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.     

PEARL MILLET (PENNISETUM AMERICANUM (L.) LEEKE) AND GRAIN SORGHUM (SORGHUM BICOLOR (L.) MOENCH) ULTRASTRUCTURE

Ultrastructural features of pearl millet (Pennisetum americanum (L.) Leeke) and grain sorghum (Sorghum bicolor (L.) Moench) caryospses were investigated with thin sections of the dry, mature grain in the transmission electron microscope, and fractured kernels in the scanning electron microscope. The pericarp of those grains is comprised of three distinct layers: epicarp, mesocarp of parenchyma cells, and endocarp of compressed cross and tube cells. Mesocarp cells of grain sorghum contain starch granules embedded in a cytoplasmic matrix. The major constituent of sorghum and millet aleurone cells are aleurone grains (protein bodies) and lipid bodies. Subaleurone cells contain a much higher proportion of protein bodies than starch granules, and the protein bodies are structurally distinct from those in the aleurone. The germ scutellar ultrastructures of the two grains were similar; protein bodies, lipid bodies, epidermal cells and parenchyma cells of the germ are described.     

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.     

Germ-line cysts are formed during oogenesis in Erpobdella octoculata (Annelida,Clitellata, Erpobdellidae)

Abstract

Erpobdella octoculata (Clitellata, Hirudinea, Erpobdellidae) has paired ovarian sacs, each containing several rod-shaped structures termed ovarian bodies. Oogenesis takes place within the ovarian bodies. We show that in the apical part of the bodies the germ-line cells form syncytial cysts of cells interconnected by stable intercellular bridges. Germ-line cyst architecture is broadly similar to that of other clitellate annelids; that is, each germ cell has only one intercellular bridge connecting it to the anuclear cytoplasmic mass, the cytophore. Unlike germ-line cysts described in other leech species, the cytophore in cysts of E. octoculata is poorly developed, taking the form of thin cytoplasmic strands. Oogenesis in E. octoculata is meroistic because the germ cells forming the cysts (cystocytes) have diverse fates, i.e., nurse cells and oocytes appear. One large ramified cell (apical cell) occurs within the apical part of the ovarian body. We compare the ultrastructure of the apical cell found in E. octoculata with that of apical cells described recently in some hirudiniform leeches. The germ-line cysts as well as the oocytes are enveloped by somatic follicular cells. As in other leeches, the follicular cells surrounding the growing oocytes have cytoplasm perforated by intracellular canals. In view of the many similarities between E. octoculata ovarian bodies and the ovary cords described in glossiphoniids and especially in hirudiniform leeches, we suggest that the ovarian bodies found in E. octoculata are in fact modified ovary cords.     

Ovary cord structure and oogenesis in Hirudo medicinalis and Haemopis sanguisuga (Clitellata, Annelida): remarks on different ovaries organization in Hirudinea

In Hirudo medicinalis and Haemopis sanguisuga, two convoluted ovary cords are found within each ovary. Each ovary cord is a polarized structure composed of germ cells (oogonia, developing oocytes, nurse cells) and somatic cells (apical cell, follicular cells). One end of the ovary cord is club-shaped and comprises one huge apical cell, numerous oogonia, and small cysts (clusters) of interconnected germ cells. The main part of the cord contains fully developed cysts composed of numerous nurse cells connected via intercellular bridges with the cytophore, which in turn is connected by a cytoplasmic bridge with the growing oocyte. The opposite end of the cord degenerates. Cord integrity is ensured by flattened follicular cells enveloping the cord; moreover, inside the cord, some follicular cells (internal follicular cells) are distributed among germ cells. As oogenesis progresses, the growing oocytes gradually protrude into the ovary lumen; as a result, fully developed oocytes arrested in meiotic metaphase I float freely in the ovary lumen. This paper describes the successive stages of oogenesis of H. medicinalis in detail. Ovary organization in Hirudinea was classified within four different types: non-polarized ovary cords were found in glossiphoniids, egg follicles were described in piscicolids, ovarian bodies were found characteristic for erpobdellids, and polarized ovary cords in hirudiniforms. Ovaries with polarized structures equipped with apical cell (i.e. polarized ovary cords and ovarian bodies) (as found in arhynchobdellids) are considered as primary for Hirudinea while non-polarized ovary cords and the occurrence of egg follicles (rhynchobdellids) represent derived condition.     

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.     

Fine-structural characteristics of female and male germ cells in Proseriata Otoplanidae (Platyhelminthes)

Fine-structural features of female germ cells differentiating within the germaria of Otoplanella baltica and Notocaryoplanella glandulosa are documented and compared with those of other free-living platyhelminths having ectolecithal eggs.In these species, encompassed in the taxon Proseriata Lithophora, insemination of the germocytes occurs within the germaria. A sperm cell, that has penetrated a germocyte differs in special features from mature male germ cells found in the testes, in parts of the male genital system, or even in other regions of the organism. The hypothesis that dense bodies correspond to acrosomal material is supported.     

Fine structure of germinatingPenicillium megasporum conidia

Summary Penicillium megasporum conidia have spore walls consisting of several layers. There is no visible change in the outer wall layers during spore germination, but the inner layers increases in thickness on only one side of the spore, resulting in a rupture of the outer wall layers and subsequently in germ tube formation. Invaginations in the plasma membrane disappear as the germ tube forms and emerges, and the nucleus migrates into the developing germ tube. Mitochondria gather at the base of the germ tube during its formation. During germination, the amount of lipid in the spore decreases and portions migrate into the germ tube. Membrane-bound, electron dense bodies are present in resting spores. These bodies decrease in size as germination proceeds, and the cytoplasm in the developing germ tube appears much more electron dense than the cytoplasm within the spore.     

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.     

Differentiation of Pluripotent Embryonic Stem Cells in the Peritoneal Cavity of Irradiated Mice

We studied the behavior and differentiation of pluripotent embryonic stem cells of R1 mice in vivo. Undifferentiated embryonic stem cells and differentiating embryoid bodies implanted in the abdominal cavity of irradiated mice were shown to form tumors containing the derivatives of all germ layers. Cells of the embryoid bodies form tumors two weeks after implantation, while undifferentiated embryonic stem cells form tumors only by week three.     

Genital primordium of the free-living marine nematode Halichoanolaimus sonorus (Chromadoria,Chromadorida)

Summary

The genital primordium of the first stage juvenile (J1) of the free-living marine nematode Halichoanolaimus sonorus (Chromadorida: Selachinematidae) was studied using transmission electron microscopy. The primordium consists of four undifferentiated cells: two primordial germ cells (PGC) 5–6 μm in diameter and two somatic cells. The PGC have a large nucleus with nucleolus. The centriole was detected in close vicinity of the PGC nucleus. Most of the cell mitochondria are in close contact with the nuclear envelope. The mitochondria are interspersed by 0.2–0.3 μm particles of an electron-dense diffuse substance devoid of surrounding membrane. Both PGC are closely attached to each other and to the neighboring somatic cells of the genital primordium. The elongated somatic cells contain nuclei devoid of nucleoli; the cytoplasm is filled with free ribosomes and contains occasional cisternae of rough endoplasmatic reticulum (RER), Golgi bodies, mitochondria, and transparent vesicles. The genital primordium is separated by a narrow space from of the intestine (dorsally) and the somatic muscles (ventrally). The PGC of H. sonorous are devoid of typical P granules known for previously studied nematodes as distinct markers of germ line cell lineage. Perinuclear particles of dense diffuse substance found in PGC of H. sonorous could be considered as germ determinants analogous to P granules.     

The structure of the germinal dense bodies (nuages) during differentiation of the male germ line of the teleost,Oryzias latipes

Summary The germinal dense body (GDB) in the teleost, Oryzias latipes, an organelle unique to the cells of germ line, is regarded as a counterpart of nuage material in amphibians and mammals. In the study described herein, GDBs in male germ line cells were examined by electron microscopy. GDBs existed continuously in the cytoplasm of primordial germ cells (PGCs), prespermatogonia, type-A spermatogonia and early type-B spermatogonia. But they became rudimentary in late type-B spermatogonia and early spermatocytes, and no longer occurred in spermatids. Differences in the morphology of GDBs of PGCs and male germ cells were also noted. In PGCs of indifferent gonads, about 50% of GDBs were amorphous bodies of fine electron-dense fibrils, whereas in spermatogonia amorphous bodies decreased in number and GDBs of strand-like structure were more frequent. The change in the morphology of GDBs began when the sex differentiation of gonads became evident, and proceeded gradually in prespermatogonia. No obvious differences in morphology of GDBs were noted between prespermatogonia in the fry at later stages of development and spermatogonia in adult fish.     

The planarian <Emphasis Type="Italic">nanos</Emphasis>-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE CARYOPSIS COAT AND THE ALEURONE CELLS

The results of a light and electron microscopic study of the caryopsis coat and aleurone cells in ungerminated, unimbibed rice (Oryza sativa) caryopses are presented. Surrounding the rice grain is the caryopsis coat composed of the pericarp, seed coat and nucellar layers. The outermost layer, the pericarp, consists of crushed cells and is about 10 μm thick. The seed coat, interior to the pericarp, is one cell thick and has a thick cuticle. Between the seed coat cuticle and endosperm are the remains of the nucellus. The nucellus is about 2.5 μm thick and has a thick cuticle adjacent to the seed coat cuticle. Interior to the caryopsis coat is the aleurone layer of the endosperm. The aleurone completely surrounds the rice grain and is composed of two cell types—aleurone cells that surround the starchy endosperm and modified aleurone cells that surround the germ. The aleurone cells of the starchy endosperm contain many aleurone grains and lipid bodies around a centrally located nucleus. The modified aleurone cells lack aleurone grains, have fewer lipid bodies than the other aleurone cells, and contain filament bundles (fibrils). Plastids of aleurone cells exhibit a unique morphology in which the outer membranes invaginate to form tubules and vesicles within the plastid. Transfer aleurone cells are not observed in the mature rice caryopsis.     

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, “Stefanidis medium” from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.     

Dry mass of Fusarium roseum spores before and after germination

The total dry mass of Fusarium roseum spores and contained lipid bodies were determined before and after spores germinated using quantitative interference microscopy. The mean for spore dry mass before germination was about 57 pg. Lipid bodies accounted for about 61% of that. Areas of lipid bodies in spores before and after germination were about 23 % but the contents of the lipid bodies accounted for only 10% of the spore dry mass after germination. The total dry mass of the spore and germ tube(s) greatly exceeded that of the spore before germination. We infer that nutrients for germ tube growth are derived from within the germinating spore and from the medium which must contain nutrients leached from non-germinating spores.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

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.