Spherical bodies present within the germinal vesicle of Podarcis sicula previtellogenic oocyte derive from the temporaneous inactivation of ribosomal genes

In the present paper we have investigated the origin of the spherical bodies (SBs) present within the germinal vesicle of about 400 microm previtellogenic oocytes in the lizard Podarcis sicula. In particular, we have attempted to clarify whether they derive from the single, large nucleolus present in early diplotenic oocyte as a consequence of ribosomal gene inactivation. We have, therefore, experimentally induced a decrease in rRNA synthesis by injecting animals with D-galactosamine or by exposing them to low temperatures. The investigations carried out have demonstrated that both treatments induce significant ultrastructural changes in the nucleolar apparatus and in particular fragmentation and the formation of SBs comparable to those observed in germinal vesicle under physiological conditions. These results indicate that the germinal vesicle of Podarcis sicula has a nucleolar apparatus that significantly changes its aspect according to its functional status and reveal that in this species, the time course of rRNA synthesis is peculiar with respect to any other vertebrate oocyte studies so far.     

Phylogeography of the Italian wall lizard, Podarcis sicula, as revealed by mitochondrial DNA sequences

In a phylogeographical survey of the Italian wall lizard, Podarcis sicula, DNA sequence variation along an 887-bp segment of the cytochrome b gene was examined in 96 specimens from 86 localities covering the distribution range of the species. In addition, parts of the 12S rRNA and 16S rRNA genes from 12 selected specimens as representatives of more divergent cytochrome b haploclades were sequenced (together about 950 bp). Six phylogeographical main groups were found, three representing samples of the nominate subspecies Podarcis sicula sicula and closely related subspecies and the other three comprising Podarcis sicula campestris as well as all subspecies described from northern and eastern Adriatic islands. In southern Italy a population group with morphological characters of P. s. sicula but with the mitochondrial DNA features of P. s. campestris was detected indicating a probably recent hybridization zone. The present distribution patterns were interpreted as the consequence of natural events like retreats to glacial refuges and postglacial area expansions, but also as the results of multiple introductions by man.     

Storage in the yolk platelets of low MW DNA produced by the regressing follicle cells.

The present work was carried out to clarify the nature and origin of the yolk DNA present in vitellogenic oocytes of the lizard Podarcis sicula. Morphological and biochemical evidences indicate that it has an intrafollicular origin, from the apoptotic bodies resulting from follicle cells regression at the end of previtellogenesis. This conclusion is reinforced by the observation that the oocyte membrane, in in vitro experiments, is unpermeable to exogenous DNA. Biochemical evidences reveal that the yolk DNA has a low (200bp) molecular weight and this suggests that it is produced by the endonucleases typically involved in apoptotic DNA laddering. Indeed, immunocytochemical analyses demonstrate that follicle cells contain significant amounts of DNAse I. In immunoblots, carried out during different periods of the ovarian cycle, the enzyme shows a MW of about 33, 66 or 100 kDa thus indicating that its activity in the follicle of Podarcis is modulated by dimerization and/or binding to regulatory factors. Mol. Reprod. Dev. 59: 422-430, 2001.     

Characterization of polymorphic microsatellite markers in the Dalmatian wall lizard Podarcis melisellensis (Squamata: Lacertidae)

We describe polymerase chain reaction primers and amplification conditions for 13 highly polymorphic microsatellite DNA loci isolated from the Dalmatian wall lizard, Podarcis melisellensis. The number of alleles per locus ranged from 12 to 41, with levels of observed heterozygosity between 0.62 and 0.94. Most of these loci were successfully cross-amplified in the closely related species P. sicula, but levels of polymorphism were always lower.     

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.     

Gene amplification proceeds by a chromosome copy mechanism

Two general mechanisms which could account for gene amplification of ribosomal DNA in the oocytes of amphibians have been tested. In one mechanism at least the first extra copy of ribosomal DNA would be derived from the chromosomal ribosomal DNA and would predict a nuclear inheritance for the amplified ribosomal DNA. In an alternative mechanism, the amplified ribosomal DNA would be derived from one or more of the amplified ribosomal DNA copies which had been released from the oocyte nucleus and transmitted as an autonomous “episome” through the germ line into the oocytes of the progeny. The latter mechanism would require a maternal inheritance for the amplified ribosomal DNA.     

Leptin presence in plasma, liver and fat bodies in the lizard Podarcis sicula: fluctuations throughout the reproductive cycle.

Leptin is a 16 kDa peptide produced by adipocytes in response to increasing fat stores and signals to the brain to stop eating and increase energy expenditure. Recent studies point out that the role of leptin is much broader and includes the regulation of reproduction. The lizard, Podarcis sicula, is an oviparous species characterized, at this latitude, by a seasonal reproductive cycle. Since in Podarcis sicula the recovery of the gonadal function coincides with the recovery of metabolic activity, we have hypothesized that leptin might be involved in the regulation of the reproductive function in this species. We have identified an immunoreactive band in the plasma of the female of Podarcis sicula, which comigrated with recombinant mouse leptin and cross reacted with polyclonal antibodies against mouse leptin. A RIA method developed by Linco (St. Louis, MO) was utilized to measure leptin concentration in plasma, liver and fat bodies of Podarcis sicula throughout the reproductive cycle. The antibody used in this kit (Linco's Multi-Species Leptin Radioimmunoassay kit) was produced in the guinea pig against human leptin but displays crossreactivity to leptin molecules of many species. The level of leptin in the plasma of Podarcis sicula was in the same range as that of mammals. Leptin levels in plasma, liver and fat bodies fluctuated during the reproductive cycle, in a way consistent with its possible role in reproduction.     

Ultrastructural Observations on the Germ Plasm in the Lizard Podarcis sicula

Ultrastructural studies on embryos and adult females of Podarcis sicula revealed fibrogranular electron-dense aggregates in the cytoplasm of primordial germ cells, oogonia, and oocytes. The ultrastructural similarities of these aggregates to fibrogranular aggregates in germ cells of some animal species and their relationship with mitochondria, free ribosomes, as well as cisternae of the rough endoplasmic reticulum strongly suggest that they correspond to the germ plasm.     

Progesterone receptor in the liver and oviduct of the lizard Podarcis sicula

In this paper we report the presence of a (3)H-Progesterone ((3)H-P) binding moiety, which has the characteristics of a true receptor, in the liver of the female of the lizard Podarcis sicula. (3)H-P binding studies show the presence of one type of binding site with an average Kd value of 6.2 +/- 2.0 nM in the cytoplasm and 6.3 +/- 1.1 nM in the nucleus. Competition experiments showed that progesterone (P) was the best competitor, while testosterone, deoxycorticosterone (DOC), corticosterone, 17 alpha-hydroxyprogesterone; R5020; RU486 and RU26988-5 were poor competitors. We have also investigated the immunological characteristics of progesterone receptor (PR) in both the liver and the oviduct of Podarcis sicula, by Western blotting using the monoclonal antibody PR22 raised against the PR isoforms A and B of chicken. One imunoreactive band of about 70 kDa was detected in cytoplasmic and nuclear extracts of both the liver and the oviduct. PR immunoreactivity was present in the liver during the quiescent phase. In the oviduct PR immunoreactivity increased from the recovery to the full grown phase. P treatment of estrogen-primed females did not affect the presence of PR in the liver, while brought about a PR increase in the oviduct. This study suggests that PR is expressed differently in the liver and the oviduct of Podarcis sicula throughout the reproductive cycle. PR might fulfill different requirements in relation to the different physiological functions of the tissue during the reproductive cycle.     

Estrogen receptor beta localization in the lizard (Podarcis s. sicula) testis

There is increasing evidence that 17beta-estradiol is necessary for normal male fertility. The aim of the present study was to characterize estrogen receptor beta (ERbeta) expression in a non-mammalian vertebrate model, the lizard (Podarcis s. sicula) testis. Immunocytochemical analysis shows that ERbeta proteins are present among germ cells in the nucleus of the spermatogonia, in primary spermatocytes and spermatids. Western blot analysis with antibodies against the ERbeta gene product revealed an isoform with a specific weight of 55 kDa. In conclusion, the widespread expression of ERbeta in the Podarcis s. sicula testis is consistent with a role for estrogens in modulating spermatogenesis in the male.     

Expression of PCNA in the testis of the lizard, Podarcis s. sicula: an endogenous molecular marker of mitotic germinal epithelium proliferation.

Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. This protein encircles DNA and can slide bidirectionally along the duplex binding to DNA polymerase delta and epsilon. It is well known that PCNA interacts with proteins involved in the cell cycle. The PCNA interactions with different cellular proteins and the importance of these interactions are discussed. To examine mitotic germinal epithelium proliferation during annual discontinuous spermatogenesis in the lizard Podarcis s. sicula, temporal and the spatial PCNA expression were investigated, and provide a useful endogenous molecular marker.     

Evolution of a centromeric satellite DNA and phylogeny of lacertid lizards.

1. The composition and phyletic distribution of a highly repetitive satellite DNA, isolated from Podarcis sicula, was studied. 2. This DNA was rich in adenine and thymine and displayed frequent adenine stretches. It was always located on the centromeric heterochromatin even in quite taxonomically distant species. 3. Southern blot hybridization of the Taq I satellite on various species of lacertid families showed a close affinity among Podarcis, Algyroides and Lacerta dugesii. 4. All the other taxa investigated did not seem to possess this repeated sequence.     

Ribosomal bodies and annulate lamellae in the oocytes of the lizardPodarcis sicula

Summary Ultrastructural studies suggest that, in the oocytes of the lizardPodarcis sicula, ribosomal bodies are structurally continuous with annulate lamellae during their organization and disaggregation. This observation may indicate the dynamic transformation of the cytomembranes of one structure into those of the other, and vice versa. Moreover, the presence of annulate lamellae has been detected for the first time in lizard oocytes. The hypothesis is advanced that ribosomal bodies and annulate lamellae, in spite of some different structural characteristics, may play a similar role during the oocyte growth.     

Organization and characterization of the keratin cytoskeleton in the previtellogenic ovarian follicle of the lizard Podarcis sicula raf

The cytokeratin (CK) cytoskeleton, previously described by immunofluorescence in the ovarian follicle of Podarcis sicula, at the electron microscope results constituted by bundles of 10 nm thick intermediate filaments containing keratin. These bundles are better evident in the cytoplasm of the pyriform cell apex pointed toward the oocyte surface and mostly in the intercellular bridges connecting fully differentiated pyriform cells to the oocyte. During the differentiation of pyriform cells, the intermediate filament bundles first appear inside the intercellular bridge, when the small follicle cells progressively enlarge after their fusion with the oocyte and assume a morphology of "intermediate" cells. The present study also reports a comparative analysis by immunolabeling, SDS-PAGE, and immunoblotting with anticytokeratins CK8, CK18, and CK19 antibodies of both the ovarian follicle and the intestine of Podarcis sicula. These antibodies, specific to the keratins of monolayered intestinal cells, react also against those expressed in the oocytes of Xenopus laevis. This study shows the presence in the ovarian follicle of this reptile only of keratin forms of homologues to the CK8 and CK18 of mammals and the lack of CK19. The same analysis carried out utilizing AE1 and AE3 antibodies, which recognize most of the acidic and basic keratins in mammals, has shown additional forms of keratins specifically expressed in the ovarian follicle (50 kDa) and in both the examined tissues (49 and 60 kDa). The reported results indicate that in the ovarian follicle of this reptile, keratins have peculiar characteristics that can be explained by the unique structural function of the cytoskeleton in this system.     

DNA amplification in sea urchin oocytes

Summary In situ hybridization of ribosomal RNA withParacentrotus lividus ovaries suggests that ribosomal DNA undergoes amplification in the mononucleolate oocytes of this sea urchin.     

Variation in the cranium shape of wall lizards (Podarcis spp.): effects of phylogenetic constraints, allometric constraints and ecology

We used geometric morphometrics to explore the influence of phylogenetic and allometric constraints as well as ecology on variation in cranium shape in five species of monophyletic, morphologically similar Podarcis lizards (Podarcis erhardii, Podarcis melisellensis, Podarcis muralis, Podarcis sicula and Podarcis taurica). These species belong to different clades, they differ in their habitat preferences and can be classified into two distinct morphotypes: saxicolous and terrestrial. We found (i) no phylogenetic signal in cranium shape, (ii) diverging allometric slopes among species, and (iii) a significant effect of habitat on cranium shape. The saxicolous species (P. erhardii and P. muralis) had crania with elongated parietals, elongated cranium bases, shortened anterior parts of the dorsal cranium, reduced chambers of the jaw adductor muscles and larger subocular foramina. These cranial features are adaptations that compensate for a flattened cranium, dwelling on vertical surfaces and seeking refuge in crevices. The crania of the terrestrial species (P. melisellensis, P. sicula and P. taurica) tended to be more elongate and robust, with enlarged chambers of the jaw adductor muscle, reduced skull bases and shortened parietals. Terrestrial species exhibited more variation in cranium shape than saxicolous species. Our study suggests that shape variation in Podarcis sp. lizards is largely influenced by ecology, which likely affects species-specific patterns of static allometry.     

Relative ribosomal RNA cistron multiplicity in oocytes and postembryonic stages of the eutelic nematodePanagrellus silusiae

Summary The number of ribosomal RNA cistrons has been measured in the total DNA extracted from L2 juvenile and adult stages of the free-living nematodePanagrellus silusiae. Saturation hybridization studies with homologous rRNA indicate that both stages have about 275 ribosomal genes per haploid equivalent. Using homologous¹²⁵I-labelled rRNA for in situ hybridization, the mean number of silver grains per DNA content for oocyte, hypodermis and gut nuclei was similar. The mean DNA contents of maturing oocyte, hypodermis and gut nuclei are about 20C, 2C, and 10C respectively. We conclude that rDNA amplification alone is insufficient to account for the variation in DNA content of oocytes and that postembryonic development in this eutelic organism occurs without a significant differential increase in the number of ribosomal cistrons per worm.Supported by the National Research Council of Canada     

Nucleolar DNA in oocytes of Salmo irideus (Gibbons)

The amplification of ribosomal genes has been studied in oocytes from Salmo irideus. In situ nucleic acid hybridization showed that the synthesis of nucleolar DNA begins in oogonium and proceeds slowly through leptotene and zygotene when a small amount of extrachromosomal nucleolar DNA is produced. In early pachytene there is a rapid build-up of nucleolar DNA demonstrable by rapid incorporation of tritiated thymidine. Synthesis stops completely in early diplotene when nucleolar DNA becomes dispersed over the inner surface of the nuclear envelope in the form of small Feulgen-positive granules. Photometric measurements of Feulgen stained nuclei showed that the final amount of amplified nucleolar DNA synthesized in each oocyte is approximately 20 g. The amplified DNA does not form a heterochromatic mass. The buoyant density of the amplified nucleolar DNA calculated from analytical centrifuge tracings in relation to DNA from Micrococcus luteus ( = 1.731 g cm^–3) is 1.715 g cm^–3 and corresponds to a G + C content of 57%. There are indications that the buoyant density of the somatic nucleolar DNA is lower than that of amplified nucleolar DNA.Similarities and differences between ribosomal gene amplifications in oocytes of Salmo irideus and the corresponding process in Xenopus are discussed.