Evidence for asynchronous mitotic cell divisions in secondary spermatogonia of Drosophila

Examination of germ cell numbers within premeiotic as well as postmeiotic cysts of various Drosophila species gave evidence against any strict synchrony of mitotic cell division in secondary spermatogonia. The evidence was based on numbers of germ cells in primary spermatocyte cysts and spermatid bundles. Each species examined had its own distribution of primary spermatocyte cyst types in pupal testes, and the most common cyst type did not necessarily contain 2, 4, 8, 16 or (2) ⁿ germ cells which implies asynchrony of the previous spermatogonial divisions. Similar but not exactly the same distributions of germ cells were found in adult spermatid bundles, if allowances were made for a 4-fold increase in germ cell number during meiosis. This observation gives support to the operation of an age-dependent factor which controls germ cell numbers within cysts [1], The data thus suggest that the commonly accepted concept of a (2) ⁿ increase of spermatogonia via synchronous mitotic divisions is not true for the species of Drosophila studied.     

White pulp compartments in the spleen of the turtle Mauremys caspica

Summary The aim of the present study was to analyze the nature of lymphoid and non-lymphoid cellular components occurring in distinct histological compartments of the splenic white pulp of the turtle, Mauremys caspica, in order to define their possible correlations with those of the spleen of higher vertebrates, principally mammals. The white pulp of M.caspica consisted of 3 clearly distinguishable regions: (1) the periateriolar lymphoid sheath, and (2) the inner and (3) the outer zones of the periellipsoidal lymphoid sheath. Reticular cells intimately associated with reticular fibres constituted an extensive meshwork in the periarteriolar lymphoid sheath which housed principally Ig-negative lyphoid cells, mature and immature plasma cells, and interdigitating cells. A few Ig-positive cells were also present in the peripheral region of the periarteriolar lymphoid sheath. The inner and outer zones of the periellipsoidal lymphoid sheath were separated by a discontinuous layer of reticular cell processes. In the inner zone, surface Ig-positive lymphoid cells predominated as well as dendritic cells, resembling ultrastructurally the mammalian follicular dendritic cells, although no germinal centres were found in the turtle spleen. Macrophages, some cytoplasmic Ig-positive cells, and Ig-negative lymphoid cells appeared in the outer zone of the periellipsoidal lymphoid sheath. These results allow us to speculate on a phylogenetic relationship between the periarteriolar lymphoid sheath and the inner and the outer zones of the periellipsoidal lymphoid sheath of the spleen of M. caspica and the periarteriolar lymphoid tissue, the lymphoid follicles and the marginal zone, respectively, of the mammalian splenic white pulp.     

The interstitial origin of germinal cells in the testis of the stickleback

The brook stickleback, Culaea inconstans (Kirtland), in common with other bony fishes, lacks a germinal epithelium in the tubules of the testis, and the tubule wall is composed of a thin, discontinuous layer of myoid cells and collagenous fibers. Labelling of germ cells with tritiated thymidine has shown that the germ cells are derived from clumps of spermatogonia in the interstitial area. Large companion cells within the lumina of the tubules extend their processes to engulf spermatogonia from the interstitium which then enter the lumen of the tubule. Subsequent development of the germ cells takes place within individual compartments formed by folds of the plasma membrane of a companion cell. The companion cell, together with its complement of germ cells, constitutes a cyst. A companion cell may surround spermatogonia in the interstitium and at the same time encompass residual sperm of the previous season within the lumen. The plasma membranes of the germ cells and the companion cells remain discrete. Mature sperm are released into the lumen of the tubule and the companion cell again extends its processes into the interstitium and engulfs more spermatogonia for the following year. Companion cells may be homologous to the Sertoli cells of higher vertebrates although their processes penetrate the interstitium during the initial stages of spermatogenesis and they do not contain a permanent stock of spermatogonia.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

Ultrastructural study of embryonic and early adult germ cells,and their support cells,in both sexes of Xiphophorus (Teleostei:Poeciliidae)

The ultrastructure of the early spermatogonia in mature testes of the platyfish, Xiphophorus maculatus, was compared to that of oogonia in mature ovaries of X. maculatus and the related X. nigrensis. Both cell types were very similar and, characterized as being large, oval to round cells containing large, central nuclei with prominent nucleoli. Abundant mitochondria with sparse transverse cristae were located at one pole or around the nucleus. Annulate lamellae and electron-dense granular material (nuage) were present. Other organelles were not prominent. A female that had received a testis graft had testicular tissue containing mature spermatozoa within the ovary, indicating that cells were present that could develop along the male line. Special crosses were carried out to obtain all-male embryos of X. maculatus and all-female embryos of X. nigrensis. The ultrastructure of the germ cells in all embryonic gonads was similar to that of the adult cells. These results suggest the presence of sexually undifferentiated germ cells in the adult gonads of both sexes. The support cells investing all of these germ cells were also similar structurally and appeared to be undifferentiated.     

Histological, histochemical, enzyme histochemical and ultrastructural investigations of the testis of Padogobius martensi between annual breeding seasons

From July to March, the testis of the spring‐spawning freshwater goby Padogobius martensi is characterized by spermatogonial proliferation. A close correlation exists among type of proliferating spermatogonia, gonado‐somatic (I_G) profiles and morphological and functional variations of the Leydig cells. The I_G reach their minimal levels by the end of summer and increase progressively but modestly during autumn and winter. Declining I_G levels are associated with proliferation of primary spermatogonia only, whereas increasing I_G levels are associated with predominant proliferation of secondary spermatogonia. Minimal I_G levels are reached when the germinal epithelium is formed by a continuum of primary spermatogonia and associated Sertoli cells. The proliferation of secondary spermatogonia begins only at this time. Spermatogenesis in autumn occurs when spermatogonial cysts contain at the most 16 cells and it rarely results in the maturation of several cysts so that the amount of sperm cells produced is either negligible or scarce. A number of degenerating cells are usually present within the spermatogonial and meiotic cysts. Leydig cells are the unique cells that display features of steroidogenic cells: mitochondria with tubular cristae, extensive smooth endoplasmic reticulum (SER), 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and glucose‐6‐phosphate dehydrogenase (G6PD) activity and sudanophilia. Light and dark Leydig cell varieties are always present. During regression, Leydig cells undergo a marked decrease in SER amount, mitochondrial sizes and number of mitochondrial cristae. In parallel, the 3β‐HSD and G6PD activities and sudanophilia decrease progressively until they become undetectable by the end of regression. In autumn, mitochondria increase in size, reaching sizes similar to those observed at the end of the spawning season in the light cells, but not in the dark cells. The SER, on the contrary, undergoes a modest and irregular increase only in a part of the Leydig cells, mostly of the light type. In parallel, the 3β‐HSD and G6PD activities increase until they become moderately intense by the end of autumn. At the end of winter, the SER is extensive and regularly dilated in both Leydig cell types, whereas mitochondria still have sizes similar to those observed in December. The 3β‐HSD and G6PD activities are strong and sudanophilia is again detectable. Sertoli cells undergo changes in shape and position in relation to the proliferation of primary spermatogonia and the development of cysts. A junction modulation occurs in association with these changes. Sertoli cells also undergo changes indicative of a decrease in activity immediately after spawning (loss of mitochondrial cristae and clarification of the mitochondrial matrix) and of an increase in activity by the end of the regressing phase (darkening of the mitochondrial matrix and increase in mitochondrial cristae, rough endoplasmic reticulum (RER) and free ribosomes). In addition, they are involved in the phagocytosis of degenerating germ cells at all stages of their development. Macrophages are found in the testis interstitium only, where they are usually adjacent to Leydig cells, myoid cells and blood capillaries and do not participate in the phagocytosis of degenerating germ cells. Myoid cells do not undergo ultrastructural changes except for an increase in the amount of heterochromatin by the end of spawning. The meaning of the autumnal spermatogenic wave and the relationships between the development of the germinal epithelium and the changes of the Leydig and Sertoli cells are discussed.     

Degeneration of germ line cells in amphibian ovary

Ogielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91 : 319–327 We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.     

Expression of the Intermediate Filament Keratin Gene,K15,in the Basal Cell Layers of Epithelia and the Hair Follicle

The intermediate filament keratin, K15, is present in variable abundance in stratified epithelia. In this study we have isolated and characterized the sheepK15gene, focusing on its expression in the follicles of sheep and mice. We show thatK15is expressed throughout the hair cycle in the basal layer of the outer root sheath that envelops the follicle. Strikingly, however, in large medullated wool follicles, a small group of basal outer root sheath cells located in the region thought to contain hair follicle stem cells areK15-negative. In the follicle bulbK15is expressed in cells situated next to the dermal papilla but not in the inner bulb cells. Elsewhere,K15is expressed at a low, variable level in the basal layer of the epidermis and sebaceous gland, often in a punctate pattern. In the esophagus of the sheepK15expression is restricted to the basal layer, in contrast to human esophagus where it is expressed throughout the epithelium. Transgenic mouse lines established with a 15-kb sheepK15gene construct exhibited faithful expression and showed no phenotypic consequences ofK15overexpression. An investigation of transgene expression showed thatK15is continuously expressed in outer root sheath cells during the hair cycle. Given its expression in the mitotically active basal cell layers of diverse epithelia and the follicle,K15expression appears to signal an early stage in the pathway of keratinocyte differentiation that precedes the decision of a cell to become epidermal or hair-like.     

Follicle cells and germ line cells both affect polarity in dicephalic chimeric follicles of Drosophila

Summary In aberrant egg follicles of the pattern mutant dicephalic (dic) the oocyte is wedged in between two groups of nurse cells, and this condition may give rise to embryos which express anterior traits at both ends. We have analysed the role of the dic genotype of the germ line cells and the surrounding somatic follicle cells in the formation of the dic follicular phenotype. By means of pole cell transplantations into Fs (1) K 1237 hosts (this cell-autonomous mutation causes degeneration of the host's germ line cells early in oogenesis), we constructed chimeras in which either the follicle cells, the germ line cells, or both were homozygous for the dic mutation. In all three combinations the dic phenotype was expressed but not in controls with dic ⁺ in both germ line cells and follicular epithelium. Since follicles with the dic phenotype may be produced if either the germ line cells or the follicle cells lack dic ⁺ gene activity we suggest that cellular interactions between both cell types are required for the correct positioning of the oocyte at the follicle's posterior pole.     

MORPHOLOGY AND FINE STRUCTURE OF FISCHERELLA AMBIGUA1

Fischerella ambigua is a branching blue-green alga, the filamentous nature of which is maintained almost entirely by sheath material. Cell division in this organism most closely resembles the septal division found in most unicellular organisms. In all filamentous blue-green algae previously examined with the electron microscope, cell division has resulted from the imagination of the plasma membrane and inner wall layer only; both the middle wall and the outer wall layers remain continuous throughout the length of the filament. In Fischerella, by contrast, the plasma membrane and the inner wall layer invaginate to produce initially 2 cells. However, the middle wall layer, outer wall layer, and sheath also invaginate to separate the daughter cells. The sheath alone remains continuous throughout the length of the filament.     

Phenotypic changes in germ cells during gonadal development of the common carp (Cyprinus carpio)

A procedure is described in which large early spermatogonia were isolated from carp testes and purified from an initial 4–5% recovery up to 60–70% using equilibrium density centrifugation on a continuous Percoll gradient. Mice were immunized with the spermatogonia via the intrasplenic route. Six hybridoma cultures, producing monoclonal antibodies (MAbs) reacting selectively with germ cells, were selected and further analysed. Reactivity with five of these MAbs was observed on primordial germ cells (PGCs) in the developing indifferent gonads at the onset of proliferation, i.e. the age of 7 weeks. One MAb, encoded WCG 6, appeared to define a new surface marker on PGCs being gradually expressed on the surface membrane between the age of 2 and 4 weeks, concomitantly with an increase in size of these mitotically silent cells. The reactivity of germ cells with five of the MAbs disappeared completely (WCG 7, 12, 15, 21) or nearly completely (WCG 6) during spermatogenesis, providing a striking difference from patterns obtained with MAbs raised previously against carp spermatozoa. Differences between male and female germ cells were not observed with the WCG-MAbs during gonad development, indicating that a common set of surface antigens is shared between germ cells of both sexes up to and including spermatogonia and oogonia.Abbreviation WCG Wageningen carp spermatogonia antibody     

Differentiation of Primary Spermatocytes to Elongated Spermatids by Mammalian FSH in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

Our previous studies (10, 11) showed that mammalian follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the initiation and promotion of spermatogenesis from secondary spermatogonia to primary spermatocytes in organ culture of testes fragments from the newt, Cynops pyrrhogaster. The present study demonstrated that FSH promoted in the same model system the differentiation of primary spermatocytes even further: to the stage of elongated spermatids. When testes fragments, consisting of somatic cells and germ cells (mostly primary spermatocytes), were cultured in a control medium for three weeks, only round spermatids and spermatogonia were observed; both the diameter of the cysts and the viability of the germ cells decreased to about 10–15% of the original level. On the other hand, when the medium was supplemented with FSH, elongated spermatids appeared by the second week; both the diameter of the cysts and the viability of the germ cells were maintained at a higher level than in the control medium. The effect of FSH was dose-dependent. However, neither transferrin, androgens (testosterone and 5α-dihydrotestosterone) nor luteinizing hormone (LH) was effective. The addition of cyanoketone, a specific inhibitor of 3β-hydroxy-Δ⁵-steroid dehydrogenase (3β-HSD) (32), to the FSH-containing medium did not prevent the differentiation promoted by FSH, indicating that it is unlikely that Δ⁴-steroid metabolites produced in fragments by FSH acted directly on germ cells. Insulin was found to improve the viability of germ cells during a 2 week of culture period. In the presence of FSH, the cells in various differentiative stages had morphological characteristics very similar to those in vivo, whereas in the absence of FSH primary spermatocytes showed abnormal features in their nuclei and cytoplasm, indicating that they were deteriorating. These results and our previous results (1–3) suggest that FSH promotes primary spermatocytes to differentiate into elongated spermatids probably by stimulating Sertoli cells to secrete factors which then act on the germ cells.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

The structure of Verson's cells in Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Testis follicles of Lepidoptera contain a large somatic cell termed Verson's cell. The present study focuses on the structure of Verson's cells and neighbouring germ cells in the Mediterranean mealmoth, Ephestia kuehniella (Pyralidae), using electron microscopy, antitubulin immunofluorescence, and phalloidin incubation for the visualization of microfilaments. Verson's cells of young larvae are connected with the follicle boundary and show large areas containing packages of glycogen particles, whereas Verson's cells of pupae lie freely within the testis follicle and are largely devoid of glycogen. Both developmental stages of Verson's cells have in common the presence of a dense cytoplasmic network of microtubules. A juxtanuclear subset of the cytoplasmic microtubule array is recognized by an antibody against acetylated microtubules. This indicates that more stable microtubules exist in this region. Microfilaments are arranged parallel to the cytoplasmic microtubules. The microtubule-microfilament-complex forms a cytoskeleton that may keep larger organelles, such as mitochondria and lysosomes, in a juxtanuclear position. Chromatin within the nuclei of Verson's cells is largely decondensed and nuclear pores are abundant. This indicates a high synthetic activity within the cells. The development of cells directly attached to Verson's cells, viz. prespermatogonia, may be controlled by the Verson's cells. Prespermatogonia, which differ in cytoplasmic density from spermatogonia further away from Verson's cells, may represent stem cells that give rise to spermatogonia and somatic cyst cells upon detachment from Verson's cells. This suggestion is compatible with the low division rate of prespermatogonia.     

Surface location and stage-specificity of differentiation antigens on germ cells in the common carp (Cyprinus carpio), as revealed with monoclonal antibodies and immunogold staining

Summary During development of juvenile and young adult carp (Cyprinus carpio, L., Teleostei) three differentiation stages were distinguished in the testis: the prespermatogenic, the early spermatogenic and the advanced spermatogenic testis. Carp testis tissue of these stages was dissociated by enzymatic digestion and viable testis cells with well preserved morphological features were obtained. The surface location and stage-specificity of differentiation antigens on these germ cells was investigated using monoclonal antibodies (MAbs) raised against carp spermatozoa. Binding of MAbs to cells was visualized with immunofluorescence as well as in the immunogold staining assay. Both methods revealed that antigenic determinants defined by seven MAbs were located on the outer surface of testis cells. Four MAbs, i.e. WCS 3, 17, 28 and 29, reacted with germ cells from both pre-spermatogenic testes (WCS 28 weakly) and spermatogenic testes. The antigenic determinants defined by three other MAbs, i.e. WCS 7, 11 and 12, appeared only after the onset of spermatogenesis. In the immunogold staining assay a post-fixation and nuclear staining procedure was developed which allowed identification of isolated germ cells, revealing clearly, for all seven MAbs, that the determinants were expressed on germ cells but not on somatic cells and, for WCS 7, 11 and 12 only, that the determinants first appeared on small spermatogonia prior to meiosis. A survey of the immunogold assay on the binding of the seven MAbs with isolated germ cells from ovaries, is included.     

Receptor-mediated and adsorptive endocytosis by male germ cells of different mammalian species

Summary The routes for adsorptive and receptor-mediated endocytosis were studied in vivo after microinjection of tracers into the lumen of the seminiferous tubules, and in vitro in isolated germ cells of different mammals. Cationic ferritin was located on the plasma membrane, in vesicles, in tubules, in multivesicular bodies and in lysosome-like granules of mouse spermatocytes. In these cells the number of multivesicular bodies varied during spermatogenesis. Spermatids and to a lesser extent residual bodies also performed adsorptive endocytosis. In the rat and monkey (Macaca fascicularis) diferric transferrin was specifically taken up by germ cells via receptor-mediated endocytosis. The labelling was observed subsequently in membrane pits, vesicles, endosome-like bodies and pale multivesicular bodies. A progressive decrease in the frequency of the labelling of the germ cells by transferrin-gold particles was observed from spermatogonia to spermatocytes and to early spermatids, which could indicate that iron is particularly required by germ cells during the mitotic and meiotic processes. Adsorptive and receptor-mediated endocytosis therefore occurs in all classes of germ cells. These endocytic processes are most probably required for germ cell division, differentiation and metabolism.     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

Germ-cell origin in the adult caecilian Ichthyophis glutinosus (Linn)

Summary In Ichthyophis glutinosus, no residual spermatogonia traceable to primordial germ cells of the embryo are seen, the primary spermatogonia of each season being formed afresh. Their only source so far as the adult is concerned, is the lining of the collecting duct and its numerous branches which ramify in the testis. No evidences of their origin from the surface epithelial cells of the testis, stromal cells or nurse cells are seen.