The ultrastructure of the nuclei and the behaviour of the sex chromosomes of human spermatogonia

Summary The nuclear structure of human spermatogonia has been studied with electron microscopical and histochemical methods. Type B spermatogonia have chromatin clumps without any special ultrastructure and several nucleoli. Five different types of nuclear bodies, and besides, a nuclear vacuole, have been observed in type A spermatogonia. Type I bodies are typical nucleoli consisting of three regions: amorphous, fibrillar and granular. Type II, III and V are considered to be atypical nucleoli. Type IV bodies are small chromatin condensations. Type I bodies are the only ones in which RNA was demonstrated by light histochemical techniques and no PAS positive material was found inside the nuclei. The absence of any special ultrastructure in the chromatin from spermatogonia, and the small mass of the chromatin condensations, show that the human X chromosome and perhaps the Y chromosome are not heteropycnotic in the interphasic nuclei of human spermatogonia.Abbreviations Used RNA ribonucleic acid - gonia spermatogonia This work has been supported by a grant (No. 2623) of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, and partially by a grant (C.M. 6522) from the Population Council.We wish to thank Professor R. E. Mancini for his suggestions during this investigation and his support for its achievement, and to Dr. J. C. Lavieri for providing the biopsies.     

Two states of active spermatogenesis switch between reproductive and non-reproductive seasons in the testes of the medaka, Oryzias latipes

Seasonal change in spermatogenesis was examined in the restricted spermatogonium‐type testes of a teleost, Oryzias latipes. Histological observation revealed that the number of each stage of germ cells during most of the non‐reproductive season, from October to January (O–J period) was nearly half of that during the reproductive season, from May to July (M–J period), except for type B spermatogonia (B‐gonia), which was actually equal. As a result, the ratio of primary spermatocytes (P‐cytes) to B‐gonia was remarkably small in the O–J period. Despite the differences between both time periods, the proliferative activity of type A spermatogonia (A‐gonia), B‐gonia, or P‐cytes was at a similar level in both periods. Moreover, in cultured testes treated with bromodeoxyuridine as a cell‐lineage tracer, P‐cytes differentiated to spermatids in 11–15 days in both M–J and O–J periods. These indicate that spermatogenesis is active in each period at a different state. In the spermatogenic testis, A‐gonial proliferation was maintained by human follicle stimulating hormone/luteinizing hormone in culture. Whereas cell death of B‐gonia and/or P‐cytes gradually increased in the M–J period in spite of those cells being constant in population sizes. In transition to the O–J period, A‐gonia and P‐cytes first decreased, which was accompanied by a decrease in proliferative activity of A‐gonia and relative increase of dead cells from B‐gonia and/or P‐cytes against live P‐cytes. These suggest that A‐gonial proliferation and cell death of B‐gonia and/or P‐cytes that is induced coordinately with B‐gonial differentiation are critical for the spermatogenic control.     

An overview of cell renewal in the testis throughout the reproductive cycle of a seasonal breeding teleost, the gilthead seabream (Sparus aurata L)

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1beta, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.     

Renewal of spermatogonia in the monkey (Macaca fascicularis)

Populations of different types of spermatogonia and their mitotic activity were analyzed in the monkey Macaca fascicularis: 3 adults aged 5-6 yr and 3 young aged 2-3 mo. Two young and two adult monkeys received injections of 3H-thymidine for radioautographic study of the relationships between Type A spermatogonia: dark Type A (Ad), pale Type A (Ap) and transition Type A (At). In the adult the number of Ad and At spermatogonia did not change significantly throughout the seminiferous epithelium cycle. The number of Ap spermatogonia doubled at Stage VII, and half divided at Stage IX to give rise to B1 spermatogonia. The durations of the seminiferous epithelium cycle and spermatogenesis were estimated as 10.5 days and 42 days respectively. In the young and adult monkeys, some Ap spermatogonia and a lesser number of At spermatogonia were labeled one h after injection of precursor. At longer intervals after injection, the number of labeled At spermatogonia increased significantly, and some Ad as well as Ap spermatogonia were also labeled. These results indicate that Ap spermatogonia are renewal stem cells, and Ad spermatogonia are reserve stem cells. The differences in labeling after isotope exposure suggest that Ap cells may give rise successively to At and Ad cells.     

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.     

Ultrastructure du tissu apical testiculaire du doryphore Leptinotarsa decemlineata Say (Coléoptère: Chrysomélidé): étude autoradiographique et cytochimique / Ultrastructure of the apical testicular tissue of the doryphore Leptinotarsa decemlineata Say (Coleopterae: Chrysomelidae): an autoradiographic and cytochemical study

Summary

The somatic ‘apical tissue’ is present in the testes of the Colorado potato beetle from the embryonic stage until the end of the reproductive life. In the vicinity of this tissue, cyst cells and primary gonia are often pycnotic. Incorporation of labelled amino acids in vivo and treatment with pronase indicate that apical cells are sites of protein synthesis. Secretions are glycosylated in the Golgi apparatus where they are also concentrated in granules. Some of them display a positive reaction to the acid phosphatase test. They are extruded by exocytosis into the intercellular spaces which they distend. We conclude that the apical secretions have a lytic activity: gonia and cyst cells are reduced to cellular residues which are accumulated in the intercellular spaces.     

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia)

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.     

Involvement of the gonadal germinal epithelium during sex reversal and seasonal testicular cycling in the protogynous swamp eel,Synbranchus marmoratus Bloch 1795 (Teleostei,Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a protogynous, diandric species. During sex reversal, the ovarian germinal epithelium, which forms follicles containing an oocyte and encompassing follicle cells during the female portion of the life cycle, produces numerous invaginations, or acini, into the ovarian stroma. Within the acini, the gonia that formerly produced oocytes become spermatogonia, enter meiosis, and produce sperm. The acini are bounded by the basement membrane of the germinal epithelium. Epithelial cells of the female germinal epithelium, which formerly became follicle (granulosa) cells, now become Sertoli cells in the developing testis. Subsequently, lobules and testicular ducts form. The swamp eel testis has a lobular germinal compartment in both primary and secondary males, although the germinal compartment in testes of secondary males resides within the former ovarian lamellae. The germinal compartment, supported by a basement membrane, is composed of Sertoli and germ cells that give rise to sperm. Histological and immunohistochemical techniques were used to describe the five reproductive classes that were observed to occur during the annual reproductive cycle: regressed, early maturation, mid-maturation, late maturation, and regression. These classes are differentiated by the presence of continuous or discontinuous germinal epithelia and by the types of germ cells present. Synbranchus marmoratus has a permanent germinal epithelium. Differences between the germinal compartment of the testes of primary and secondary males were not observed.     

Development and early differentiation of gonad in the european eel (Anguilla anguilla [L.], Anguilliformes,Teleostei): A cytological and ultrastructural study

The structure of the gonad of the European eel (Anguilla anguilla [L.]), an “undifferentiated” gonochoristic teleost, was investigated by transmission electron microscopy from 6–8 cm elvers to 22 cm yellow eels with juvenile hermaphroditic gonads. The pear-shaped gonads of 6–8 cm elvers assume, in 12–15 cm eels, a lamellar shape and enlarge by migration of germ cells, which we refer to as primary primordial germ cells. In the gonads of ∼ 16 cm eels, the primary primordial germ cells multiply, giving rise to clusters of germ cells that have ultrastructural characteristics of the primary primordial germ cells but show giant mitochondria, enlarged Golgi complexes, and round bodies not limited by membranes. We refer to these as secondary primordial germ cells. In 16–18 cm eels, syncytial clones of oogonia interconnected by cytoplasmic bridges are also observed. In 18–22-cm-long eels, the gonads contain primordial germ cells, oogonial clones, early oocyte cysts, single oocytes in early growth stages, and primary spermatogonia. Such germ cells are present in the same cross section where they are either intermingled or are in areas of predominantly female germ cells close to areas with predominantly male germ cells. These gonads are juvenile hermaphroditic and should be considered ambisexual because in larger eels they differentiate either into an ovary or into a testis. Somatic cells always envelop the germ cells following their migration into the gonad. These somatic cells first show similar ultrastructural features and then differentiate either into early Sertoli cells investing spermatogonia, or into early follicular (granulosa) cells investing the early previtellogenic oocytes. In eels ∼ 14 cm long, primitive steroid-producing cells also migrate into the gonad. In the ambisexual gonad they differentiate either into immature Leydig cells in the male areas, or into early special cells of the theca in the female areas. Nerve fibers are joined to the steroid-producing cells. Gonad development and differentiation are also associated with structural changes of the connective tissue characterized by the progressive appearance and deposition of collagen fibrils first in the mesogonadium, then in the gonad vascular region, and then in the germinal region. The collagen-rich areas are massive in the male areas and reduced in the female ones. J. Morphol. 231:195–216, 1997. © 1997 Wiley-Liss, Inc.     

FORMATION OF GERM CELL-LIKE CELLS IN THE PERITONEAL EPITHELIUM OF THE TESTES OF ESTROGEN-TREATED ADULT MALES OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER

Columnar cells of the peritoneal epithelium in slender cords of the testes were examined in normal and estradiol benzoate-treated Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, by light and electron microscopy. In normal newts, the peritoneal epithelium covering the slender cord consists of columnar cells, which contain extraordinarily large, oval or spindle-shaped nuclei with conspicuous indentations. The nucleus contains chromatin granules and the cytoplasm is filled with numerous tonofilaments. The primordial germ cells are scattered throughout the slender cord, and each cell is surrounded by a few follicle cells. Between the germ cells and follicle cells there are microvilli-like processes. The nucleus of primordial germ cells is multilobate and has electron lucent areas, dispersed chromatin and several electron-dense nucleoli. In the lighter cytoplasm, the nuage material is found very near to nuclear pores, and is frequently seen among the mitochondria. The nucleolus-like body is not associated with other organelles. The primary spermatogonia have bilobate nuclei. It is remarkable that most of the cytoplasmic organelles are found in the deep nuclear indentations. The nuage material and nucleolus-like body are well developed in the cytoplasm. After treatment of newts with estradiol benzoate for one year, four types of cells can be distinguished in the peritoneal epithelium. One type is quite different from the columnar cells. These newly appeared cells are large and light in appearance. Their nucleus is highly lobate, and contains dispersed chromatin and several nucleoli with compact electron dense material in its periphery. The cells are characterized by the presence of nuage material and nucleolus-like bodies in the cytoplasm. There are microvilli-like processes between these cells and adjacent elongated cells. These ultrastructural characteristics of the light cells are very similar to those of primordial germ cells and/or primary spermatogonia in normal testes. These findings suggest that the light cells which appear in the peritoneal epithelium of the testes on administration of estrogen may be germ line cells.     

Fine structural study of the acrosome formation in the starfish Marthasterias glacialis (Echinodermata, Asteroidea)

The fine structure of the spermatogenic cells in the starfish Marthasterias glacialis was studied regarding acrosome formation. The main finding in the spermatogenesis of M. glacialis is that the formation of the pro-acrosomal vesicles seems to be initiated in late spermatogonia. Small dense bodies resulting from the division of large granulofibrillar masses were also observed in the cytoplasm of late spermatogonia. During spermiogenesis the inner acrosomal vesicle membrane becomes coated first with dense materials originated from the cytoplasmic dense bodies and then with cisternae of endoplasmic reticulum. Both coating materials are incorporated in the periacrosomal space of the mature acrosome. Besides being involved in the genesis of the periacrosomal material, cytoplasmic dense bodies were also seen in close relationship with intercellular bridges and midpiece structures of spermatids. These findings are discussed in comparison with other echinoderm spermatogenesis.     

Response of the human testis to long-term estrogen treatment: Morphology of Sertoli cells,Leydig cells and spermatogonial stem cells

Summary The present investigation is concerned with the morphological changes observed in human testicular tissue following prolonged estrogen administration. Testicular material obtained from 11 transsexual patients who had been submitted to long-term estrogen treatment prior to sex-reversal surgery was studied by means of light- and electron microscopy.The testes of all patients examined present a more or less uniform appearance: There are narrow seminiferous cords surrounded by an extensively thickened lamina propria. They contain Sertoli cells and spermatogonia exclusively. There is no evidence of typical Leydig cells.The persisting spermatogonia show the characteristic features of pale type-A spermatogonia, whereas dark type-A spermatogonia are almost completely eliminated from the epithelium. In view of the fact that spermatogonia that survived radiotherapy and treatment with various noxious agents have recently been regarded as the stem cells of the human testis, it is suggested that also the majority of those spermatogonial types that are less sensitive to disturbances of the endocrine balance may consist of stem cells. The present results, therefore, corroborate the concept that the stem cells of the human testis may be derived from pale type-A spermatogonia or the variants of this cell type.Sertoli cells display two types of ovoid nuclei. In contrast to untreated material the nuclei lie adjacent to the basal lamina, and organelles and telolysosomes are confined to the apical cytoplasm. The apico-basal differentiation of mature cells, therefore, is not observed. Moreover, typical organelles and inclusions of mature cells are absent, as are the junctional specializations. Thus, Sertoli cells have transformed into immature cells, resembling precursors prior to puberty.Fibroblast-like cells in the interstitial tissue, which display strongly lobulated nuclei, a well-developed smooth endoplasmic reticulum, lipid droplets, and numerous inclusions are assumed to represent dedifferentiated Leydig cells.Since after estrogen treatment serum testosterone and gonadotropin levels are known to be reduced, it appears that the morphological changes correlate well with the endocrine status.     

Ultrastructure of the testis in Synbranchus marmoratus (Teleostei,Synbranchidae): the germinal compartment

Synbranchus marmoratus, is a protogynic diandric species in which two types of males, primary and secondary, are found. In both types, the germinal compartment in the testes is of the unrestricted lobular type, but in secondary (sex reversed females) males the lobules develop within the former ovarian lamellae. In the present study, the germinal compartment was examined in both types of males using light microscopy as well as scanning and transmission electron microscopy. Germinal compartment is limited by a basement membrane and contains Sertoli and germ cells. During maturation, processes of Sertoli cells form the borders of spermatocysts containing isogenic germ cells. Characteristically, type A and type B spermatogonia have a single nucleolus and grouped mitochondria associated with dense bodies or nuage. Type B spermatogonia, spermatocytes and spermatids are joined by cytoplasmatic bridges and are confined within spermatocysts. Secondary spermatocytes are difficult to find, indicating that this stage is of short duration. Biflagellated spermatozoa have a rounded head, no acrosome, and possess a midpiece consisting of two basal bodies, each of which produces a flagellum with a typical 9+2 microtubular composition. No associations occur between sperm and Sertoli cells. There were no differences between spermatogenesis in primary and secondary males in this protogynic, diandric fish.     

Origin of Germ Cells and Early Differentiation of Gonads in the Starfish, Asterina pectinifera

The origin of the germ cells and the development of the genital system in the annually spawning starfish, Asterina pectinifera , were studied by light and electron microscopy. Characteristic germ cells were first characterized in gonads after spawning: the gonia are larger than somatic cells, have large nuclei (with electron-lucent nucleoplasm), and show mitochondrial aggregation associated with nuage (electron-dense bodies). In young starfish without gonads similar cells were detected in the haemal sinus, where they were termed primordial germ cells (PGCs). Brachiolariae and metamorphosed juveniles had a cellular cluster in the coelomic epithelium, near the hydroporic canal. The cluster was comprised of cells endowed with the above-mentioned characteristics of the germ cells. The germ cell counts indicated that PGCs migrate from the aboral haemal sinus near the hydroporic canal, through the haemal sinus to the gonads, where they settle, proliferate, and differentiate into gonia.     

Ultrastructure of developing germ cells in the fetal human testis

Electron microscopic studies of the testis were performed on 12 human embryos and fetuses between 9 and 30 weeks post conceptionem. According to their ultrastructural features, the fetal germ cells could be divided into the following three stages of differentiation: 1) gonocytes, 2) intermediate cells, and 3) fetal spermatogonia. Sertoli cells were present among the germ cells in all the testes studied. The gonocytes showed the highest nucleo-cytoplasmic ratio. Their round nucleus contained a centrally located, prominent nucleolus. The cytoplasm displayed a well developed Golgi apparatus, lipid droplets and parallel arrays of short cisternae of the rough surfaced endoplasmic reticulum (rER). Microfilaments were numerous, particularly just beneath the cell membrane. The intermediate cells were found to extend several cytoplasmic processes and to contain a moderate number of long, branched and/or widened rER cisterna which were frequently connected to the perinuclear cisterna. Intermediate cells were often connected to one another by intercellular cytoplasmic bridges. The fetal spermatogonia also displayed cytoplasmic bridges. These cells showed the lowest nucleo-cytoplasmic ratio and more condensed nuclear chromatin. The mitochondria were situated close to the nucleus. Many of them were connected by a cementing substance. Lipid droplets and rER cisternae were rare in these cells. Infoldings of the inner nuclear membrane were often present in the gonocytes and in the intermediate cells, but were rarely observed in the fetal spermatogonia. Glycogen particles, polyribosomes, and chromatoid bodies ("nuage") were present in all the three germ cell types. With the maturation of the fetus, the number of gonocytes was found to decrease, whereas the number of fetal spermatogonia increased. The Sertoli cells also changed their ultrastructure, showing an increase in the number of rER cisternae, as well as of microfilaments, lipid droplets, and secondary lysosomes.     

Ultrastructural changes in the spermatogonia and spermatocytes of Poecilia reticulata during spermatogenesis

Summary The structure of guppy (Poecilia reticulata) spermatogonia and spermatocytes has been studied using electron microscopy. The spermatogonia, situated at the apex of the seminiferous tubule, are almost all surrounded by a network of Sertoli cells; they have very diffuse chromatin and one or two large nucleoli. The cytoplasm contains relatively few organelles, although annulate lamellae are found. The mitochondria have few cristae and are concentrated at one pole of the cell; they are sometimes found with intermitochondrial cement. These spermatogonia are separated from each other, having no intercellular bridges or inclusion in Sertoli cells, and are relatively undifferentiated; they correspond to stem cells. The spermatogonia beneath the apex are organized into cysts. First-generation spermatogonia are more dense and heterogeneous, their nuclei becoming smaller and their chromatin becoming denser during successive generations. In spermatocytes, the synaptinemal complex exists as a modified form until metaphase. The concentration of organelles in the cytoplasm increases and the organelles become more diversified as spermatogenesis progresses. Many cytoplasmic bridges are observed (several per cell), indicating that the cells remain in contact after several divisions. These changes in germ cell structure have been related to some of the characteristic features of spermatogenesis in guppy, e.g. the large number of spermatogonial generations and the complexity of spermiogenesis.     

Origin of nucleolus-like bodies found in the nucleoplasm and cytoplasm of Vicia faba meristematic cells

Cytohistochemical staining and RNase-gold labelling have been applied to root-tip meristematic cells of Vicia faba to study the origin and biological significance of 2 types of inclusions: one seen in the nucleoplasm and the other in the cytoplasm of early telophase cells. They have been termed "dense bodies" and "cytoplasmic nucleolus-like bodies" (NLB), respectively. Both types of inclusions respond positively to silver staining and ribonucleoprotein (RNP) staining in a similar fashion to nucleolus. Interestingly, the dense bodies label heavily with the RNase-gold complex, as does the nucleolus, while the cytoplasmic NLB have no affinity with the label. In most cases, the dense bodies label more heavily than the nucleolus. Light microscope surveys reveal that the dense bodies sometimes appear to be released from the surface of the nucleolus. On the other hand, prenucleolar material showing the same silver staining and RNP preferential staining characteristics as the dense bodies begin to accumulate on the surface of chromosomes in mid-anaphase. This material does not label with RNase-gold. These data are discussed in terms of the hypothesis that the dense bodies are derived from the nucleolus by direct budding or fragmentation, and the cytoplasmic NLB are composed of prenucleolar material that failed to attach to chromosomes.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

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).     

Germ cell sex prior to meiosis in the rainbow trout

Germ cells make two major decisions when they move from an indeterminate state to their final stage of gamete production. One decision is sexual commitment for sperm or egg production, and the other is to maintain mitotic division or entry into meiosis. It is unclear whether the two decisions are made as a single event or separate events, because there has been no evidence for the presence of germ cell sex prior to meiosis. Here we report direct evidence in the fish rainbow trout that gonia have distinct sexuality. We show that dazl expression occurs in both male and female gonia but exhibits differential intracellular distribution. More strikingly, we show that boule is highly expressed in male gonia but absent in female gonia. Therefore, mitotic gonia possess sex, sperm/egg decision and mitosis/meiosis decision are two independent events, and sperm/egg decision precedes mitosis/meiosis decision in rainbow trout, making this organism a unique vertebrate model for mechanistic understanding of germ cell sex differentiation and relationship between the two decisions.