Immunocytochemical and electrophoretic distribution of cytokeratins in the regenerating epidermis of the lizard Podarcis muralis

Using immunocytochemistry at light- and electron-microscope levels, we studied the distribution of three monoclonal antibodies (AE1, AE2, AE3) specific for mammalian alpha-keratins in regenerating lizard epidermis. We also characterized the keratins expressed during this process by immunoblotting after electrophoretic separation. The AE1 antibody is localized in the basal and suprabasal layers of prescaling and scaling epidermis. During the first stages of scale neogenesis, the AE1 antibody also marks the differentiating oberhautchen and beta-layer, but it disappears from these layers as they mature. This antibody does not stain the prekeratinized and keratinized outermost layers in the hinge region. The AE2 antibody labels the superficial wound epidermis, prekeratinizing and keratinized beta- and alpha-layers, but not basal and suprabasal cells. The AE3 antibody labels all living and keratinized epidermal layers, although AE3 immunoreactivity decreases and disappears as the beta-layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron-dense areas within the beta-layer, suggesting retention of alpha-keratins. In the stages of tail regeneration examined, immunoblotting with the three antibodies used for the immunolocalization gives a pattern similar to that of the normal epidermis, except distally, where the process of scale differentiation begins. In this region, in addition to the keratin forms discovered in the normal and in proximal regenerating epidermis, an intense low molecular weight band at 40-41 kDa, positive to all three antibodies, is clearly detectable. Furthermore, in the distal region AE1 and AE3 antibodies, but not the AE2, recognize a weak band at 77-78 kDa not present in the normal and proximal epidermis. The localization and the possible role of the different keratins in the regenerating epidermis is discussed.     

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.     

Changes in ovarian follicles and in vitro sex hormone release in the lizard Podarcis sicula sicula

An in vitro superfusion method was used to test sex hormone release from different kinds of ovarian follicle (growing follicles, postovulatory follicles, and atretic follicles) in the lizard Podarcis sicula sicula. Sex hormone output changes with the stage of follicle evolution and sexual cycle. Previtellogenetic follicles prevail in early-spring quiescent ovaries and secrete mainly progesterone, which is probably utilized at that phase to delay ovarian resumption. In the active ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17β-estradiol, which is necessary for sustaining vitellogenin synthesis by the liver and oviduct growth. As follicles become ripe, progesterone production is resumed, and it increases in young postovulatory follicles. This is in line with the functions assigned to the hormone at that phase of the sexual cycle, i.e., the induction of oocyte maturation and the regulation of egg retention in the oviduct. Postovulatory follicles can also synthetize 17β-estradiol. After oviposition, this hormone, which is secreted by the old postovulatory follicles, can reinitiate vitellogenin synthesis, allowing the development of a new oocyte set. Our data confirm that active, although ephemeral, corpora lutea are also formed in oviparous species. A limited contribution to ovarian sex steroid production derives also from atretic follicles, at least at the early stages of the breeding cycle. © 1993 Wiley-Liss, Inc.     

Endothelin-B (ET(B)) receptor distribution in tissues of the lizard Podarcis sicula

Although the structural and pharmacological properties of endothelin (ET) receptors have been studied, little is known concerning their physiological significance, even if each subtype is supposed to have a distinct physiological action. Thus, to further elucidate the physiological function of this receptor, we examined the presence and distribution of the endothelin-B receptor (ET(B)) subtype in tissues of the lizard Podarcis sicula, using immunoblotting and immunohistochemistry. Immunoblotting indicated that, although the ET(B) receptor appears to be ubiquitous, it is present at different levels in the tissues examined. Furthermore, immunohistochemistry showed that this receptor is very abundant in endothelial cells of all tissues, suggesting that there is an ET(B)-mediated autocrine system of endothelin, which plays an important role in the regulation of endothelial cell function. On the other hand, the presence of ET(B) immunoreactivity also in endocrine systems such as adrenal and thyroid glands suggests an involvement also in the paracrine system of these organs.     

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.     

Putative steroid-binding receptors and nonreceptor components and testicular activity in the lizard Podarcis sicula sicula.

Labelled testosterone- and oestradiol-binding molecules have been found in the cytosol and nuclei of lizard testes. DNA-cellulose affinity chromatography was used to separate putative sex-steroid-binding receptors (adhering molecules) and nonreceptor components (nonadhering molecules). A putative androgen receptor (Kd: 10(-10) mol l-1; 3-9 fmol g-1 tissue) was found mainly in the nuclei of testicular cells when actively undergoing spermatogenesis. This suggests that, as in higher vertebrates, testosterone is implicated in spermatogenetic step regulation (meiosis and spermiogenesis) in lizard testis. In the cytosol, testosterone-binding molecules (Kd: 10(-9) mol l-1; 384-784 fmol g-1 tissue) with several properties of androgen-binding proteins are present from autumn to spring. The behaviour of these molecules is consistent with the role assigned to androgen-binding proteins as androgen reservoir. A putative oestrogen receptor is present throughout the sexual cycle, except during the culmination phase (breeding). The putative oestrogen receptor may be involved in the regulation of the first spermatogenetic step (spermatogonia multiplication) and in the induction of post-reproductive refractoriness. This phase is present in temperate-zone lizards. These studies show that the evaluation of sex-steroid-binding molecules is useful in considering the relationships between sex hormones and spermatogenetic activity in the testes of lizards.     

Testicular steroidogenic enzymes in the lizard Podarcis sicula during the spermatogenic cycle

Steroidogenic Acute Regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), 5α-Reductase (5α-Red), P450 aromatase are key enzymes involved in steroidogenesis. Recently, we showed the expression and the localization of P450 aromatase in Podarcis sicula testis during the different phases of the reproductive cycle, showing its involvement in the control of steroidogenesis, particularly in 17β-estradiol synthesis. Now, we have investigated the presence and distribution of the other enzymes involved in steroidogenesis, i.e. StAR, 3β-HSD, 17β-HSD and 5α-Red, during three significant periods of the reproductive cycle: summer stasis (July–August), autumnal resumption (November) and reproductive period (May–June). We demonstrated for the first time that all these enzymes are always present in somatic cells (Leydig and Sertoli) and germ cells (spermatogonia, spermatocytes I and II, spermatids and spermatozoa) of Podarcis testis, mainly in spermatids and spermatozoa. The present results strongly suggest that in Podarcis testis both somatic and germ cells could be involved in local sex hormone synthesis and that 5α-Red and P450 could carry out a pivot role.     

alpha and beta spectrin distribution during the differentiation of pyriform cells in follicles of lizard Podarcis sicula

Using alpha and beta spectrin mammalian antibodies on Western blotting, we demonstrated that lizard ovarian follicles contain two isoforms of alpha spectrin, Mr 94 and 134 kDa, and a 230 kDa beta spectrin, and that their pattern modifies in relation to pyriform cell differentiation. In fact, a positive immunoreaction is firstly evident within follicular epithelium of previtellogenic follicles when small cells differentiate into pyriform cells via intermediate cells. Later on, immunostain is present in pyriform cells and in the oocyte cortex that previously appears unstained. It is noteworthy that immunostain is also present on small cells located in contact with the oocyte membrane, but not on those located under the basal lamina and among pyriform cells, not engaged in pyriform cell differentiation. During the subsequent stages of previtellogenic phase, spectrin immunostain over the follicular epithelium and in the oocyte cortex does not change. By contrast, in vitellogenic follicles, when the follicular epithelium is constituted only by small cells, immunostain is evident at the level of the oocyte cortex and the cytoplasm of regressing pyriform cells. The present data strongly suggest that the alpha and beta spectrin pattern put in evidence during the different phases of lizard oocyte growth is related to the differentiation of small into pyriform cells, where such protein may guarantee a relationship between surface glycoproteins (Andreuccetti et al., 2001: Anat Rec 263:1-9), and the cytoskeleton distribution (Maurizii et al., 2000: Raf Mol Reprod Dev 57:159-166). Furthermore, the distribution of spectrin mRNA, similar to that observed for the protein, demonstrates that spectrin, once synthesized within pyriform cells, is transferred through intercellular bridges in the oocyte cortex, thus confirming that pyriform cells are nurse that significantly are involved in the oocyte growth. Finally, the present data demonstrate that alpha spectrin of lizard ovarian follicles has Mr quite different from those so far reported and may constitute a new group of isoforms. This important result will be the focus of future experiments. Mol. Reprod. Dev. 67: 101-107, 2004.     

Vitellogenin precursors in the liver of the oviparous lizard,Podarcis sicula

In reptiles, as in the other oviparous vertebrates, vitellogenin (VTG) synthesis is stimulated in the liver by ovarian estrogens. In this article, the presence of VTG precursors was detected in liver subcellular fractions of the oviparous lizard, Podarcis sicula, in the reproductive period. The rough endoplasmic reticulum (RER) and the smooth microsomal fraction (SMF), which includes smooth endoplasmic reticulum and Golgi complex, were separated by means of two different sucrose gradients. The successful separation was controlled at the electron microscope. The contents of the different compartments were extracted by means of n-octyl-beta-D-glucopiranoside detergent and subjected to SDS-PAGE. Western Blotting with homologous anti/VTG antibody revealed two immunoreactive proteins of about 84 and 70 kDa in the RER, and four proteins of about 180, 150, 60, 50 kDa in the SMF; all these proteins appeared phosphorylated and glycosylated. The differences in the molecular weight of these VTG precursors are 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.     

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.     

Response of the thyroid gland of the lizard Podarcis sicula to endothelin-1

Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET) induced hormonal functions in various tissues. We have studied the interaction of ET-1 with thyroid gland of the lizard Podarcis sicula. The effects of ET-1 administration on the plasma levels of the thyroid hormones (T3 and T4) and TSH were stimulatory. Morphological changes in the thyroid after treatment with ET-1 were also detected: the height of the epithelial cells slightly increased and the apical surface acquired microvilli protruding into the follicular lumen. The colloid filled up the lumen and showed a rich peripheral vacuolation. In conclusion, a modulatory role in the control of the reptilian thyroid gland is suggested for ET-1. This is the first report on the interaction of ET-1 with the thyroid gland of reptilian.     

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.     

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.     

Fine observation on nerves colonizing the regenerating tail of the lizard Podarcis sicula

During the regeneration of lizard tail, nerves sprouting from ganglia and the spinal cord invade the blastema as far as the apical epidermis. Electron microscopical observations reveal axons storing dense granules (dg) and dense core vesicles (dcv) which are concentrated in nerve terminals or in axoplasmatic regions. In the regenerating spinal cord (SC) these terminals resemble aminergic-peptidergic endings and grow as far as the distal portion of the SC, which is made up of irregularly arranged ependymal cells. Some axons storing dcv contact blastematic cells and other nerve terminals show a plasma membrane incomplete or broken. Whether this latter aspect is due to fixation artifacts or physiological rupture is unknown. Nerves containing dcv and a few dg also originate from spinal ganglia innervating the regenerating tail. The accumulation of material into these endings is probably slow and a possible trophic influence on the regeneration of lizard tail is discussed.     

Effects of leptin administration on the endocrine pancreas and liver in the lizard Podarcis sicula

In this study, we investigated the presence of leptin receptor in pancreatic islets and the effect of exogenous leptin administration in Podarcis sicula on glucose metabolism. Our data show the presence of leptin receptor immunoreactivity in the endocrine pancreas suggesting that leptin may act at a peripheral level as previously postulated in mammals. The effects of short- and long-term and dose-response treatment with supraphysiological concentrations of leptin on circulating levels of insulin, glucagon and glucose in the blood have been evaluated. Taken together, our results indicate that leptin treatment was followed by an increase in insulin, glucagon and glucose in the blood, depending on the dose of leptin. Moreover, leptin treatment brought about a decrease of glycogen and the appearance of tyrosine-phosphorylated proteins in the liver. This study shows that in the lizard P. sicula leptin is involved in glucose metabolism.     

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.     

Lipovitellins and phosvitins of the fertilized eggs during embryo growth in the oviparous lizard Podarcis sicula

In the lizard Podarcis sicula, the major vitellogenin (VTG)-derived yolk proteins, lipovitellins and phosvitins, were extracted from the yolk globules of laid and fertilized eggs at different periods of incubation up to 44 days close to hatching. Embryonic development was almost over at this time. Yolk proteins were isolated by precipitation in saturated (NH(4))(2)SO(4), separated on SDS-PAGE and detected by Western blotting with homologous polyclonal anti/VTG antibody. Two lipovitellins of 110 and 116 kDa were always present in the yolk of laid eggs after 1, 10, 18, and 44 days from oviposition. Both these proteins were glycosylated and were recognized by the anti/VTG antibody; their N-terminal sequences were analyzed. Four phosvitins were detected in freshly laid eggs, but their number decreased during incubation, and after 44 days only a single protein of approximately 6.5 kDa was present. The results indicated that, in this lizard, during embryonic development, lipovitellins remain unchanged, whereas the phosphorylated components of yolk undergo continuous degradation.     

Estrogen-induced Akt-1 activity in the lizard (Podarcis s. sicula) testis

There are always more evidences indicating that 17beta-estradiol (E2) is to be necessary for normal male fertility. Here we report the expression of the most ubiquitously expressed member of the akt family of genes, akt1, in the lizard (Podarcis s. sicula) testis. We have used a nonmammalian vertebrate model (the lizard P. s. sicula) to investigate the regulation of the serine/threonine kinase Akt activity, implicated in the control of cell proliferation, survival, and metabolism, in the testis during the annual sexual cycle and to study whether E2 exerts a role in the spermatogenesis through Akt-1 activity. Immunocytochemistry analysis show that Akt-1 proteins are present in the spermatogonia (SPG), and spermatocytes (SPC), and spermatids (SPT). The annual E2 profile shows a progressive increase during the active spermatogenesis (from April to June) and a peak in the month of August (spermatogonial mitosis). In parallel, Akt-1 (molecular weight 60 kDa) are highly phosphorylated during the period of active spermatogenesis and in post-refractory period (August) compared with the winter stasis (from November to March). Present results demonstrate that E2 treatment induces the activation of Akt-1, and this effect is counteracted by the anti-estrogen ICI 182-780.