Development Of Germ Cells And Their Relations To Embryonic Stem Cells

During embryonic development, germ cells first emerge at a specific location segregated from somatic cell development in both vertebrates and invertebrates (7-9). This physical segregation of germ cells has been hypothesized to allow germ line specification to occur with a minimal influence from somatic cell development (8). In mammals, germ cell development has been best studied in mice. Mouse germ cells are first recognized at the base of allantois in the extraembryonic mesoderm at approximately 7 days postcoitum (dpc) as a cluster of approximately 50 cells that exhibit the alkaline phosphatase activity (7-9). These fetal germ cells are called primordial germ cells (PGCs). PGCs are then transferred from the extraembryonic tissue to the embryo per se at approximately 8.5 dpc and migrate through the hindgut while rapidly proliferating. These cells further migrate through the dorsal mesentery into the genital ridges (fetal gonads) at approximately 10.5 dpc. PGCs continue to proliferate in the genital ridge until approximately 12.5 dpc when the sex differentiation becomes morphologically evident. On 13.5 dpc, approximately 25,000 PGCs can be found in the genital ridge (10); thus, PGCs increase 500-fold in number from the time of their emergence. At the initiation of sex differentiation, female and male germ cells take different developmental pathways (Fig. 1). In females, PGCs enter meiosis and then become arrested at meiotic prophase. In males, PGCs encapsulated in the testicular cords (fetal seminiferous tubules) become mitotically arrested. The arrested stage continues until birth in both sexes. After birth, female germ cells are periodically recruited for meiotic maturation, whereas male germ cells initiate mitosis. In the male, these diploid postnatal germ cells are called spermatogonia. Spermatogonia undergo mitosis and sequentially differentiate before committing to meiosis (11). In mice, first meiotic male germ cells (spermatocytes) appear around 10 days of age and first spermatozoa, approximately 35 days of age (12,13). The male gametogenesis (spermatogenesis) continues throughout life.

The mechanism of germ cell specification, migration, proliferation/survival, and sex-dependent differentiation during embryonic development remains elusive and is beyond the scope of this chapter (see refs. 8,9,14-17 for detail). However, the process of germ cell development depicts a unique characteristic of germ line stem cells. Because all female germ cells enter meiosis on sex differentiation, they lose self-renewal potential before birth, resulting in the loss of stem cells in the postnatal female germ line. In contrast, a population of self-renewing cells (stem cells) remains in the male germ line throughout life. This is the foundation of continuous spermatogenesis and the regeneration of sper-matogenesis following testicular insults, including sterilizing cancer therapy (5,18). Consequently, the number of gametes during the reproductively active periods in males far exceeds that in females. Such a sex-dependent difference in the existence of stem cells cannot be seen in other types of stem cells.

PGCs are a transient cell type and are not true stem cells under a strict stem cell definition. These cells are rather "precursors" of germ line stem cells, because the cells that have characteristics identical to PGCs do not exist in normal postnatal mammals, reflecting the lack of extended self-renewal activity of PGCs. If fact, many embryonic cells are precursors. For example, although the cells in the inner cell mass of blastocysts are the origin of embryonic stem cells

Fetal Mesodermal Progeniter

Fig. 1. The life cycle of germ cells. Fertilization of an egg by a sperm triggers the embryonic development. The cells in the ICM (shown as a dark part) of the blastocyst are the origin of all cell types in the body as well as ES cells. PGCs are first found in the extraembryonic mesoderm and translocate into the embryo per se. Then, they migrate toward the embryonic gonads while actively proliferating. These proliferating PGCs are capable of transforming to totipotent EG cells. After the migration into the gonads, male germ cells enter mitotic arrest, whereas all female germ cells enter meiosis, followed by the arrest at the meiotic prophase. After birth, male germ cells reinitiate mitosis and undergo meiosis and spermiogenesis, which is a complex morphologic transformation of haploid spermatids to sperm. Mitosis, meiosis, and spermiogenesis continue throughout life in the male germ line (spermatogenesis). It is important to note that a population of stem cells exists in the male germ line, whereas it is absent in the female germ line. (Modified from ref. 95 with permission.)

Fig. 1. The life cycle of germ cells. Fertilization of an egg by a sperm triggers the embryonic development. The cells in the ICM (shown as a dark part) of the blastocyst are the origin of all cell types in the body as well as ES cells. PGCs are first found in the extraembryonic mesoderm and translocate into the embryo per se. Then, they migrate toward the embryonic gonads while actively proliferating. These proliferating PGCs are capable of transforming to totipotent EG cells. After the migration into the gonads, male germ cells enter mitotic arrest, whereas all female germ cells enter meiosis, followed by the arrest at the meiotic prophase. After birth, male germ cells reinitiate mitosis and undergo meiosis and spermiogenesis, which is a complex morphologic transformation of haploid spermatids to sperm. Mitosis, meiosis, and spermiogenesis continue throughout life in the male germ line (spermatogenesis). It is important to note that a population of stem cells exists in the male germ line, whereas it is absent in the female germ line. (Modified from ref. 95 with permission.)

(ES cells; Chapter 1), they do not self-renew but rather disappear during normal embryonic development.

Although PGCs are a transient cell population, these cells have significant potential to be a source of stem cells, not only for male germ line stem cells but also for pluripotent embryonal carcinoma (EC) cells and totipotent embryonic germ (EG) cells (see Chapter 1). From the 1950s to the 1970s (reviewed in ref. 19), Leroy Stevens at the Jackson Laboratory intensively studied teratomas/ teratocarcinomas (hereafter, only the term "teratocarcinomas" will be used for simplicity). Using mice, he showed that the implantation of blastocysts or of the genital ridge (10.5-12.5 dpc) into adult testes resulted in the formation of tera-tocarcinomas, in which a wide range of differentiated tissues can be observed (see Chapter 1). He identified PGCs as the origin of teratocarcinomas that arise after implanting genital ridges into adult testes (20). Teratocarcinomas were found to contain the stem cells that were later isolated and called EC cells (see Chapter 1). The self-renewal potential and pluripotency (potential to differentiate into multiple, but not all, cell types) of EC cells was proven by Barry Pierce, thus confirming the stem cell properties of these cells (21). Ralph Brinster later demonstrated by injecting EC cells into blastocysts that these tumor stem cells could be integrated into the normal developmental process to produce chimeric mice without causing tumorigenesis (22), although EC cells do not enter the germ line. Therefore, PGCs are the source of pluripotent EC cells that can contribute to the generation of chimeric mice.

In 1992, Matsui et al. (23) and Resnick et al. (24) reported that mouse PGCs transformed to cells that morphologically resembled ES cells when cultured in vitro with a cocktail of growth factors. These cells, called EG cells, were derived by culturing proliferating PGCs (8.5-12.5 dpc) with leukemia inhibitory factor (LIF), Steel factor (also called c-Kit ligand), and basic fibroblast growth factor (bFGF). Using similar culture techniques, EG cells have also been obtained from pigs and humans (25,26). The studies using mice have shown that EG and ES cells are similar not only in their morphology, but also in their function. As with ES cells, EG cells can be maintained in undifferentiated states indefinitely in vitro and will generate teratocarcinomas in vivo (23). When injected into blas-tocysts, both ES and EG cells are integrated into the normal developmental process of host mice and contribute to both somatic and germ cell lineages (27). Therefore, ES and EG cells are both totipotent (differentiate into all cell types), although they are apparently not identical (28). Another characteristic shared by both of these tumor stem cells is that they are derived from transient cell types that express Oct-3/4 transcription factor: ES cells from the inner cell mass/ epiblast and EG cells from PGCs (8,9,29,30) (see Chapter 1).

Although mouse PGCs have the potential to transform into totipotent stem cells, ironically these cells have never been successfully used for cloning by nuclear transplantation (31,32). Mice can be successfully cloned using the nuclei of many somatic cell types (33). However, no reports have shown live birth of offspring cloned from PGC nuclei (31,32). Yamazaki et al. have shown that embryos cloned using nuclei of 10.5-dpc PGCs can develop normally until mid-gestation but die shortly thereafter (32). Furthermore, embryos cloned from later-stage PGCs never develop normally (32). These results might indicate that the genomes of germ cells may be programmed in a specific manner. For example, it is well known that the patterns of DNA methylation and imprinting in the germ line are distinct from those of somatic cell lineages (34,35). Therefore, it is possible that genomic modifications specific to the germ line render the reprogramming of the germ cell genome difficult in cloning by nuclear transplantation.

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Pregnancy Diet Plan

Pregnancy Diet Plan

The first trimester is very important for the mother and the baby. For most women it is common to find out about their pregnancy after they have missed their menstrual cycle. Since, not all women note their menstrual cycle and dates of intercourse, it may cause slight confusion about the exact date of conception. That is why most women find out that they are pregnant only after one month of pregnancy.

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