Morphological and molecular responses in ovaries of Mytilus galloprovincialis collected in two different sites of the Naples Bay.

Mytilus galloprovincialis female specimens were collected from two mussel farms located in two sites next to Castel dell'Ovo, a historical complex located in the Naples Bay. Such sites were named, respectively, A-area and B-area for the different microbiological parameters so that mussels from A-area can be sold without purification, whereas mussels from B-area must be purified before sale. The mussels were collected during the nonreproductive (summer 2009) and reproductive periods (autumn 2009). Gonadosomatic index, structural organization of the ovary, presence of apoptosis, estrogen receptors expression, as well as the bisphenol A (BPA) content in the ovaries, were evaluated. Ovaries from specimens collected in area B showed a different and significant distribution of the investigated biomarkers as well as of BPA content in respect to those measured in the A-area specimens, confirming that mussels are valid sentinel organisms to biomonitor in the Naples bay too.

Effects of nonylphenol on vitellogenin synthesis in adult males of the spotted ray Torpedo marmorata.

The aim of this investigation was to assess the effects of nonylphenol (NP), an oestrogen-like environmental pollutant, on the vitellogenin (VTG) synthesis in adult males of the aplacental viviparous cartilaginous fish Torpedo marmorata. The VTG recovery in males is considered a biomarker of xeno-oestrogenic pollution as this lipophosphoglycoprotein is physiologically induced by oestrogens only in females of oviparous and ovoviparous vertebrates. Using in situ hybridization and immunohistochemistry, T. marmorata males injected with nonylphenol showed the presence of VTG in the liver and the kidney. In particular, vtg messenger (m)RNA and VTG protein were expressed in the liver, whereas in the kidney cells only the presence of VTG was recorded. By contrast, no expression for VTG was detected in the testis. These results demonstrate that in T. marmorata NP induces the expression of vtg only in the liver; the presence of VTG in the kidney and its absence in the testis are discussed.

Developing follicles of the spotted ray Torpedo marmorata express different glycoside residues in relation to granulosa differentiation and vitelline envelope formation.

Lectins constitute a class of proteins/glycoproteins that specifically bind to terminal glycoside residues. The present investigation aimed to identify lectin-binding sites in developing follicles of Torpedo marmorata. Using eleven lectins (WGA, GSI-A4, GSI-B4, PSA, UEA-I, PNA, MPA, Con-A, DBA, LCA, BPA, SBA), we demonstrated that the biochemical nature and the distribution of carbohydrate residues significantly change during oogenesis in the granulosa cells and the vitelline envelope. In fact, a progressive appearance of surface glycoproteins bearing terminated ss-GlcNAc O-linked side chains was observed in the granulosa during the differentiation of pyriform-like cells from the small ones via intermediate cells simultaneously with a significant reduction of the D-Gal chains present in their nucleus. Glycoproteins bearing ss-GlcNAc O-linked side chains were first evident on the surface of small cells in contact with the oocyte, then on the intermediate ones, and finally on pyriform-like cells. The distribution pattern of such glycoproteins over the differentiated granulosa cells remained unchanged during the subsequent stages of the oocyte growth so granulosa cells preserved the same sugar distribution pattern. Furthermore, a progressive loss of D-Gal residues was evident in the nucleus of granulosa cells. In fact, staining for D-Gal was intense in the nucleus of small follicle cells and progressively reduced till disappearing in differentiated pyriform-like cells. Conversely, the small follicle cells located under the basal lamina were devoid of ss-GlcNAc residues, and the nuclear content in D-Gal remained unchanged. This finding strongly suggests that surface glycoproteins containing ss-GlcNAc residues, and the nuclear content in D-Gal might be related to the differentiation of pyriform-like cells. The present investigation also demonstrates that the content of the sugar residues of the vitelline envelope (VE) changes during oocyte growth, suggesting that pyriform-like cells may contribute to its formation.

Surface glycoproteins bearing alpha-GalNAc terminated chains accompany pyriform cell differentiation in lizards.

The present investigation demonstrates that in squamate reptiles, as already reported for Podarcis sicula (Andreuccetti et al., 2001), the differentiation of pyriform cells from small, stem follicle cells is characterized by the progressive appearance on the cell surface of glycoproteins bearing alpha-GalNAc terminated O-linked side chains. Using a lectin panel (WGA, GSI-A4, GSI-B4, PSA UEA-I, PNA, Con-A, DBA, LCA, BPA, SBA), we demonstrated that, during previtellogenesis, the pattern of distribution of DBA binding sites over the follicular epithelium dramatically changes. In fact, binding sites first appear in follicular epithelium at the time that small cells begin to differentiate; in such follicles, labeling is evident on the cell surfaces of small and intermediate cells. Later on, as the differentiation progresses, the binding sites also become evident on the cell surface of pyriform cells. Once differentiated, the pattern of the distribution of DBA binding sites over the follicular epithelium does not change. By contrast, during the phase of intermediate and pyriform cell regression, DBA binding sites gradually decrease, so that the monolayered follicular epithelium of vitellogenic follicles, constituted only by small cells, shows no binding sites for DBA. It is noteworthy that binding sites for DBA are present on small cells located in contact with the oocyte membrane, but not on those located under the basal lamina or among pyriform cells, and therefore not engaged in the differentiation into pyriform cells. This finding demonstrates that, in squamates, the pattern of distribution of alpha-N-GalNAc containing glycoproteins significantly changes during previtellogenesis, and that these modifications are probably related to the differentiation of small stem cells into highly specialized pyriforms.

An ultrastructural study of germ cells during ovarian differentiation in Torpedo marmorata.

An ultrastructural investigation, performed on embryos, neonates, subadult and adult females, demonstrated that in Torpedo marmorata oogenesis occurs very early in life and continues, in its proliferative phase, also after birth. Clusters of early meiotic cells were already evident in the ovarian cortex of 6-cm-long embryos, as well as in the ovary of newborns and three-month-old young. Conversely, in the ovaries of subadult and adult females, all the germ cells present were organized into follicles, and no clusters of oogonia and early meiotic cells were generally found in the cortex, except for one adult female where clusters of germ cells not organized in follicles were found in the cortex. These data demonstrated that, in Torpedo marmorata, oogenesis is immediate, and, as oogonia persist after birth, more similar to that of mouse, monkey, rabbit, and ferret (Mauleon Arch Anat Microsc, 1967; 56:125-150; Byskov and Hoyer 1994) than to that of human, rat, pig, and guinea pig (Byskov and Hoyer 1994). Such a pattern is in agreement with the reproductive strategy of Torpedo, a scantly prolific species with low uterine fecundity. The presence of meiotic cells that are not organized in follicles in one adult female might be consistent with the large individual variability characterizing cartilaginous fishes. The possibility that such a character is typical of mature females should be rejected as oogonia and early meiotic cells were not found inside the totally sectioned gonads of subadult and adult females.

Pyriform cell differentiation in Podarcis sicula is accompanied by the appearance of surface glycoproteins bearing alpha-galNAc terminated chains.

The present histochemical and cytochemical study using a lectin panel (WGA, GSI-A4, GSI-B4, PSA UEA-I, PNA, LCA, Con-A, DBA, MPA, BPA) has demonstrated that, in Podarcis sicula, the differentiation of small follicle cells into pyriform cells by means of intermediate cells is accompanied by the appearance of glycoproteins bearing alpha-GalNAc terminated O-linked side chains on the cell surface. The distribution of DBA- and MPA-binding sites over the follicular epithelium changed during the different stages of oocyte growth. DBA- and MPA-binding sites first appeared at the beginning of folliculogenesis within the zona pellucida (ZP) and on the surface of small cells, i.e., the stem cells of pyriform cells. Afterward, labeling was evident on the cell surfaces of intermediate cells and, later on, also of pyriform cells. On the other hand, no labeling was detected on the small cells located under the basal lamina, which, reportedly, do not differentiate into pyriform cells (Filosa et al. J. Embryol. Exp. Morphol., 1979; 15:297-316). Once pyriform cells were differentiated, the distribution of DBA- and MPA-binding sites over the follicular epithelium remained unchanged until intermediate and pyriform cells underwent apoptosis (Motta et al. J. Exp. Zool., 1996; 276:233-241) and the follicular epithelium transformed into a monolayer composed of small follicle cells only (Filosa Mon. Zool. Ital., 1973; 7:151-165). During this stage of oocyte growth, DBA and MPA labeling gradually decreased to completely disappear in the follicular epithelium of vitellogenic follicles. It is noteworthy that the observed changes in the distribution of DBA- and MPA-binding sites represent the first evidence recognized by lectins of a gradual modification of surface glycoprotein distribution over the follicular epithelium in the ovarian follicles of nonmammalian vertebrates so far studied. Finally, the zona pellucida (ZP), characterized by the presence of GalNAc, GluNAc, Man, and Gal, was demonstrated to be first synthetized by the oocyte and later on by the follicle cells.

Intercellular bridges between granulosa cells and the oocyte in the elasmobranch Raya asterias.

In the present ultrastructural study intercellular bridges, connecting somatic granulosa cells to oocyte, have been detected for the first time and their modifications have been followed during Raja oogenesis. Intercellular bridges make their first appearance in small previtellogenic follicles as connecting devices between small cells and the oocyte. Later on, when the follicular epithelium becomes polymorphic and multilayered, for the presence of small, large, and pyriform-like cells, intercellular bridges link the oocyte and the different granulosa cells. Intercellular bridges contain ribosomes, whorl of membranes, mitochondria and vacuoles. Such cytoplasmic components are present also in the cell apex of large and pyriform-like cells thus suggesting, in agreement with other species (Motta et al. J. Exp. Zool., 1996;276:223-241) they may flow toward the oocyte. In this regard the presence of intercellular bridges during the oogenesis of cartilagineous fish may represent a crucial event of the active cooperation between granulosa cells and the oocyte.

Structural and functional modifications of the nucleolus during previtellogenic oocyte growth in the lizard Podarcis sicula.

In the present study we analyse the nature and the functional significance of the spherical and fibrillo-granular structures appearing in the oocyte nucleus of the lizard Podarcis sicula, following the disappearance of the typical nucleolus. By LM and TEM approaches, we demonstrate that the fibrillo-granuli, containing DNA, RNA and nucleolar proteins, are micronucleoli transcriptionally active and that their DNA is probably derived from nucleolar fragmentation. By contrast, we could not explain the origin and role of the so-called spherical bodies, appearing earlier in oocyte growth; these, in fact, do not contain nucleic acids or nucleolar proteins and do not incorporate uridine. Different possible explanations of their significance are discussed.

Localization of egg-surface carbohydrates and fertilization in Discoglossus pictus (Anura).

The distribution of carbohydrates on the unfertilized egg surface in regions receptive or refractory to sperm penetration was analysed in the anuran amphibian Discoglossus pictus by means of a panel of lectins at the level of both the light and electron microscope. Results showed that a gradient of fucosyl-containing glycoconjugates was present at the pre-determined site of sperm entrance and topographically overlapped a similar gradient in sperm entry previously reported. Of a number of simple and complex carbohydrates examined for their ability to inhibit fertilization, fucoidan, ascophyllan and fucose affected the sperm-egg interaction. These sugars, although not decreasing sperm motility or triggering the acrosome reaction, rendered the sperm unable to penetrate the egg jelly coats. The possible involvement of fucosyl-containing glycoconjugates in the sperm-egg interaction in Discoglossus pictus is discussed.

Silver enhancement of colloidal gold particles in deplasticized semithin epoxy sections.

A simple method for light microscope screening of pre-embedded, gold-labeled samples, by silver enhancing deplasticized semithin epoxy sections is presented. This method is of great help in visualizing the topography of gold labeled sites at low magnification and for selecting labeled sample regions for subsequent electron microscope analysis on serial ultrathin sections. The method was developed during a study of lectin binding sites on the egg surface of the anuran amphibian Discoglossus pictus.

An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative squamata.

An electron microscopic study of the differentiation of pyriform cells and their contribution to oocyte growth in three lizards (Tarentola mauritanica, Cordylus wittifer, Platysaurus intermedius) and one colubrid snake (Coluber viridiflavus) revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte (see also Hubert: Bull Soc Zool Fr 102:151-158, 1977; Filosa et al: J Embryol Exp Morphol 54:5-15 1979; Klosterman: J Morphol 192:125-144, 1987). Once differentiated, pyriform cells display ultrastructural features indicative of synthetic activity, including abundant ribosomes, Golgi membranes, vacuoles, mitochondria, and lipid droplets. These cellular components extend to the apex of the cell at the level of the intercellular bridge, suggesting that constituents of pyriform cells may be transferred to the oocyte. Furthermore, we demonstrate for the first time that pyriform cells incorporate exogenous yolk. The yolk is segregated inside maturing yolk granules that form in the pyriform cell in the same manner as described for vitellogenic oocytes in non-mammalian vertebrates (see Wallace: Developmental Biology, A Comprehensive Synthesis 127-177, 1985). It is the first clear evidence that pyriform cells and the oocyte may fulfill similar vitellogenic functions. The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocyte cooperate.

Ribosomal gene amplification in oocytes of the lizard Podarcis sicula.

In order to provide cytological evidence of amplification, Podarcis sicula oocytes were studied by cytophotometry, thymidine incorporation and in situ DNA-DNA hybridization. Our results show that DNA replication is completed during the preleptotene stage, the leptotene oocytes having the typical 4C nuclear DNA content. Between the zygotene and the mid-pachytene stages further DNA synthesis occurs with consequent increase of the ribosomal nuclear DNA content. These results and the variations in nucleolar organization observed during differentiation give clear evidence of the existence of ribosomal gene amplification in Podarcis sicula oocytes.

Structural modifications of the nuclear components during lizard oogenesis in relation to the differentiation of the follicular epithelium.

This paper deals with an electron microscope study of nucleolar ultrastructural modifications that occur in the oocytes of the lizard Podarcis sicula during ovarian follicle differentiation. In small diplotene oocytes around which a monolayered follicular epithelium forms, the nucleolus appears as a fibrillo-granular structure. Afterwards, simultaneously with the beginning of pyriform cell differentiation inside the granulosa, the nucleolus progressively condenses and breaks into fragments, forming dense spherical bodies. In larger follicles, with well differentiated pyriform cells, a typical nucleolus is no longer detectable in the oocyte nucleus. These ultrastructural modifications suggest a possible impairment of the oocyte nucleolus in ribosome organization. A possible involvement of pyriform cells in supplying ribosomes to the growing oocyte is discussed.

Regulation of oocyte number during oocyte differentiation in the lizard Podarcis sicula.

This paper concerns the differentiation process of germ cells from oogonia to primary follicles in the lizard Podarcis sicula. The study was carried out at the morphological level and using a cytophotometric analysis for determining the number of differentiating germ cells undergoing degeneration. The progressive disorganization of the germ cell clusters during the early diplotene stage and the role played by the prefollicular cells during this process are described. Oocyte degeneration has been observed between the mid-zygotene and the early diplotene stages. When the primary follicle (oocyte plus follicular cells) is being formed, the degeneration process stops and the oocyte undergoes regular growth and ovulation.

Cell junctions during the early development of the sea urchin embryo (Paracentrotus lividus).

Thin sections, lanthanum tracer and the freeze-fracture technique revealed the presence of different types of cell junctions in early sea urchin (Paracentrotus lividus) embryos. During the first four cleavage cycles, which are characterized by synchrony of cell division, sister blastomeres were connected only by intercellular bridges, formed as a result of incomplete cytokinesis; no trace of other junctions was found at these stages. From the 16-cell stage onwards, septate junctions and gap junctions began to appear between blastomeres. It is postulated that cell-cell interactions may provide a mechanism for the propagation of signals necessary for the coordination of cell proliferation and differentiation.

Calcium ultrastructural localization in Xenopus laevis eggs following activation by pricking or by calcium ionophore A 23187.

In the egg of Xenopus laevis a cortical network of smooth endoplasmic reticulum (SER) surrounds and interconnects each cortical granule (CG) (Campanella and Andreuccetti, '77). This network is a possible intracellular site of calcium storage to be called into action for CG exocytosis. In our experiments, Xenopus eggs, unfertilized or activated by pricking or by calcium ionophore A 23187, have been fixed in osmium-pyroantimonate for calcium localization. Our data show that deposits can be detected only in activated eggs. The calcium chelator edetate (EGTA) and x-ray microprobe analysis demonstrate that they contain calcium. Deposits are found on liposomes and on all intraovular cytomembranes, which therefore appear to be possible sites of calcium sequestration. In the case of ionophore-activated eggs, deposits are detectable independently of the presence of extracellular calcium. These data show that in Xenopus at activation an intracellular liberation of calcium occurs similar to that described in other species. Furthermore, the fact that antimony deposits are observed only after activation makes Xenopus eggs appropriate material in which to follow the temporal and spatial sequence of appearance of the deposits during the early stages of activation. Our results show that antimony deposits appear first in SER vesicles between the plasma membrane and CGs and then spread to the rest of the egg cytomembranes. These data corroborate our hypothesis that in Xenopus the cortical SER network is the first intracellular site where calcium is released at activation. The possible mechanism of calcium release and propagation along the egg cortex is discussed.

The modifications of cortical endoplasmic reticulum during in vitro maturation of Xenopus laevis oocytes and its involvement in cortical granule exocytosis.

In Xenopus laevis eggs, cisternae shells which surround cortical granules (CG) are part of a cortical endoplasmic reticulum (ER) network. In this paper the origin of such ER shells has been studied in full-grown, progesterone-exposed Xenopus oocytes. Furthermore, the possible role of the cortical ER in the activation process has been investigated by pricking maturing oocytes. It has been shown that in full-grown ovarian oocytes ER CG shells are absent and ER cisternae are extensively and randomly distributed throughout the peripheral cytoplasm, where they appear to be continuous with annulate lamellae (AL). Following hormone treatment, the AL completely disaggregate and the ER cisternae gradually migrate to the cortex where they surround the CG constituting the typical cortical network described in uterine eggs. Furthermore, it has been found that 8 h after progesterone treatment (before the first polar body extrusion) the response to pricking (CG exocytosis) occurs only at the animal half; there is no observable response in the vegetal half. At this time ER shells surround CG only in the animal hemisphere. A complete CG exocytosis occurs following the first polar body emission, when the cortical ER is well organized in the whole oocyte cortex. The correlation between the differentiation of the cortical ER and the arousal in the oocyte of the ability to respond to a pricking stimulus is discussed in the light of an involvement of the cortical ER in the propagation of CG exocytosis.