Cytokeratin cytoskeleton in the differentiating ovarian follicle of the lizard Podarcis sicula Raf

By immunoblotting and immunocytochemical techniques, we characterized the cytokeratins previously localized by us in the previtellogenic ovarian follicle of Podarcis sicula. Our results show that these cytokeratins correspond to those expressed in the monolayered epithelia. In fact, the immunoblotting analysis showed that the NCL-5D3 antibody, specific for human low molecular weight cytokeratins expressed in monolayered epithelia, reacted with the cytokeratins extracted both from the ovary and from the monolayered intestinal mucosa of Podarcis sicula. Furthermore, this antibody, in this reptile as in humans, clearly immunolabeled sections of corresponding tissues. The organization of the cytokeratin cytoskeleton in the main steps of the ovarian follicle differentiation was also clarified. The reported observations suggest that in Podarcis sicula, the cytokeratin cytoskeleton is absent in the early oocytes. It first appears in the growing oocytes as a thin cortical layer in concomitance with its becoming visible also in the enlarging follicle cells. In the larger follicles, this cytoskeleton appears well organized in intermediate cells and in particular in fully differentiated pyriform cells. In both these cells a cytokeratin network connects the cytoplasm to the oocyte cortex through intercellular bridges. At the end of the previtellogenic oocyte growth, the intense immunolabeling of the apex in the regressing pyriform cells suggests that the cytokeratin, as other cytoplasmic components, may be transferred from these follicle cells to the oocyte. At the end of the oocyte growth, in the larger vitellogenic oocytes surrounded by a monolayer of follicle cells, the cytokeratin constitutes a heavily immunolabeled cortical layer thicker than in the previous stages. Mol. Reprod. Dev. 48:536–542, 1997. © 1997 Wiley-Liss, Inc.     

alpha-Tubulin and acetylated alpha-tubulin during ovarian follicle differentiation in the lizard Podarcis sicula Raf

During most of the previtellogenic oocyte growth, the follicular epithelium of the lizard Podarcis sicula shows a polymorphic structure, due to the presence of different follicle cells. These include small cells which divide and move from the periphery of the follicle to the oocyte surface, intermediate cells which represent an initial step in the process of cell enlargement, and large pyriform cells engaged in the transport of different materials to the oocyte through intercellular bridges. We have studied, by immunolocalization and immunoblotting, the localization of alpha-tubulin and its acetylated form in different follicle cells and in the oocyte during the main steps of ovarian follicle differentiation. Our results indicate that alpha-tubulin is present in all follicle cells at different stages of ovarian follicle differentiation, while its acetylated form is detectable exclusively in the small proliferating and migrating follicle cells. In pyriform cells, alpha-tubulin is localized around the nucleus, extends to the cell apex, and crosses the zona pellucida into the oocyte cortex. The presence of acetylated tubulin in the small follicle cells may be related to the proliferation and/or migration of these cells. The absence of acetylated tubulin form in the cytoplasm of intermediate and pyriform cells can be related to the colocalization of alpha-tubulin with the keratin cytoskeleton in these cells, as detected by confocal microscopy. We have also identified the colocalization of alpha-tubulin with keratin in the cortical region of the oocyte, in particular when the cortex is engaged in the uptake of the yolk proteins.     

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.     

Vasa protein is localized in the germ cells and in the oocyte-associated pyriform follicle cells during early oogenesis in the lizard <Emphasis Type="Italic">Podarcis sicula</Emphasis>

The vasa gene, first identified in Drosophila, is a key determinant for germline formation in eukaryotes. Homologs of vasa have been identified and linked to germline development, in many invertebrates and vertebrates. Here, we analyze the distribution of Vasa in early germ cells (oogonia and oocytes) and previtellogenic ovarian follicles of the lizard Podarcis sicula. During most of its previtellogenic growth, the oocyte in this lizard species is structurally and functionally integrated through intercellular bridges with special follicle cells called pyriform cells. The pyriform cells function similarly to Drosophila nurse cells, but are somatic in origin. In the oogenesis of P. sicula, Vasa is initially highly detected in the oogonia, but its levels decrease in early stage oocytes before the onset of pyriform cell differentiation. In the later stages of oogenesis, the high level of Vasa is related with the nurse function of the pyriform follicle cells. These observations suggest that cells of somatic origin are engaged in the synthesis of Vasa in the oogenesis of this lizard.     

Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae)

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid‐filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron‐lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs. J. Morphol., 2010. © 2010 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.     

Expression of vitellogenin receptor in the ovarian follicles during the reproductive cycle of the spotted ray Torpedo marmorata Risso 1880

The aim of this investigation was to identify the encoding sequence of vitellogenin receptor gene (vtgr), and its expression during the oogenesis in the spotted ray, Torpedo marmorata, in different phases of reproductive cycle. From an ovarian cDNA of vitellogenic female, we obtained a fragment of 581?bp, which corresponds to a partial sequence encoding the vitellogenin receptor (VTGR) in Torpedo (accession number: gi/193244760). This sequence shows a high identity with the VTGR of other vertebrates, particularly Leucoraja erinacea (89% identity) and Squalus acanthias (84% identity). We also showed that vtgr mRNA expression in the ovary modifies during the oogenesis and throughout the reproductive cycle. Indeed, in immature females, whose ovary contains only previtellogenic follicles, vtgr mRNA occurred in the oocyte cortex as well as within intermediate and pyriform cells. In mature females, whose ovary contains pre- and vitellogenic follicles, vtgr mRNA was detectable not only in the oocyte cortex and in intermediate and pyriform cells but also in small follicle cells present in the follicular epithelium of vitellogenic follicles. In ovulating females, that, as pregnant ones, show pre-and vitellogenic follicles, vtgr mRNA was evident in the oocyte cortex only, whereas in pregnant females, no vtgr mRNA was evident. The role of VTGR in the control of Torpedo vitellogenesis is discussed.     

Differential expression of ZPC in the bovine ovary,oocyte, and embryo

Using nonradioactive in situ hybridization (ISH), the mRNA encoding the zona glycoprotein bZPC was localized in bovine ovaries, oocytes, and embryos. In the ovary, the distribution of the mRNA was correlated with the developmental stage of the follicle. Whereas in primordial and primary follicles the mRNA was predominantly seen in the oocyte, it was found in both the oocyte and the follicle cells of secondary and tertiary follicles. In 2-day-old embryos produced by in vitro fertilization (IVF), no mRNA encoding ZPC could be demonstrated. Immunoblotting using monospecific polyclonal antibodies against porcine ZPC revealed a distinct band at a molecular weight of 47 kD in the ovarian cortex of cows, calves, and fetuses as well as in bovine follicle cells. Immunohistochemistry using the ZPC antibody displayed a strong signal in the zona pellucida of bovine oocytes and 2- to 6-day-old embryos as well as in the follicle cells. Our results show that during follicular development bovine ZPC is synthesized by the oocyte of the primary follicle and by both the oocyte and the follicle cells of the secondary and tertiary follicle. After fertilization, the synthesis of the zona protein is finished. Mol. Reprod. Dev. 49:435–443, 1998. © 1998 Wiley-Liss, Inc.     

Light and electron microscope studies on ovarian follicles in the lizard Chalcides ocellatus

The development of ovarian follicles in a skink has been studied with light and electron microscopy. In early stages the previtellogenic oocyte has a follicular covering (granulosa) comprising only two cell types, small cells and pyriform cells. A complex microvillous interdigitation between follicle cells and oocyte is present from very early stages but regresses as a mature size is reached. The outer thecal layer differentiates into distinct interna and externa as growth proceeds. Occasional biovular follicles are formed. Pyriform cells establish direct continuity with the oocyte via cytoplasmic bridges which traverse the layer of microvilli interdigitating in the zona pellucida. Such bridges appear most frequently just before the onset of yolk deposition; the organelles and cytoplasmic constituents presumed to be transferred across them may stimulate this activity. As the follicles grow, the pyriform cells shrink and disappear to leave just the small cells forming the single layered granulosa. There is asynchrony in recruitment and/or early growth rates of follicle crops and uniformity of oocyte size appears only as vitellogenesis nears completion (with up to five oocytes, about 1 cm in diameter, on each side). Yolk deposition may involve transformation of golgi vesicles or pinocytotic vesicles but there is no evidence to show mitochondria as foci for deposition.     

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.     

Studies on the annual reproductive cycle of the female cobra,Naja naja. IV. OVARIAN HISTOLOGY

Morphological changes of the ovary of the Chinese cobra, Naja naja, throughout the annual reproductive cycle are described. A single clutch of between 6 and 22 eggs is produced in late June. From July to the following April the ovary remains quiescent and contains small previtellogenic, hydration stage follicles. The growth of an ovarian follicle from a primary oocyte to maturation and ovulation is estimated to take three years. The histology of the germinal epithelium and the follicular granulosa shows seasonal changes correlated with the growth of the oocyte. During the quiescent period, the germinal epithelium lacks mitotic activity, but during April, when yolk deposition and rapid growth of the preovulatory follicles take place, the germinal epithelium shows intense mitotic activity. The growth of the smallest hydration stage follicles, and the occurrence of cytoplasmic bridges between the pyriform cells of the granulosa and the developing oocyte, also appear to increase during this period. The possible function of the pyriform cell is discussed and the literature on the origin and fate of these cells in the squamate ovary is reviewed. Postovulatory follicles (corpora lutea) and two types of atresia are described and compared with what is known of these structures in other reptiles.     

Localization of prolyl endopeptidase mRNA in small growing follicles of porcine ovary

Prolyl endopeptidase (EC3.4.21.26) has been considered a unique intracellular enzyme catalyzing internal peptide bond hydrolysis of Pro-X. In this study, the distribution of prolyl endopeptidase activity and its mRNA was investigated in the follicles of porcine ovary. Both follicular fluid and granulosa cell fractions from small follicles showed higher activity than those from large follicles. Molecular cloning and Northern blot analysis suggested that only one species of prolyl endopeptidase gene was expressed in the ovary. In addition, in situ hybridization study revealed that the prolyl endopeptidase mRNA expresssion was more noticeable in the granulosa cell layers of small ovarian follicles than in those of large follicles, suggesting its importance in the early stage of follicular development. Mol. Reprod. Dev. 50:121–127, 1998. © 1998 Wiley-Liss, Inc.     

Oocyte growth and follicular hierarchy may be locally controlled by an inhibin-like protein in the lizard Podarcis sicula.

The lizard Podarcis shows an ovarian annual cycle with three to four ovulatory waves between April and July (reproductive period). In August to September, a refractory stage occurs, followed by a nonreproductive period (October to March), during which the oocytes undergo slow growth and prepare themselves for vitellogenesis and ovulation. In the reproductive period, only a certain number of oocytes start growing, giving rise to a follicular hierarchy, which is controlled by still unknown mechanisms. In the present paper, immunoreactive inhibin was detected in previtellogenetic follicles of the reproductive period, and in particular, in the pyriform cells of the follicular epithelium. As the follicle grew and the pyriform cells disappeared, immunostaining shifted to the oocyte cytoplasm. The smaller follicles did not show any immunoreactivity. In the nonreproductive period, no follicles were labeled. We conclude that in the reproductive period, inhibin characterizes the follicles destined to ovulation and might be one of the main factors controlling follicular hierarchy.     

Ovarian histology of the placentotrophic Mabuya mabouya (Squamata, Scincidae)

Ovarian structure and folliculogenesis of females at different reproductive stages are described for the viviparous placentotrophic lizard Mabuya mabouya. The small ovaries have a thin wall formed by the ovarian epithelium and a thin tunica albuginea. One to two germinal beds that contain numerous oogonia and developing primordial follicles are derived from the ovarian epithelium and are next to the ovarian hilum. The ovarian cortex contains follicles at different stages of development, corpora lutea, and atretic follicles. The yolk nucleus and Balbiani complex were not evident in the ooplasm of previtellogenic follicles. The follicular epithelium of these follicles is polymorphic, as in other species of Squamata, but the larger cells are spherical and monolayered rather than pyriform. The zona radiata of the preovulatory follicles is less developed than in lecithotrophic species. These features suggest a decrease in metabolic and absorptive processes during follicular growth. In preovulatory follicles (1.5-1.8 mm diameter), primordial yolk vacuoles and small cortical granules are deposited in the ooplasm instead of fatty yolk platelets, so that only one stage of vitellogenesis is observed. Polyovular atretic follicles occur in some females. Follicular atresia is minimal for preovulatory follicles, but is more frequent in follicles with polymorphic epithelia. In the corpus luteum, the luteal tissue is formed from granulosa cells and luteolysis occurs during the late gastrula -- late neurula embryonic stages. Thus, the maintenance of gestation from the pharyngula to preparturition stages seems to be related to secretion of extraluteal progesterone, possibly of placental origin. These observed ovarian features are related to the high degree of placental complexity of this species and show that the evolution of advanced placentotrophy in species of this lineage has been accompanied by concomitant changes in ovarian function.     

alpha-Spectrin has a stage-specific asymmetrical localization during Xenopus oogenesis

Xenopus oocyte organization largely depends upon the cytoskeleton distribution, which is dynamically regulated during oogenesis. An actin-based cytoskeleton is present in the cortex starting from stage 1. At stages 4-6, a complex and polarized cytoskeleton network forms in the cytoplasm. In this paper, we studied the distribution of spectrin, a molecule that has binding sites for several cytoskeletal proteins and is responsible for the determination of regionalized membrane territories. The localization of alpha-spectrin mRNA was analyzed during Xenopus oogenesis by in situ hybridization on both whole mount and sections, utilizing a cDNA probe encoding a portion of Xenopus alpha-spectrin. Furthermore, an antibody against mammalian alpha-spectrin was used to localize the protein. Our results showed a stage-dependent mRNA localization and suggested that spectrin may participate in the formation of specific domains in oocytes at stages 1 and 2 and 4-6. Mol. Reprod. Dev. 55:229-239, 2000.     

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.     

Isolation,characterization and molecular cloning of cathepsin D from lizard ovary: Changes in enzyme activity and mRNA expression throughout ovarian cycle

During vitellogenesis, the oocytes of oviparous species accumulate in the cytoplasm a large amount of proteic nutrients synthetized in the liver. Once incorporated into the oocytes, these nutrients, especially represented by vitellogenin (VTG) and very low‐density lipoprotein (VLDL), are cleaved into a characteristic set of polypeptides forming yolk platelets. We have studied the molecular mechanisms involved in yolk formation in a reptilian species Podarcis sicula, a lizard characterized by a seasonal reproductive cycle. Our results demonstrate the existence in the lizard ovary of an aspartic proteinase having a maximal activity at acidic pH and a molecular mass of 40 kDa. The full‐length aspartic proteinase cDNA produced from total RNA by RT‐PCR is 1,442 base pairs long and encodes a protein of 403 amino acids. A comparison of the proteic sequence with aspartic proteinases from various sources demonstrates that the lizard enzyme is a cathepsin D. Lizard ovarian cathepsin D activity is maximal in June, in coincidence with vitellogenesis and ovulation, and is especially abundant in vitellogenic follicles and in eggs. Ovarian cathepsin D activity can be enhanced during the resting period by treatment with FSH in vivo. Northern blot analysis shows that cathepsin D mRNA is exceedingly abundant during the reproductive period, and accumulates preferentially in previtellogenic oocytes. Mol. Reprod. Dev. 52:126–134, 1999. © 1999 Wiley‐Liss, Inc.