Maria Gabriella Maurizii

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 α‐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 β‐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 β‐ and α‐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 β‐layer matures. The ultrastructural study shows that the AE2 and AE3, but not the AE1, antibodies specifically label small electron‐dense areas within the β‐layer, suggesting retention of α‐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. J. Morphol. 246:179–191, 2000.

Doubly Uniparental Inheritance of Mitochondria As a Model System for Studying Germ Line Formation

Background Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. Methodology/Principal Findings We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. Conclusions/Significance In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.

Paternally Transmitted Mitochondria Express a New Gene of Potential Viral Origin

Mitochondrial ORFans (open reading frames having no detectable homology and with unknown function) were discovered in bivalve molluscs with doubly uniparental inheritance (DUI) of mitochondria. In these animals, two mitochondrial lineages are present, one transmitted through eggs (F-type), the other through sperm (M-type), each showing a specific ORFan. In this study, we used in situ hybridization and immunocytochemistry to provide evidence for the expression of Ruditapes philippinarum male-specific ORFan (orf21): both the transcript and the protein (RPHM21) were localized in spermatogenic cells and mature spermatozoa; the protein was localized in sperm mitochondria and nuclei, and in early embryos. Also, in silico analyses of orf21 flanking region and RPHM21 structure supported its derivation from viral sequence endogenization. We propose that RPHM21 prevents the recognition of M-type mitochondria by the degradation machinery, allowing their survival in the zygote. The process might involve a mechanism similar to that of Modulators of Immune Recognition, viral proteins involved in the immune recognition pathway, to which RPHM21 showed structural similarities. A viral origin of RPHM21 may also support a developmental role, because some integrated viral elements are involved in development and sperm differentiation of their host. Mitochondrial ORFans could be responsible for or participate in the DUI mechanism and their viral origin could explain the acquired capability of M-type mitochondria to avoid degradation and invade the germ line, that is what viruses do best: to elude host immune system and proliferate.

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. Mol. Reprod. Dev. 57:159–166, 2000.

Vasa protein is localized in the germ cells and in the oocyte-associated pyriform follicle cells during early oogenesis in the lizard Podarcis sicula

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.

Vasa expression in spermatogenic cells during the reproductive-cycle phases of Podarcis sicula (Reptilia, Lacertidae)

The vasa gene encodes a DEAD-box ATP-dependent RNA helicase that regulates the translation of multiple mRNAs involved in germ line differentiation. This protein has been deeply studied in many animals, but few data are available to date on reptiles. In this work, we sequenced a portion of Podarcis sicula vasa gene (Ps-vasa), developed a specific antibody and verified its specificity. Using anti-Ps-Vasa and confocal microscopy, we studied Vasa expression in male germ cells during the reproductive cycle of P. sicula: during full gonadal activity (spring), during regression of gonadal activity (summer) and during slow autumnal recrudescence. We also analyzed Vasa expression in young testes when the walls of the seminiferous tubules were forming. The aim was to verify if Vasa is involved in the process of male germ cell differentiation in all phases of the reproductive cycle. In adult testes, during full gonadal activity and during recrudescence, Vasa staining was detected from spermatogonia to spermatids. Vasa spots were also observed in the nucleus of germ cells supporting its function in different cellular compartments. No Vasa staining was observed in mature spermatozoa during the spring and mid-late November. The seminiferous epithelium analyzed in the summer appeared reduced with only spermatogonia, all Vasa-immunostained, some in division to replace germ cells. In immature testes, the seminiferous epithelium contained only spermatogonia and spermatocytes. The clear immunostaining in their cytoplasm revealed that Vasa is already expressed in juvenile male gonads, suggesting a role in the differentiation process since P. sicula early developmental stages.

The germarium of panoistic ovarioles of Bacillus rossius (Insecta Phasmatodea): Larval differentiation

Summary In newly hatched larvae of Bacillus rossius (Insecta Phasmatodea) viewed with light and electron microscopes, ovarioles appear as bag-like structures; during the first instar they initially assume a bell-shaped appearance. Afterwards, they begin to elongate and their vitellarium essentially consists of a row of oocytes with a clear growth gradient. This typical ovariole morphology becomes more evident in the subsequent instars up to the fourth. The germarium appears as a region interposed between growing oocytes of the vitellarium and somatic cells of the terminal filament. Light and electron microscope observations indicate that the structure of the germarium does not markedly change during the different instars: it contains, besides somatic prefollicular cells, germ elements arrested in the “diffuse” stage which precedes diplotene of growing oocytes. Only in newly hatched larvae, squash preparations of the germarium also show germ cells in earlier meiotic prophase (zygo-pachytene). The observati...

First evidence of Vasa expression in differentiating male germ cells of a reptile

The vasa gene has a central role in germ-cell development, and is conserved in animals. Vasa homologs usually exhibit germ-line-specific expression, so vasa mRNA and protein are generally utilized as germ-line markers. The gene encodes a member of the DEAD-box family of ATPdependent RNA helicases that appears to regulate the translation of multiple mRNAs involved in germ-line differentiation (Gustafson and Wessel, 2010). During spermatogenesis, for example, the presence of Vasa was well documented in the cytoplasm of differentiating germ cells in many vertebrates and invertebrates (Gustafson and Wessel, 2010). We used a species-specific anti-Vasa antibody to analyze, by confocal microscopy, Vasa expression during spermatogenesis in Podarcis sicula. The aim of the study was to assess if Vasa is involved in male germ line differentiation in this lizard, since no data are available to date on reptiles. Vasa wasspecifically expressed inP. siculamalegametes from theearly to the last stages of development, but was not detectable in mature spermatozoa residing in the lumen of the tubule (Fig. 1), as reported in other animals (Toyooka et al., 2000). In P. sicula spermatids, Vasa-stained spots aggregate into a single, large cytoplasmic body located in a notch of the nucleus (Fig. 1, inset). This structure may correspond to nuage material—the chromatoid body (CB) in spermatogenic cells—that is identifiable in the posterior part of developing spermatids in mice and rats. In these mammals, the CB is displaced toward the caudal pole of the nucleus of early elongating spermatids, where the bulk of the CB material condenses into a dense sphere, eventually forming a ring around the base of the developing flagellum (Shang et al., 2011). Because of its maintenance during male gamete development, the CB was proposed to have a role in spermatid cytodifferentiation. Indeed, as the egg germ plasm, the nuage-like CB includes proteins involved in the stabilization and translation of germ-cellspecific transcripts (Shang et al., 2011)—one of which is Vasa, considering that vasa-mutant mice exhibit a deficiency in post-meiotic progression before CB formation (Toyooka et al., 2000 and references therein). In P. sicula, Vasa staining remains at one site of the spermatid elongated nucleus until the last stages of differentiation. At the periphery of the lumen of the P. sicula seminiferous tubule, for example, some germ cells similar to spermatozoa, but with a less-strongly packed chromatin, show intense Vasa staining at one extremity of the nucleus (Fig. 1); we believe these are spermatids in the last stages of differentiation, shortly before spermiation, the process during which sperm are released from the supporting somatic Sertoli cells into the lumen of the seminiferous tubule. The presence of Vasa until later stages of spermatid differentiation suggests that this protein may play an important role in the differentiation of P. sicula male germ cells.

Structural modifications of the fat body in the stick insect Bacillus rossius during larval development

Abstract The fat body of the female stick insect, Bacillus rossius (Rossi), was examined ultrastructurally in developing larvae. Two different fat body tissues are present in first instar larvae: A remnant of an embryonic tissue, and a newly differentiated tissue appearing only after hatching, the larval fat body. The embryonic fat body consists of large cells with numerous lipid droplets of saturated fatty acids. They are randomly dispersed in a dense cytoplasm packed with free‐ribosomes. The larval fat body includes cells highly enriched in glycogen and with numerous unsaturated lipid droplets. With the proceeding of development, the embryonic fat body is destined to disappear. By contrast, the larval fat body undergoes progressive decrease in glycogen content, and the cytoplasm gradually fills with numerous Golgi apparatus and cisternae of the rough endoplasmic reticulum. Lipid droplets remain in the larval fat body until the transition to the adult stage takes place. With the attainment of the stage I...

The germarium of panoistic ovarioles of Bacillus rossius (Insecta Phasmatodea): Structure and function during imaginal life

Summary In adult females of Bacillus rossius (Insecta Phasmatodea) the germarium, localized at the ovariole tip just below the terminal filament and above the vitellarium, progressively reduces in size and eventually disappears at the end of the ovulatory period. The observations with light and electron microscopes show that in the end-chamber most germ cells are arrested in a post-pachytenic diffuse stage, which just precedes diplotenic oocyte growth. These observations also indicate that the reduction in size of the germarium of ovulating females should probably be ascribed to a progressive and extensive activation of the resting germ cells. The average number of ovulated eggs per ovariole (6.7±0.9) is consistent with this view. However, occasional findings of lepto-zygotenic germ cells in some preovulatory ovarioles of adult females do not completely rule out the persistence of scarce undifferentiated germ elements (oogonia) in the larval germarium at the onset of adult life. Furthermore, the reduction...