Primordial germ cells and early stages of oogenesis in Ophryotrocha puerilis (Polychaeta,Dorvillediae)

Summary Each setigerous segment of the protandric polychaete Ophryotrocha puerilis contains two primordial germ cells. A ventral furrow in the gut wall together with the peritoneal lining of the gut forms a genital blood vessel. The gonocytes are located within the peritoneum of this genital blood vessel. At sexual maturity the gonocytes undergo a proliferation cycle, the first division of which gives rise to a cell which is extruded into a forming outpocketing of the coelomic lining. The stem cell remains within the peritoneum. Inside the forming gonad the detached cell goes through a series of four mitotic divisions. The resulting 16 cells are interconnected by cytoplasmic bridges. These bridges are arranged in a very regular pattern which allows the mitotic cycles to be followed. While remaining still within the gonad the 16 cells begin to synthesize yolk and to take up exogenous yolk precursors. At this stage a differentiation into oocytes and nurse cells becomes visible. The oocytes deposit yolk platelets of the definitive size whereas the polyploid nurse cells produce only small yolk bodies that are passed to the adjacent oocytes. In a later stage the cell bridges between adjacent nurse cells are cut and pairs of one oocyte and one nurse cell are released to the coelomic cavity during breakdown of the gonadal sac. Oocyte-nurse cell-complexes then freely float in the coelomic fluid. The proliferation of gonadal cells is well synchronized within one segment. In anterior segments, however, gonadal proliferation usually begins earlier than in posterior segments but smaller oocytes in posterior segments catch up within a few days. Finally a batch of oocytes is produced in which all the oocytes are of the same size (120 m). The origin of the primordial germ cells remains unknown.     

Ultrastructural observations on oogenesis in Drosophila

The ultrastructure of the follicle cells and oocyte periplasm is described during the stages of oogenesis immediately prior to, during, and immediately subsequent to, vitellogenesis. A number of features have not been described previously in Drosophila. Some yolk appears prior to pinocytosis of blood proteins. However, most of the protein yolk forms while the periplasm is filled with micropinocytotic invaginations and tubules derived from the oolemma. These tubules retain the internal layer of material characteristic of coated vesicles and are found to fuse with yolk spheres. No accumulation of electron-dense material in the endoplasmic reticulum or Golgi of the oocyte is found. Both trypan blue and ferritin are accumulated by the oocyte. The follicle cells have an elaborate endoplasmic reticulum during the period of maximum yolk accumulation. Adjacent cells are joined at their base by a zonula adhaerens, forming a band around the cells, and by plaques of gap junctions. Gap junctions are also present between nurse cells and follicle cells. During chorion formation, septate junctions also appear between follicle cells, adjacent to the zonula adhaerens.     

Yolk formation in an annelid (Ophryotrocha puerilis, polychaeta)

The polychaete Ophryotrocha does not show a distinct breeding season. Egg masses are produced throughout the year (continuous breeder sensu Olive and Clark, 1978). A female specimen may contain up to three different generations of oocytes with oocyte growth and maturation in each batch being well synchronized. Oogenesis takes about 18 days from proliferation of the oogonia to mature eggs. In each segment pairs of sister cells interconnected by cytoplasmic bridges are located in outpocketings of the ventral mesentery which form the gonad wall. Presumptive oocytes and nurse cells are not easily distinguished at that time. Vitellogenesis is initiated while both oocytes and nurse cells are still in the ovary. Mitochondria, multivesicular bodies (transformed mitochondria ?), dense bodies, preformed yolk bodies of smaller size and lipid droplets are probably passed through the cytoplasmic bridge from the nurse cell to the oocyte. Yolk formation includes different mechanisms and materials of different origin. Autosynthetic yolk formation predominates during the first intraovarial growth phase. After detachment of oocyte-nurse cell-complexes from the gonad pinocytotic activity of nurse cells and particularly oocytes, increases considerably. The existence of coated vesicles suggests that external sources of yolk precursors contribute to yolk formation. Prior to oocyte maturation the remnants of the nurse cell are incorporated by oocytes.     

Vitellogenesis in Varroa jacobsoni,a parasite of honey bees

Reproduction in Varroa jacobsoni occurs only in cells of the capped honey bee brood. Female mites were sampled at different times after cell sealing and ovaries containing a vitellogenic oocyte of the first gonocycle were examined under an electron microscope. It was found that the cytoplasmic connection between the lyrate organ and the oocyte persists far into the vitellogenic growth phase. In addition, a large amount of yolk material is taken up from the haemolymph. All ultrastructural features characteristic of vitellogenesis, such as microvilli, coated pits, vesicles and growing yolk platelets, are present. If more than four Varroa females live in an overcrowded brood cell, they appear to be in stress conditions and their vitellogenic oocytes may become atretic. Alterations typical for oocyte degradation and oosorption were observed in such situations.     

Establishment and movement of egg regions revealed by the size class of yolk platelets in Xenopus laevis

Sizes of yolk platelets were measured in sections of oocytes and embryos in Xenopus. It was found that the average size of the largest group of platelets in cells differed between germ layers of neurulae. It was small (3 to 5 m) in the ectoderm, medium-sized (5 to 8 µm) in the mesoderm, and large (over 8 m) in the endoderm. Platelets of these size classes formed layers in egg, the yolk gradient, by the end of oocyte maturation. The yolk gradient contained products of the mitochondrial cloud and a part of the germinal vesicle material at certain positions. The layers of small, medium and large platelets in the egg changed their locations to distribute to the ectoderm, mesoderm and endoderm of neurulae, respectively. The yolk layers in the egg thus represented different prospective fates, and a figure describing the locations of these layers could be regarded as a fate map for the one-cell stage. Most of the marginal blastomeres of embryos at cleavage stages consisted of a few parts with different prospective fates. Results were discussed with reference to available fate maps for cleavage stage embryos.     

The ultrastructure of the ovarian follicle of medaka,Oryzias latipes

Summary The follicular cells in the oocytes of Oryzias latipes were studied by electron microscopy in order to clarify the fine structure, and the role of the cells during yolk formation and ovulation. The smallest follicles were observed during the early phase of peri-nucleolus stage of the oocyte. The cells have flattened nuclei, and perikarya with undeveloped organelles. But when the oocytes attain diameter of about 250 (yolk vesicle stage), both types of endoplasmic reticula are present. Moreover, the microvilli of the plasma membrane of oocyte as well as the follicles protrude into the pore canals of the zona radiata. In the oocytes of yolk stage the rough-surfaced endoplasmic-reticulum is typically developed and observed around the nuclei. Other organelles (lysosomes, mitochondria and Golgi) increase in number. The relation between the changes of cytoarchitecture in the follicles and yolk formation is discussed.At 17.00 p.m. on the day preceding ovulation the microvilli withdraw somewhat. Ribosomes are attached to the vesicular and cisternal endoplasmic reticula. When the oocytes attain complete maturation (24.00 p.m. at near ovulation), striking changes of the follicles are observed. The microvilli are almost withdrawn. In the degenerating follicles the lamellar structure is formed, and lipids are deposited at the center. At this time the contents of lysosomes have mostly disappeared.     

Gamete Structure and Fertilization in the Barents Sea Sponge Leucosolenia complicata

The ultrastructure of male and female gametes of asconoid sponge Leucosolenia complicata(Calcispongiae, Calcaronea), a hermaphrodite species that reproduces in autumn, is described. The mature sponge's oocytes were up to 70 m in diameter, had no coatings, and contained a nucleus about 31 m in diameter with large nucleoli (up to 6.6 m). There were vacuoles with fibrillar contents typical of calcareous sponges in ooplasm. During vitellogenesis, a cluster of a great number of nurse cells developed above each oocyte from transformed choanocytes. Mature spermia of L. complicatalooked like orbicular cells about 2.5 m in diameter, with no acrosome or tail. The spermium nucleus (diameter about 2.2 m) was formed by incompletely condensed chromatin and was surrounded with a thin layer of cytoplasm of nonuniform thickness. In the thick layer of cytoplasm beyond the ribosomes, there were two or three mitochondria, dictyosomes, and electron-dense protein bodies lying freely under the nucleus. Fertilization occurred with the aid of a carrier cell. During spawning (mass release of spermia), any nurse cell complex can seize a spermium and transform into a carrier cell in situ. The transformation of a seized spermium into a spermiocyst was connected with the rapid isolation of the spermium nucleus from the protein body. Fertilization began with the penetration of the protein body into the oocyte cytoplasm. Only after this did the spermium's nucleus penetrate into the oocyte.     

Accumulation of yolk in a caecilian (Gegeneophis ramaswamii) oocyte: A light and transmission electron microscopic study

There is a paucity of information on the female reproductive biology of the caecilian amphibians when compared with the other vertebrate groups. Hence, the accumulation of nutrient reserves in the form of yolk and formation of yolk platelets were studied in Gegeneophis ramaswamii, adopting light microscopic histological and transmission electron microscopy analysis. Previtellogenic as well as vitellogenic follicles were observed in appropriate preparations. On the basis of the source and the routes of entry, we identified four types of yolk precursor materials, precursors 1 to 4. The earliest material appearing in the oocyte consists of abundant lipid vesicles during the previtellogenic phase, i.e., much before the follicular epithelium is fully established. This is a contribution from the oocyte mitochondria, which we identified as yolk precursor material 1, and it is autosynthetic. Once the follicle cell‐oocyte interface is fully established, there is an accumulation of the principal component of the heterosynthetic yolk by sequestration from the blood through the intercellular spaces between follicle cells in a pinocytic process. This we identified as yolk precursor material 2. There was also an indication of a lipidic yolk material synthesis in the follicle cells sequestered from maternal blood through the follicle cells in an endocytic process in which the macrovilli of follicle cells and the microvilli of the oocyte play a role. This we identified as yolk precursor material 3. Contribution to the yolk of peptidic, glycosidic, and/or lipidic material synthesized in the vitellogenic oocyte was also indicated. This we identified as yolk precursor material 4. The sequential development of intercellular associations and indications of synthesis/sequestration of the yolk have been traced. Thus, we report the mechanistic details of synthesis/sequestration of the yolk materials in a caecilian. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

The development of Spongilla lacustris from the oocyte to the free larva (Porifera,Spongillidae)

Summary During June and July oocytes appear in well-developed specimens of Spongilla lacustris. These differentiate from archeocytes, and during the first growth phase they reach a diameter of ca. 50 m. At this time each oocyte is enclosed in a single-layered follicle epithelium, which is retained until emergence of the larva.In the second phase the oocytes grow to about 220 m by phagocytosis of trophocytes. When phagocytosis has come to an end, there is a distinct layering of the yolk material that has formed within the cytoplasm of the oocyte. Small yolk granules surround the centrally located nucleus, and peripheral to these is a layer of larger spheres of yolk.Cleavage is totally equal to unequal. Some blastomeres are binucleate. In the 15-cell staged micro- and macromeres appear.The embryo consists of uniform cells with high yolk content; at the periphery they are slightly flattened rather than spherical. In this stage of development the first scleroblasts appear.Further development to the young larva is marked by the appearance of a cavity (the larval cavity) lined with pinacocytes. The cavity expands to occupy about half the volume of the larva at emergence, becoming hemispheric in shape. The cells at the periphery of the larva form a columnar, single-layered, multiseriate ciliated epithelium with teardrop-shaped nuclei.The emerging larva breaks through its follicle and the wall of the excurrent canal system; occasionally larvae can be found in the canals. At this time the larva has developed a few flagellated chambers, which may already be integrated into the primordia of the excurrent canal system. The previously discernible scleroblasts have now formed isolated spicules, which may adhere to form spicule-spongin complexes.     

Ultracytochemical localization and microprobe quantitation of calcium stores in the insect oocyte

Summary Detection of calcium in the follicles of Galleria mellonella (Lepidoptera) was performed using two cytochemical methods. Calcium precipitation was obtained either with ammonium oxalate (AO) or with N,N-naphtaloyl-hydroxylamine (NHA). In both cases the X-ray on line analysis monitored the presence of calcium in the oocytes, which was correlated with the accumulation of yolk spheres. concentration of calcium in oocytes filled with yolk and treated with AO amounted to 9 mmoles per 1,000 g tissue we weight. This value is similar to that calculated previously for follicles untreated with any reagent and prepared for the analysis by the freeze-drying technique (Przelcka et al. 1980).Examination of the ultrastructure of oocytes treated with NHA revealed calcium precipitate at the follicular epithelium/oocyte interface, in endocytotic canaliculi and vesicles formed by the oocyte plasma membrane, in ooplasm, and in yolk spheres. In oocytes treated with AO, the calcium-precipitate intermingled with the precipitate produced by the osmium alone. The presumed cause of this phenomenon is discussed.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary Each ovarian follicle of Triops cancriformis is four-celled; these cells (one oocyte and three nurse cells) are interconnected by cytoplasmic bridges. In the course of differentiation, the nurse cells are early recognizable; they increase in size more than the oocyte and their nuclei contain many nucleoli. For the first time in Arthropoda, yolk globules are reported to be present in nurse cell cytoplasm; these globules arise from the smooth endoplasmic reticulum. The functional significance of the intercellular bridges and the trophic role of the nurse cells are discussed.The authors are grateful to Dr. Bruno Sabelli for his support and to Mr. Francesco Monte for his technical assistance     

A cytochemical study of oocyte growth in four species of millipedes

Summary The cytochemistry of oocyte growth was investigated in four species of millipedes; Narceus americanus, Oxidus gracilis, Cheiropus plancus, and a Pleuroloma species, probably P. cala. The oocyte of all four species produced a yolk nucleus which arose in contact with the nuclear membrane, subsequently detached, migrated into the central ooplasm and disrupted coincident with the appearance of protein yolk granules in the oocyte cytoplasm. Since the follicular epithelium did not display any morphological or cytochemical manifestations of secretory activity, it is suggested that direct incorporation of exogeneous proteins into yolk may play a lesser role in vitellogenesis in these forms than in insects and many other animal groups. Ooplasmic RNA levels achieved a maximum before vitellogenesis was initiated and then decreased. Similarly the nucleoli increased in size and RNA content up to the point at which cytoplasmic RNA levels began to decline. Basic proteins associated with RNA were present in the oocyte cytoplasm and the nucleoli. These achieved a peak concentration in the same size oocytes as RNA, but the intensity of staining decreased more rapidly than that of RNA during subsequent growth. The concentration of nucleoplasmic protein in oocytes of all four millipede species increased in the germinal vesicles during the course of oocyte growth. Coincident with the initiation of vitellogenesis, a class of cytoplasmic inclusions was developed which have been called concentric ring bodies. These inclusions consist of concentric layers of organic matrix material with crystallized calcium salts sandwiched between. These probably represent calcium reserves which are utilized in the formation of the exoskeleton by the embryo.Presented in partial fulfillment of the requirements for the Master of Science degree to the Graduate School of Arts and Sciences of the University of Florida.This work was supported by the following U.S. Public Health Service grants: Pathology Training Grant 5-T1-GM-1142-03, and to R. R. C. HD-1499-04 and Career Development Award K-3-HD-6176-04.     

Further studies on the ring canal system of the ovarian cystocytes of Drosophila melanogaster

Summary An ultrastructural study was made of the ring canal system which connects the sister ovarian cystocytes that arise in the germaria of wild type Drosophila melanogaster females. It was discovered that during an oogonial mitosis both chromosomes and spindle are enclosed by a multilayered, perforated membrane system derived (at least in part) from the nuclear envelope. The cytokinesis of stem line oogonia takes place through the formation of a cleavage furrow. A second method of formation of plasma membrane is found in the case of cystocytes. It involves the production along the plane of division of a plaque of interconnected vesicles and tubules and later the coalescence of nearby tubules to form continuous sheets of membrane which segregate the cytoplasms of the sister cells. However, these remain connected by a canal which is enclosed by a ring-shaped rim that is completed prior to the plasma membrane to which the rim is subsequently attached. It is postulated that the rim represents a transformed midbody. As development proceeds the canal becomes wider, its rim becomes thicker, and the inner circumference of the rim becomes coated with a thick deposit having different cytochemical properties than the rim itself. Cystocyte divisions produce sister cells which differ in that one receives all previously formed canals; the other none. In the case of the last division (and perhaps in earlier ones as well) the sister cell receiving all previously formed canals also receives more cytoplasm than its sister. As the cells of the cluster grow, the canals remain close together. This finding suggests that when new plasma membrane is synthesized, it is added in areas remote from the canals. An investigation of the positioning in three dimensions of the fifteen canals of a newly formed, 16 cellcluster suggests that the spindles produced at one division are never parallel to those formed at the subsequent division. This continual shifting of the axes of the spindles at consecutive divisions presumably results in the branching chains of cells which characterize a cystocyte cluster. The possession of a unique pattern of cortical structures by two cystocytes is accompanied by the nuclear synthesis of synaptonemal complexes. The other fourteen cystocytes differentiate into nurse cells. In the most posterior portion of the germarium one of the two potential oocytes switches to the nurse cell developmental pathway. This switched off oocyte and the definitive oocyte grow at rates which differ greatly and are correlated to the amount of contact between their surfaces and certain follicle cells. As development proceeds centrioles accumulate in the oocyte, and most of these are thought to have been carried from the nurse cells into the oocyte in the nutrient stream.The authors are grateful to Richard Z. Belch and James E. Bradof for their conscientious assistance and to E. John Pfiffner for preparation of the inked drawings and construction of the Polyform models. This research was supported by the National Science Foundation grant GB7457.     

Electron microscopic and autoradiographic studies on vitellogenesis in Necturus maculosus

Electron microscope studies on Necturus maculosus oocytes ranging in size from 1.1–1.5 mm in diameter indicate the primary proteinaceous yolk to arise within structures referred to in other amphibian oocytes as yolk precursor sacs or bodies. The origin of these yolk precursor sacs appears to result from the activity of the Golgi complexes which form multivesicular and granular-vesicular bodies, the limiting membrane of which is at times incomplete. During differentiation, the yolk precursor sacs contain small vesicles similar in size to Golgi vesicles, larger vesicles similar to vesicular elements of the agranular endoplasmic reticulum and, on occasion, a portion of a mitochondrion. The interior of these sacs becomes granular, perhaps by a dissolution of the components just described, and soon becomes organized into a crystalline configuration. In oocytes 2.0–2.5 mm in diameter, an extensive micropinocytotic activity begins, continues throughout vitellogenesis, and constitutes the primary mechanism for the formation of secondary yolk protein. Numerous coated and smooth-surfaced vesicles, as well as electron-dense and electronlucent ones, fuse in the cortical ooplasm to form progressively larger yolk platelets.     

Ultrastructure of oogenesis of two oviparous demosponges: Axinella damicornis and Raspaciona aculeata (Porifera)

We investigated the cytology of the oogenic cycle in two oviparous demosponges, Axinella damicornis and Raspaciona aculeata, during 2 consecutive years both by light and electron microscopy. Oocytes of both species were similar in their basic morphological features but differences were noticed in time required to complete oocyte maturation and mechanisms of acquisition of nutritional reserves. The oogenic cycle of A. damicornis extended for 7-8 months in autumn-spring, while that of R. aculeata did it for 3-5 months in summer-autumn. Yolk of A. damicornis was predominantly formed by autosynthesis. Oocytes endocytosed bacteria individually and stored them in groups in large vesicles. Bacteria were digested and lipidic material was added to the vesicles to produce a peculiar granular yolk hitherto unknown in sponges. Scarce cells carrying heterogeneous inclusions were observed in the perioocytic space, and were interpreted as putative nurse cells. Such cells were presumably releasing lipid granules to the perioocytic space. In contrast, large numbers of nurse cells were found surrounding the oocytes of R. aculeata. They transported both lipid granules and heterogeneous yolk bodies to the oocytes. R. aculeata also produced some of their yolk by autosynthesis. The involvement of nurse cells in the vitellogenesis of R. aculeata shortened the oocyte maturation, whereas a largely autosynthetic vitellogenesis in A. damicornis prolonged the duration of oogenesis.     

Ultrastructure of the oocyte and embryo of the calcified sponge,Petrobiona massiliana (Porifera,Calcarea)

Summary In the spongePetrobiona massiliana, a Calcarea related to pharetronid fossils, the oocyte and the embryo both receive an unusual amount of maternal nurse cells. Yolk granules are large and display a lamellar structure throughout the entire growth period, which allows them to be used as markers of the oocyte reserves. The cruciform cells (cellules en croix) of the embryo appear to degenerate at an early stage. These features are compared to those found in other Calcarea.     

Oogenesis in a paedogenetic dipteran insect under normal conditions and after experimental elimination of the follicular epithelium

Summary Paedogenetically developing eggs of the gall midgeHeteropeza pygmaea are not deposited, but develop in the hemocoel of the mother larva. The nurse chamber remains present in the cleaving egg, and the follicular epithelium does not form a chorion but envelops the growing egg during embryonic development. It is possible to obtain naked eggs, i.e. eggs lacking the follicular epithelium, which are able to develop up to the blastoderm stage but remain spherical instead of assuming an elongated shape. Oogenesis of normal and naked eggs has been studied at the ultrastructural level with special reference to the nurse chamber. It is shown that the nurse chamber nuclei develop large nucleoli during oogenesis, indicating that the nurse chamber supplies the oocyte with ribosomal RNA (rRNA). The dense bodies in the nurse chamber may represent an intermediate stage in the transport of the rRNA from the nurse chamber to the oocyte; they are probably not related to the polar granules in the oocyte. It is also shown that the intercellular bridge joining the nurse chamber to the oocyte disappears shortly before cleavage initiation. During egg cleavage the follicular epithelium surrounds the nurse chamber, which degenerates and is gradually absorbed by the growing egg plasmodium. Naked cleaving eggs are never attached to a nurse chamber or to relics of it. Naked oocytenurse chamber complexes frequently aggregate, which may indicate a role of the follicular epithelium in follicle separation during normal development.     

In Vivo Study of Vitellogenin-Gold Transport in the Ovarian Follicle and Oocyte of Xenopus laevis

The transport of injected vitellogenin (VTG)-gold in the ovarian follicle and developing oocyte in Xenopus is described. The gold particles reached the extracellular spaces of the theca and interfollicular spaces within 1 and 2 hr, respectively, after a tracer injection at 20°C. The tracers moved through channels between the constitutive cells of both the capillary endothelium and the follicle cell layer.
Compartments in the peripheral cytoplasm of vitellogenic oocytes at stage IV, which relate to yolk formation, seemed to be segregated as follows: (a) internalization compartment consisting of coated pits and vesicles of the oolemma covering the oocyte "macrovilli", (b) transport compartment of endosomes and multivesicular endosomes in the oocyte cortex, and (c) crystallization compartment of primordial yolk platelets (PYP) in the sub-cortical region. The gold particles appeared in the internalization and transport compartments at 3–6 hr after the tracer injection and in the cystallization compartment at 12–18 hr. The VTG, internalized by receptor-mediated endocytosis, was transferred from coated vesicles to multivesicular endosomes by vesicle-to-vesicle fusion. VTG crystallization took place in globular-shaped PYPs of about 1 μm. At 24 hr after the tracer injection, the gold particles appeared in completely crystallized yolk platelets, most of them clustered in the superficial layer and some integrated into the crystals.     

Morphogenesis of the micropylar apparatus in ovarian follicles of the fungus gnatBradysia tritici (syn.Sciara ocellaris)

Summary The ultrastructure and morphogenesis of the micropylar apparatus (MPA) have been studied in follicles of the fungus gnatBradysia tritici. The MPA is formed by a group of follicle cells located at the anterior pole of the single large nurse cell. In principle, the MPA consists of two thickened plates made of vitelline membrane material, the lower (LMP) and upper micropylar plate (UMP). The former is synthesized by 3 follicle cells, the latter by 4 different follicle cells. The micropylar channel system consists of a central channel with a single outer orifice and three branches which reach the plasma membrane of the oocyte. The branches are moulded by cellular extensions of the LMP-forming cells which are sandwiched between the two growing micropylar plates. Microtubuli and microfilaments were identified parallel to the long axis of the cellular extensions. At the time of MPA synthesis the nurse cell is still large and hence the MPA-forming cells have no contact to the oocyte. At the end of oogenesis when the regression of the nurse cell is completed, the MPA becomes connected to the other parts of the egg shell. At this time an ultrastructurally homogeneous region forms in the adjacent ooplasm (cytoplasmic cone). The possible relevance of these cytological observations for the control of development is discussed.