Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.     

A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells

Phosphoglycerate kinase 2 (PGK2) is a germ cell-specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affinity chromatography with the 3′-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, several associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH-type splicing regulatory protein (KSRP) were identified. KSRP, a protein of ~75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the ~75-kDa KSRP, a ~52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3′-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid–binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.     

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.     

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development.     

The chromatoid body: a germ-cell-specific RNA-processing centre

The chromatoid body, a unique cloud-like structure of male germ cells, moves dynamically in the cytoplasm of haploid spermatids, but its function has remained elusive for decades. Recent findings indicate that microRNA and RNA-decay pathways converge to the chromatoid body. This highly specialized structure might function as an intracellular focal domain that organizes and controls RNA processing in male germ cells.     

Neuroglia in the corpus callosum of the primate, slow loris (Nycticebus coucang coucang)

The corpus callosum of adult slow loris consists of a mixed population of several cell types, i.e. free subependymal cells, oligodendrocytes, astrocytes and microglia. The free subependymal cell is rather small and slender with a somewhat patchy nucleus. It shows scanty cytoplasm with free ribosomes. Oligodendrocytes vary both in nuclear and cytoplasmic densities and can be divided into three classes: light, medium dense and dark types. Their cytoplasm contains microtubules, rough endoplasmic reticulum and Golgi saccules. Astrocytes are pale cells with large amount of filaments in their cytoplasm. Microglia are small cells with granulated nuclei. The cells often show large cytoplasmic protrusions containing the usual cell organelles and lipofuscin bodies in their cytoplasm. Lastly, cells with typical features of neurons are occasionally encountered among the white matter.     

Morphogenesis and fate of the residual body in human spermiogenesis

Summary In the human testis the formation of the residual body of the spermatid and its morphological changes during and after spermiation were studied by means of electron microscopy. The caudal cytoplasmic mass of the late spermatid contains a Golgi complex, mitochondria, annulate lamellae, a chromatoid body, flower-like structures, ribosomes, a few large vacuoles, myelin-like membrane profiles and sporadic lipid droplets. When, by detachment of the caudal cytoplasm from the free spermatozoon, the residual body is formed, the chromatoid body has disappeared; the mitochondria are clustered peripherally; the ribosomes appear as a single complex in contact with a large vacuole containing granular material; in place of the Golgi complex aggregations of vesicles are present. The lipid droplets remain unchanged. The residual bodies or their fragments are either extruded via the seminiferous tubular lumen into the excurrent ducts or they are engulfed by Sertoli cells where in the supranuclear region the successive steps of decomposition can be observed. The participation of the various constituents in the disintegration of the residual body is discussed. In contrast to other mammalian species, in man the sporadic lipid droplets seem to be of minor importance in the fate of the residual body.     

Germ cell-specific DNA and RNA binding proteins p48/52 are expressed at specific stages of male germ cell development and are present in the chromatoid body

Proteins homologous to the Xenopus oocyte mRNA binding proteins mRNP₃₊₄ and designated p48/52 have been identified in male mouse germ cells (1993: Dev Biol 158:90–100). Western and Northwestern blots of extracts from testes and isolated germ cells indicate that p48/52 are present during meiosis but reach their highest levels postmeiotically at a time when many mRNAs are stored. Here we analyze the cellular and subcellular distribution of p48/52 in rat and mouse testes by LM and EM immunocytochemistry using an anti-mRNP₃₊₄ antibody. Immunolabeling was found to be predominantly cytoplasmic and specific to germ cells at certain periods during their development. p48/52 were first detected in early pachytene spermatocytes at stage V of the seminiferous cycle and progressively increased during the remainder of meiotic prophase to a post-meiotic peak in steps 1–8 round spermatids; thereafter, labeling gradually declined as elongated spermatids underwent nuclear condensation and elongation. A proportionally higher concentration of cytoplasmic immunolabeling was found within the lacunae of the anastomotic granulofilamentous network of the chromatoid body. The pattern of synthesis of these mRNA binding proteins together with their association with the chromatoid body suggests a role as germ cell-specific mRNA stabilizing and/or storage proteins. © 1996 Wiley-Liss, Inc.     

Ultrastructural observations on the germ line of Xenopus laevis

Summary The development of the male germ line in Xenopus laevis has been examined by electron microscopy. Findings have been compared to the parallel process in the female. Three structures unique to the germ line were found in both male and female cells: a fibrillar nuclear region free of DNA; largely proteinaceous masses of nuage material; and a chromatoid body. Germ plasm bodies of the egg and early embryo appear to represent a form of nuage material. The finding of a structure which can be identified as a chromatoid body in the female germ line is unique, as is its presence in sexually undifferentiated primordial germ cells. The chromatoid body in Xenopus, unlike that in mammals, does not persist in the spermatozoon. Instead, it dissociates into a series of coated vesicles during spermatogenesis. The chromosomal ultrastructure of meiotic prophase stages in Xenopus is similar in both sexes until diplotene, when male bivalents condense and enter meiotic metaphase instead of entering the extended lampbrush stage characteristic of the female. The multiple nucleoli present in gonia are lost at the onset of meiotic prophase, but no obvious mechanism for this process was observed.The author would like to thank Drs. Joseph Gall and Bernard Tandler for their helpful suggestions during the course of this investigation. The author is a postdoctoral fellow of the National Institutes of Health, U.S.A. This research was supported by N.I.H. Grants 51823 and 12427.     

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.     

The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

Formation and ultrastructural organization of gonads in Oikopleura gracilis Lohmann, 1896 (Urochordata: Appendicularia)

This study deals with the formation and ultrastructural organization of the gonads in a common species of appendicularian, Oikopleura gracilis, from Peter the Great Bay. Light microscopy observations show that the gonads develop from a transparent primordium that is located in the basolateral part of the gonad cavity; the primordium increases in size in the process of development and differentiates into the testis and ovary. The testis is covered by a single layer of ultrastructurally uniform follicular epithelium and contains a population of proliferating male gonocytes. The ovary contains two types of germ line nuclei, which are large polyploid nuclei that belong to the auxiliary cells and small meiotic nuclei of the oocytes. The two nuclei types, together with a common cytoplasm, form a syncytium of the ovary, or the coenocyst. As in the dioecious Oikopleura dioica, the coenocyst of O. gracilis produces naked oocytes that are devoid of a type III follicular membrane. The coenocyst is covered by a single-layered follicular epithelium, in which two cell types can be distinguished ultrastructurally. Thus, the synchronous maturation of sex products in O. gracilis is achieved by the formation of the germ-line syncytium in the testis and the coenocyst in the ovary, which generates a large number of simultaneously ripening oocytes that are competent for fertilization.     

Mouse Tudor Repeat-1 (MTR-1) is a novel component of chromatoid bodies/nuages in male germ cells and forms a complex with snRNPs

Characteristic ribonucleoprotein-rich granules, called nuages, are present in the cytoplasm of germ-line cells in many species. In mice, nuages are prominent in postnatal meiotic spermatocytes and postmeiotic round spermatids, and are often called chromatoid bodies at the stages. We have isolated Mouse tudor repeat-1 (Mtr-1) which encodes a MYND domain and four copies of the tudor domain. Multiple tudor domains are a characteristic of the TUDOR protein, a component of Drosophila nuages. Mtr-1 is expressed in germ-line cells and is most abundant in fetal prospermatogonia and postnatal primary spermatocytes. The MTR-1 protein is present in the cytoplasm of prospermatogonia, spermatocytes, and round spermatids, and predominantly localizes to chromatoid bodies. We show that (1) an assembled form of small nuclear ribonucleoproteins (snRNPs), which usually function as spliceosomal complexes in the nucleus, accumulate in chromatoid bodies, and form a complex with MTR-1, (2) when expressed in cultured cells, MTR-1 forms discernible granules that co-localize with snRNPs in the cell plasm during cell division, and (3) the deletion of multiple tudor domains in MTR-1 abolishes the formation of such granules. These results suggest that MTR-1, which would provide novel insights into evolutionary comparison of nuages, functions in assembling snRNPs into cytoplasmic granules in germ cells.     

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes.

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.     

The relationship between the nuage and the chromatoid body during spermatogenesis in the Rat

Summary Cytoplasmic structures ultrastructurally similar to the nuage are present in the cytoplasm of all spermatogenic cells in adult rats. The nuage is a discrete organelle which should not be confused with the chromatoid body. In step 7–8 spermatids transient contact is established between the nuage and the chromatoid body. This indicates a very specific recognition of the nuage by the chromatoid body. It is suggested that the nuage and the chromatoid body are separate cell organelles the functions of which are somehow related to each other.     

Ultrastructural localization of RNA in the chromatoid bodies of undifferentiated cells (neoblasts) in planarians by the RNase–gold complex technique

Undifferentiated cells of planarians (Platyhelminthes, Turbellaria), also called neoblasts, are totipotent stem cells, which give rise to all differentiated cell types, while maintaining their own density by cell proliferation. Neoblasts are the only somatic cells of planarians bearing chromatoid bodies in their cytoplasm; these organelles disappear as differentiation takes place. Studies on germinal cells of several groups of organisms have shown that chromatoid bodies contain substantial amounts of RNA. To test its presence in neoblasts, we have used an RNase–gold technique. We found chromatoid bodies labeled with RNase–gold particles. Heterogeneity in the density of the label, may be correlated with the functionality and complexity of these organelles. The gold marker was also present over the nucleus and rough endoplasmic reticulum, but mitochondria, secretory granules, and the extracellular space were devoid of label. This specific localization of RNA in planarian chromatoid bodies supports earlier findings on germ cells and embryonic cells in a variety of organisms, indicating that chromatoid bodies are information-storage structures, essential during the process of cell differentiation. © 1993 Wiley-Liss, Inc.     

Cytoplasmic inclusions specific to the sea urchin germ line

We have studied primordial germ cells and gamete-producing cells of both sexes of the sea urchin Lytechinus pictus using electron microscopy. Two fibrogranular inclusions are present in most of the stages examined and are specific to the germ-line cells. One of the inclusions consists of amorphous material of undefined shape and size while the other type consists of spherical bodies 0.1 μm in diameter. The latter are named goniosomes and are scattered individually in the cytoplasm or in ordered arrays of up to 50 units. Both goniosomes and amorphous material are often associated with mitochondria. A third type of inclusion, the bipartite body, is found only in oocytes and may be a specific product of oogenesis.