responsible both prenatally and postnatally.

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