A novel set of structures within the elasmobranch,ovarian follicle

Elasmobranch fishes produce some of the largest oocytes known, exceeding 10 cm in diameter. Using various microscopy techniques we investigated the structural adaptations which facilitate the production of these large egg cells in three species of shark: the Atlantic sharpnose shark, Rhizoprionodon terraenovae, dusky smoothound, Mustelus canis and the little gulper shark, Centrophorus uyato. The ovarian follicle of elasmobranchs follows the typical vertebrate pattern, with one notable exception; the zona pellucida reaches extreme widths, over 70 μm, during early oogenesis. Contact between the follicle cells and the oocyte across the zona pellucida is necessary for oogenesis. We describe here a novel set of large, tube‐like structures, which we named follicle cell processes that bridge this gap. The follicle cell processes are more robust than the microvilli associated with the follicle cells and the oocyte plasma membrane and much longer. During early oogenesis the follicle increases in size relatively quickly resulting in a wide zona pellucida. At this stage the follicle cell processes appear taut, uniform and radially oriented. As oogenesis continues the zona pellucida narrows and the follicle cell processes change their orientation, appearing to wrap around the oocyte. The presence of the contractile protein actin within the follicle cell processes and their change in orientation may well be an adaptation for maintaining the integrity of these large oocytes. The follicle cell processes also contain electron dense material, identical to material found within the follicle cells, suggesting a role in the transport of metabolites to the developing oocyte. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Microtubule organization and nucleation in the differentiating ovarian follicle of the lizard Podarcis sicula

We analyzed the organization of the microtubular cytoskeleton and the distribution of centrosomes at the different stages of differentiation of the ovarian follicle of the lizard Podarcis sicula by examining immunolabeled α‐ and γ‐tubulins using confocal microscopy. We observed that in the follicular epithelium the differentiation of the nurse pyriform cells is accompanied by a reorganization of the microtubules in the oocyte cortex, changing from a reticular to a radial pattern. Furthermore, these cortical microtubules extend in the cytoplasm of the connected follicle cells through intercellular bridges. Radially oriented microtubules were still more marked in the oocyte cortex during the final stages of oogenesis, when the yolk proteins were incorporated by endocytosis. The nucleation centres of the microtubules (centrosomes) were clearly detectable as γ‐tubulin immunolabeled spots in the somatic stromal cells of the germinal bed. A diffuse cytoplasmic immunolabeling together with multiple labeled foci, resembling the desegregation of the centrosomes in early oogenesis of vertebrates and invertebrates, was revealed in the prediplotenic germ cells. In the cytoplasm of growing oocytes, a diffuse labeling of the γ‐tubulin antibody was always detectable. In the growing ovarian follicles, immunolabeled spots were detected in the mono‐layered follicle cells which surrounded the early oocytes. In follicles with a polymorphic follicular epithelium, only the small follicle cells showed labeled spots. A weak and diffuse labeling was observed in the pyriform cells while in the enlarging intermediate cells the centrosomes degenerated like in the early oocytes. Our observations confirm that in P. sicula most of the oocyte growth is supported by the structural and functional integration of the developing oocyte with the pyriform nurse cells and suggest that their fusion with the oocyte results in an acquirement by these somatic cells of characteristics typical of the germ cells. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

How the ovarian follicle of Podarcis sicula recycles the DNA of its nurse,regressing follicle cells

In Podarcis sicula specialized follicle cells send reserve materials to the previtellogenic oocyte via intercellular bridges. Immediately before the onset of vitellogenesis this transferring becomes particularly massive so that the cell volume significantly reduces, meanwhile in the nucleus the morphological alterations typical of apoptosis appear. To clarify why these follicle cells are not simply fully resorbed by the oocyte and to determine whether their DNA is discarded or recycled, we carried out a series of morphological and biochemical investigations. The finding that large macromolecular scaffolds are formed and that these are able to retain the DNA until it is extensively cut by two different endonucleases suggests that regression of the follicle cells is programmed and that the fate of their DNA is strictly controlled. Following its genetical neutralization via fragmentation, the DNA is apparently recycled by being transferred into the oocyte via the intercellular bridges, that, in fact, remain open until the very late stages of cell regression. The small DNA fragments reaching the oocyte cytoplasm would not interfere with meiosis completion but could significantly contribute to the stock of reserve materials to the advantage of the growing oocyte and/or developing embryo. Mol. Reprod. Dev. 51:421–429, 1998. © 1998 Wiley-Liss, Inc.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.     

Immunocytochemical localization of a follicle specific protein of the hawkmoth Manduca sexta

A follicle specific protein (FSP-I) from the hawkmoth Manduca sexia, has been localized in developing follicles by immuno-fluorcscence and immuno-gold labeling techniques. At the light microscopical level, the protein was demonstrated to be present in both the basolateral and apical parts of foilicular epithelial cells, as well as in clearly defined, spherical compartments in the cortex of the developing oocyte. Immuno-gold labeling at the electron microscopical level revealed the localization of FSP-I in cndoplasmic compartments of the foilicular epithelial cells, in the extracellular matrix of the follicle and in endocytic compartments of the oocyte. Our results indicate that M. sexta FSP-I is synthctizcd and secreted by the foilicular epithelial cells, after which it is taken up by the developing oocyte through endocytic routes.     

Observations of follicle cell processes in a holocephalan

The presence of follicular cellular processes (FCP) that cross the zona pellucida, has been recorded in the ovarian follicles of Callorhinchus callorhynchus. This constitutes the first report describing the presence of these structures in a species of the Holocephali. Considering that FCPs have only previously been reported in the Selachii, these findings suggest that FCPs could have been lost by the Batoidea after their divergence, around 280 M B.P.     

Sperm storage in male elasmobranchs: A description and survey

Two basic types of spermatozoan aggregates, spermatophores and spermatozeugmata, found in 14 different species of sharks, one species of skate, and one species of chimaera (holocephalan), were investigated using light and scanning electron microscopy. Spermatophores, aggregates (usually 1,000–6,000 μm in diameter and larger) of randomly clumped sperm embedded in and surrounded by an eosinophilic matrix, were found in Alopias superciliosus, Odontaspis taurus, Carcharodon carcharias, Isurus oxyrinchus, and Lamna nasus. Three types of spermatozeugmata, sperm structures without a surrounding capsule or matrix, are described. The first, clumps of 60–200 sperm unbound in a supporting matrix, are found in Squalus acanthias and Hydrolagus colliei. In the second type, single-layered spheres are formed of sperm clumps with the sperm heads bound in a common supporting matrix. These are found in Carcharhinus limbatus and Carcharhinus plumbeus. The third type of spermatozeugmata are large multilayered, compound structures formed by the accretion of several single-layered aggregates. These multilayered structures characteristically are found in Carcharhinus falciformis, C. limbatus, Carcharhinus obscurus, C. plumbeus, Carcharhinus porosus, Prionace gluaca, Rhizoprionodon terraenovae, and Sphyrna lewini. Sperm aggregates of all types are stored between the septa and in the lumen of the terminal ampulla of the epididymis. In their various forms they are the final product of the mature male elasmobranch reproductive tract. In a male with mature claspers, the presence of sperm aggregates is a more reliable indicator of maturity and sexual activity than is clasper condition alone. © 1994 Wiley-Liss, Inc.     

Protein incorporation by isolated amphibian oocytes : IV. The role of follicle cells and calcium during protein uptake

When Xenopus oocytes are manually dissected from the ovary, they are generally invested by a layer of follicle cells. These cells frequently retract from and eventually slough off the surface of the oocyte, even though protein uptake by the oocyte continues at the same rate. Autoradiographs indicate that 3H-vitellogenin is incorporated at the surface of the oocyte regardless of the presence or absence of overlying follicle cells. Treatment of the oocyte with EDTA or Ca-free medium causes a rapid loss of both the follicle cells and ability of the oocyte to incorporate external protein. The two processes occur at different rates, however, and if treated oocytes are incubated in Ca-containing saline for a sufficient time, they partially recover their ability to incorporate protein. From these observations we have concluded that (1) follicle cells do not seem to play a role during protein incorporation by Xenopus oocytes in vitro; (2) the lack of external calcium inactivates the normal protein incorporation process at the oocyte surface; (3) normal function can be partially regenerated in Ca-containing media.     

Teeth outside the mouth: The evolution and development of shark denticles

Vertebrate skin appendages are incredibly diverse. This diversity, which includes structures such as scales, feathers, and hair, likely evolved from a shared anatomical placode, suggesting broad conservation of the early development of these organs. Some of the earliest known skin appendages are dentine and enamel-rich tooth-like structures, collectively known as odontodes. These appendages evolved over 450 million years ago. Elasmobranchs (sharks, skates, and rays) have retained these ancient skin appendages in the form of both dermal denticles (scales) and oral teeth. Despite our knowledge of denticle function in adult sharks, our understanding of their development and morphogenesis is less advanced. Even though denticles in sharks appear structurally similar to oral teeth, there has been limited data directly comparing the molecular development of these distinct elements. Here, we chart the development of denticles in the embryonic small-spotted catshark (Scyliorhinus canicula) and characterize the expression of conserved genes known to mediate dental development. We find that shark denticle development shares a vast gene expression signature with developing teeth. However, denticles have restricted regenerative potential, as they lack a sox2+ stem cell niche associated with the maintenance of a dental lamina, an essential requirement for continuous tooth replacement. We compare developing denticles to other skin appendages, including both sensory skin appendages and avian feathers. This reveals that denticles are not only tooth-like in structure, but that they also share an ancient developmental gene set that is likely common to all epidermal appendages.     

Ultrastructural study of the follicle cells in the freshwater gastropod Viviparus viviparus L.

Summary The morphology and the role of the follicle cells of Viviparus viviparus were examined by means of light and electron microscopy. The follicle cells appear to contain glycogen and fat, and often lysosomes or heterogeneous inclusions. Therefore, they seem to be active in phagocytosis and storage. They are probably involved in the nutrition of the oocyte. Their role in the formation of a selectively permeable barrier is discussed.The authors thank Drs. H.H. Boer, M. de Jong-Brink and J. Wijdenes of the Free University of Amsterdam for their assistance in the translation of this paper     

Ultrastructural differentiations in the developing follicle cortex of Locusta migratoria,with special reference to vitelline membrane formation

Summary Electron microscopic studies on developing follicles of Locusta migratoria show the vitelline membrane to be composed of two ultrastructurally distinguishable components: The vitelline membrane bodies (VMBs) and, in addition, fine granular material, cementing the VMBs together. VMBs form first in the oocyte-near zone within the oocyte-follicle cell space. Subsequently, the second vitelline membrane substance is secreted between the VMBs through apical protrusions of the follicle cells. The possible origin of the VMBs is discussed.Yolk uptake in Locusta seems to occur predominantly by pinocytosis. During oocyte development the oocyte membrane is enlarged by numerous microvilli and folds. In addition pinocytotic vesicles are pinched off. It is supposed that the latter loose their coat and eventually transform into large proteid yolk spheres.This work was supported by the Volkswagenstiftung, HannoverI wish to thank Prof. Dr. H. Emmerich, Techn. Hochschule Darmstadt, for valuable discussions     

Follicular epithelial cell apoptosis of atretic follicles within developing ovaries of the mosquito Culex pipiens pallens

Follicular atresia, the degeneration of developing follicles, is always incident to normal oogenesis in both oviparous and viviparous animals. Photo- and electron-microscopic observation of degenerating follicles within developing ovaries taken from blood-fed Culex pipiens pallens mosquitoes showed gradual degradation of the internal structures including yolk granules in the oocyte. The epithelial cells, which sometimes incorporated yolk granules from the oocyte along with the shrinkage of the follicle, gradually lost their uniform columnar shape, while their integrity as a covering layer remained. In situ active caspase analysis detected active enzymes in these epithelial regions. In the latest stages of atresia where either the nurse cells or oocyte were lost, the follicle was mainly comprised of irregularly shaped epithelial cells, and some of these cells' nuclei contained condensed chromatin peripherally, one of the characteristics of apoptotic cells. Also terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling treatment indicated that DNA fragmentation occurred in these follicles. It seems likely that in atretic follicles the epithelial cells survive to play key roles in the event, and then finally undergo their own apoptotic cell death so as to give the developmental site to the next follicle in the same ovariole.     

Gap junctions between the follicle cells and the oocyte during oogenesis in an insect,Tribolium destructor/Coleoptera

Summary Oocyte-follicle cell gap junctions inTribolium occur in all oogenetic stages studied. During early previtellogenesis the junctions are found exclusively between lateral membranes of oocyte microvilli and the membrane of prefollicle cells. In late previtellogenesis and vitellogenesis the junctions are located between the tips of oocyte microvilli and the flat membranes of the follicle cells. During previtellogenesis gap junctions are infrequent, whereas in the phase of yolk accumulation their number increases considerably, exceeding 17 junctions/m² of the follicle cell membrane. It could be shown by microinjection of a fluorescent dye that gap junctions are in a functional state during vitellogenesis. Possible roles of heterologous gap junctions in oogenesis are discussed.     

BMP signaling dynamics in the follicle cells of multiple Drosophila species

The dorsal anterior region of the follicle cells (FCs) in the developing Drosophila egg gives rise to the respiratory eggshell appendages. These tubular structures display a wide range of qualitative and quantitative variations across Drosophila species, providing a remarkable example of a rapidly evolving morphology. In D. melanogaster, the bone morphogenetic protein (BMP) signaling pathway is an important regulator of FCs patterning and dorsal appendages morphology. To explore the mechanisms underlying the diversification of eggshell patterning, we analyzed BMP signaling in the FCs of 16 Drosophila species that span 45 million years of evolution. We found that the spatial patterns of BMP signaling in the FCs are dynamic and exhibit a range of interspecies' variations. In most of the species examined, the dynamics of BMP signaling correlate with the expression of the type I BMP receptor thickveins (tkv). This correlation suggests that interspecies' variations of tkv expression are responsible for the diversification of BMP signaling during oogenesis. This model was supported by genetic manipulations of tkv expression in the FCs of D. melanogaster that successfully recapitulated the signaling diversities found in the other species. Our results suggest that regulation of receptor expression mediates spatial diversification of BMP signaling in Drosophila oogenesis, and they provide insight into a mechanism underlying the evolution of eggshell patterning.     

Do sharks exhibit heterodonty by tooth position and over ontogeny? A comparison using elliptic Fourier analysis

Tooth morphology is often used to inform the feeding ecology of an organism as these structures are important to procure and process dietary resources. In sharks, differences in morphology may facilitate the capture and handling of prey with different physical properties. However, few studies have investigated differences in tooth morphology over ontogeny, throughout the jaws of a single species, or among species at multiple tooth positions. Bull (Carcharhinus leucas), blacktip (Carcharhinus limbatus), and bonnethead sharks (Sphyrna tiburo) are coastal predators that exhibit ontogenetic dietary shifts, but differ in their feeding ecologies. This study measured tooth morphology at six positions along the upper and lower jaws of each species using elliptic Fourier analysis to make comparisons within and among species over their ontogeny. Significant ontogenetic differences were detected at four of the six tooth positions in bull sharks, but only the posterior position on the lower jaw appeared to exhibit a functionally relevant shift in morphology. No ontogenetic changes in morphology were detected in blacktip or bonnethead sharks. Intraspecific comparisons found that most tooth positions significantly differed from one another across all species, but heterodonty was greatest in bull sharks. Additionally, interspecific comparisons found differences among all species at each tooth position except between bull and blacktip sharks at two positions. These morphological patterns within and among species may have implications for prey handling efficiency, as well as in providing insight for paleoichthyology studies and reevaluating heterodonty in sharks.     

l-Serine transport in growing and maturing mouse oocytes

Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth. Serine transport continued when oocytes resumed meiosis but ceased partway through first meiotic metaphase, remaining quiescent in mature eggs in second meiotic metaphase. The serine transporter was sodium dependent and inhibited by alanine, cysteine, leucine, or histidine, and had a Michaelis–Menten constant (K_m) for serine of 200 µM. Unexpectedly, exposing cumulus cell-enclosed oocytes to the physiological mediator of meiotic arrest, natriuretic peptide precursor Type C, substantially stimulated serine transport by the enclosed oocyte. Finally, in addition to transport by the oocyte itself, cumulus cells also supply serine to the enclosed oocyte via gap junctions within intact cumulus–oocyte complexes.     

Synthesis and secretion of egg-specific protein from follicle cells of the silkworm,Bombyx mori

The synthesis and secretion of egg-specific protein (ESP) were investigated using the follicle cells isolated from the developing ovary of the silkworm, Bombyx mori. The follicle cells were isolated manually from a follicle into a cell layer by thoroughly extruding the oocyte contents through a small hole. Whole follicles and isolated follicle cells were incubated in vitro with [³⁵S]methionine, and ESP and its precursors were immunochemically isolated using antiserum raised to ESP. The isolated follicle cells incorporated label into ESP but the incorporation rate was about one-fifth of that found in whole follicles. About 20% of the total radioactivity of ESPs were recovered from the incubation medium of the isolated follicle cells while only trace activity (<2%) was found in the incubation medium of whole follicles. These results clearly showed that follicle cells synthesize and release ESP to be taken up by the developing oocyte.     

Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2-4, and 4-6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed.     

Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: Lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2–4, and 4–6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed.