A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).

https://rupress.org/jcb/article-pdf/74/1/68/1072582/68.pdf

Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization.

3 The sertoli cell

The spermatozoa of most invertebrates (e.g., sea urchins) and nonmammalian vertebrates (e.g., fishes and amphibians) have full capacity to fertilize eggs upon leaving the testis. Testicular spermatozoa of mammals, on the other hand, do not possess the ability to do so. Their fertilizing capacity develops as they pass through the epididymis (Young, 1931; Nishikawa and Waide, 1952; Blandau and Rumery, 1964; Bedford, 1966; Orgebin-Crist, 1967). This process, apparently “unique” to mammals, is referred to as the epididymal maturation of spermatozoa. Even after their maturation, however, spermatozoa require an additional phase of maturation or capacitation within the female genital tract before they are able to fertilize eggs (Austin, 1951, 1952; Chang, 1951a). Thus, epididymal maturation and capacitation are two extra steps that mammaliam spermatozoa must take before they can effect fertilization. In this chapter, I will discuss how mammalian spermatozoa prepare themselves for fertilization and how the spermatozoa and eggs interact during fertilization. The process and mechanisms of sperm transport in the female genital tract will not be dealt with extensively here. Readers are referred to Bishop (1961, 1969), Blandau (1969), Bedford (1970b, 1972b), Thibault (1972, 1973a), Zamboni (1972), Blandau and Gaddum-Rosse (1974), Hafez and Thibault (1975), Overstreet and Katz (1977), Overstreet and Cooper (1978, 1979b), Overstreet et al. (1978), Shalgi and Kraicer(1978), Cooper et al. (1979) and Hunter (1975, 1980). The rejection or elimination of extra spermatozoa by the fertilized egg, one of the most fascinating events in fertilization, will not be discussed here. Instead, readers are referred to the chapter by Dr. Wolf in this volume. The physiology of egg activation has been described and discussed to some extent by Gwatkin (1977) and Yanagimachi (1978a). There are numerous reviews dealing with general aspects of mammalian fertilization. The following are recommended to aid in grasping the outline of mammalian fertilization: Austin and Bishop (1957), Austin and Walton (1960), Austin (1961, 1968), Blandau (1961), Piko (1969), Thibault (1969), Bedford (1970a,b, 1972b), Gwatkin (1976, 1977), Yanagimachi (1977, 1978a), Bedford and Cooper (1978) and Hunter (1980).

Mechanisms of Fertilization in Mammals

Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles.

Publisher Summary This chapter discusses germ plasm and the differentiation of the germ cell line. Germ plasm is defined as a substance present in the cytoplasm of gametes, which is segregated into specific cells during blastulation and determines that those cells shall become the progenitors of the germ cell line during subsequent development. The composition of germ plasm is discussed. The histochemical evidence for the presence of RNA in polar granules of insects and germinal plasm of amphibians indicates that RNA is present only in eggs and early embryos, while a protein component is present throughout the germ cell cycle. Good morphological and experimental evidence for the presence of germ plasm is available for only a few animal species. The germinal cells of chaetognaths were present in early stages of embryonic development and identified a group of cells at the bottom of the archenteron cavity in gastrulas of Sagitta as the primordium of the germ line. Experimental studies are given to determine the role of germinal plasm in anuran eggs by altering the cytoplasm in which it is contained.

Germ plasm and the differentiation of the germ cell line.

Ultrastructural observations on germ cells in human fetal ovaries

A certain degree of synchronization of germ cell differentiation is a general feature of mammalian oogenesis. In the rat (3, 10), mouse (4), and rabbit (15, 16), germ cell differentiation is highly synchronized in that there is a direct correspondence between fetal age and predominant stage of cellular activity. In the guinea pig (11), monkey (2), and man (1), the synchronized pattern is less apparent because of a considerable overlapping of mitosis, meiosis, and degeneration. Correpondence between age and stage of differentiation of a significant percentage of germ cells is found even in these species, however. In the course of an electron microscopic study of ovarian development in the newborn rabbit, we have observed that the synchronization of the oogenetic process finds expression not only in the close relationship between fetal age and stage of cellular differentiation, but also in the maturation of the germ cells in groups and in the synchronous differentiation of all the cells in each group. Such synchronization appears to be related

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Intercellular bridges and synchronization of germ cell differentiation during oogenesis in the rabbit

Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.

Ultrastructure of germ cell development in the human fetal testis.

Ovarian Development: The Functional Importance of Germ Cell Interconnections

Testicular differentiation, as evidenced by the appearance of a distinct tunica albuginea and formation of plate-like aggregates of germ cells and mesenchymal cells, occurs at 16 days in the rabbit fetus. The germ cells at this stage of development are isolated large, round to oval cells that are easily distinguished from the smaller elongated mesenchymal elements surrounding them. The germ cell nuclei have a smooth spherical contour and a prominent eccentric nucleolus. Cytoplasmic organelles are sparse, consisting of scattered polyribosomes and spherical or ovoid mitochondria distributed singly in the cell. At 18 days, the cell groups form into distinct cords of germ cells and supporting cells, the earliest stage of seminiferous tubule formation. Between 22 and 25 days, proliferating germ cells show increasing condensation of nuclear chromatin and increased nucleolar complexity. The cytoplasm contains groups of mitochondria, dense polyribosomal complexes, lipid aggregates, scattered smooth vesicles and prominent Golgi complexes. Intercellular bridges appear between germ cells on day 22 and increase in number on succeeding days. During the last week of fetal development, germ cells become aligned in rows at the periphery of the primitive tubules. Their arrangement at this time resembles that of spermatogonia in the adult testis. However, the fetal “prespermatogonia” lack some of the characteristic ultrastructural features of adult spermatogonia.

Bernard Gondos

Papers

Ultrastructural observations on germ cells in human fetal ovaries

Intercellular bridges and synchronization of germ cell differentiation during oogenesis in the rabbit

Ultrastructure of germ cell development in the human fetal testis.

Ovarian Development: The Functional Importance of Germ Cell Interconnections

Ultrastructure of developing germ cells in the fetal rabbit testis