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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., (Copyright 2001 Wiley-Liss, Inc.)

Recombinant purine nucleoside phosphorylase (PNPase) from Escherichia coli was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate-liberating enzymes. The E. coli enzyme was overexpressed in E. coli by inserting the genomic fragment containing the deoD gene downstream of the isopropyl beta-d-thiogalactoside-inducible promotor of pSE380 expression vector. The recombinant protein was purified to approximately 90% homogeneity and with a yield of approximately 9000 units of activity/L of culture, using an efficient one-column procedure. A continuous spectrophotometric assay coupling P(i) release to the phosphorolysis of the nucleoside analogue 7-methylinosine (m(7)Ino) was recently described. Here, we report the steady-state kinetic parameters of the recombinant E. coli PNPase catalyzed reaction with m(7)Ino and P(i) as substrates and compare these parameters with those of a bacterial PNPase commercially available for use in coupled assays. Under the assay conditions described, the recombinant E. coli protein is active at higher pH values and is stable up to a temperature of approximately 55 degrees C and following multiple freeze-thaw cycles. It is activated by high ionic strength but loses some activity following dialysis or concentration under pressure. Finally, a new procedure for the synthesis of m(7)Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase-coupled P(i) assays more attractive., (Copyright 2001 Academic Press.)

To determine whether oogonial proliferation and oocyte recruitment are under control of hypophyseal and/or ovarian factors, we carried out a series of investigations using Podarcis sicula, a lizard inhabiting the temperate lowlands of Europe in which oocyte recruitment occurs throughout the year, as animal model. Germinal beds containing oogonia and oocytes in prefollicular stages were cocultured with different ovarian compartments in presence/absence of FSH, and the effects of different treatments were evaluated by counting the number of prelepto-leptotene oocytes. Results revealed that oocyte recruitment from the pool of oogonia is under the control of a factor released by follicle cells while FSH has an indirect effect on modulating oogonial proliferation. SDS-PAGE analyses carried out on media conditioned by follicles suggest that the factor involved in the control of oocyte recruitment may be a small protein (about 21 kDa) and that its release is dependent on the period of the ovarian cycle but apparently not on the circulating levels of FSH.

Objective: To elucidate the molecular basis of G6PD deficiency in the Han and Li nationalities in Hainan, China., Methods: Polymerase chain reaction and restriction enzyme digestion were used to screen the mutations 1388G-->A, 1360C-->T, 1024C-->T, 592C-->T, 517T-->C, 493A-->G, 487G-->A, 392G-->T and 95A-->G. Single strand conformation polymorphism analysis was used to screen the other mutations followed by DNA sequencing to characterize the mutations of the samples with abnormal SSCP bands., Results: Of the fifty-nine Han cases with G6PD deficiency, fourteen with 1388G-->A (23.7%), three with 871G-->A(5.1%), one with 835A-->T(1.7%), one with 517T-->C (1.7%), three with 392G-->T(5.1%), and four with 95A-->G(6.8%) were found. Of the thirty-two Li cases with G6PD deficiency, six with 1388G-->A(18.8%), three with 871G-->A(9.4%), and two with 95A-->G(6.3%) were found. A new mutation 835A-->G which causes the substitution of Ala for Thr at 279 in a Han case was identified and named as G6PD Haikou. The enzyme activity of the variant is about 10% of the normal and lower than the activity of the variant 835A-->T with about 40% of the normal. Analysis of the 3D model of human G6PD has revealed that the hydroxyl group of Thr at 279 is a group in maintaining the interaction of the G6PD subunits., Conclusion: The most common mutations of G6PD deficiency in Han and Li nationalities in Hainan are similar. Compared with the mutation spectrum of G6PD gene in the populations in other regions of China, the results indicate that some G6PD gene mutations are widespread in the populations of different regions in the southern part of China. The hydroxyl group of the Thr at 279 of human G6PD may be a necessary group for maintaining the interaction of the G6PD subunits and the enzyme activity.

Full-length zebrafish cDNAs encoding two aspartic proteinases were cloned and sequenced. One of the two cDNAs was a 1708 bp product with an open reading frame of 398 amino acid residues corresponding to a cathepsin D. The other was a 1383 bp product encoding a polypeptide chain of 416 amino acids homologous to nothepsin, an aspartic proteinase first identified by us in the liver of Antarctic Notothenioidei. Gene expression assessed by RT-PCR and northern blot hybridization of RNA from different tissues showed that the expression was tissue- and sex-specific. Whereas the cathepsin D gene was expressed in all the tissues examined independently of the sex, the nothepsin gene was expressed exclusively in female livers.

The study of environmental factors affecting vertebrate reproduction has long interested both developmental and evolutionary biologists. Although photoperiod has been considered to be an important environmental parameter for vertebrates such as birds, temperature is probably a primary external factor responsible for reproductive cyclicity in reptiles. In spite of the progress made in the understanding of reptilian reproductive strategies and adaptations, much remains to be learned about the interplay between endocrine physiological factors, such as hormones, and environmental parameters. In this report, we have examined the effects of in vivo administered FSH on oocyte recruitment during the most significant periods of the reproductive cycle of the lizard, Podarcis sicula. The results show that when FSH is administered in proximity to the reproductive period, it stimulates oocyte growth and ovulation; when the hormone is administered at the beginning of the winter stasis it affects ovarian activity without inducing ovulation. Ovarian adenylate cyclase activity is moderately sensitive to in vitro FSH stimulation during the pre- and post-reproductive periods. The sensitivity to hormone stimulation increases significantly during the reproductive period and winter stasis. We have also tested the hypothesis that environmental temperature affects the responsiveness of ovarian adenylate cyclase to FSH stimulation. For such a purpose, we exposed animals to 28 degrees C or 4 degrees C in different periods of the ovarian cycle. The results show that, whenever the temperature applied mimics the thermal regime of the coming season, adenylate cyclase sensitivity to FSH shifts towards levels that anticipate the natural responsiveness.

This article describes a new organelle found in the cytoplasm of the growth stage fish oocytes. In particular, we describe its organization at the morphological level and investigate its composition by different cytochemical and immunocytochemical approaches with both light and electron microscope. The conclusion is that the body is a peculiar protein scaffold functioning as a temporary trap for the storage of rRNA in the mid to late growth stage oocytes. Its presence would be related to the reorganization of the mass of amplified rDNA in micronucleoli and to the consequent temporary stop in the rRNA synthesis., (Copyright 2000 Wiley-Liss, Inc.)

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.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-kappaB activation, in response to tumor necrosis factor-alpha treatment, is strongly reduced in human HeLa cells overexpressing G6PD.

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.

We report on the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Southern Italy (Campania region). Thirty-one unrelated G6PD-deficient males were analysed at DNA level for the presence of G6PD gene mutations. Nine different G6PD variants were identified, eight of which have already been described (Mediterranean, Seattle, two different A-, Santamaria, Cassano, Union and Cosenza). G6PD Mediterranean, Santamaria, A- and Union were associated with haemolytic episodes. G6PD Seattle, which is polymorphic in several populations, Cassano and Cosenza appeared to be asymptomatic. A new variant (G6PD Neapolis) is reported here. The 467(Pro-->Arg) substitution responsible for G6PD Neapolis is discussed in the light of the current 3D model of human G6PD and in comparison with other natural mutations which occur in the proximity of residue 467.

Gonadotropins (FSH and LH) affect several mammalian gonadal functions. In particular, FSH stimulates oogonial proliferation and oocyte growth, while LH regulates ovulation and progesterone secretion. In lacertilian reptiles, gonadal function is also regulated by pituitary gonadotropins, but which hormone controls ovarian activities and the mechanisms of action are unknown. The present study aimed to clarify mechanisms of action of pituitary gonadotropins on the ovary of Podarcis sicula (Lacertilia). The data demonstrate that mammalian gonadotropins FSH and LH produce a threefold stimulation of adenylate cyclase activity in follicular membranes, while hCG and TSH are less effective, causing a twofold increase in adenylate cyclase activity. Neurotransmitters such as dopamine, serotonin, and catecholamines have no effect on enzyme activity. The action of mammalian FSH and LH on the ovary mimics the effect of homologous hormones: in lizard ovaries incubated in vitro in the presence of isolated homologous pituitary glands, the intracellular cAMP level increased by 50% with respect to control ovaries. Mammalian gonadotropins appear homologous to lizard gonadotropin(s): Southern blot analyses show that the lizard genome contains nucleotide sequences homologous to those encoding for mammalian beta FSH and beta LH. Both homologous and heterologous desensitization of adenylate cyclase activity occurs in the lizard ovary. In fact, responsiveness of adenylate cyclase to gonadotropin stimulation is abolished in animals 2 hr after in vivo treatment with FSH. Sensitivity to gonadotropin stimulation is restored 2 weeks after the beginning of the in vivo treatment. Desensitization was also observed in ovaries incubated in vitro with mammalian FSH or with isolated pituitary glands.

The homeobox gene goosecoid (gsc) and the winged-helix gene Hepatic Nuclear Factor-3beta (HNF-3beta) are co-expressed in all three germ layers in the anterior primitive streak and at the rostral end of mouse embryos during gastrulation. In this paper, we have tested the possibility of functional synergism or redundancy between these two genes during embryogenesis by generating double-mutant mice for gsc and HNF-3beta. Double-mutant embryos of genotype gsc(-/-);HNF-3beta(+/-) show a new phenotype as early as embryonic days 8.75. Loss of Sonic hedgehog (Shh) and HNF-3beta expression was observed in the notochord and ventral neural tube of these embryos. These results indicate that gsc and HNF-3beta interact to regulate Shh expression and consequently dorsal-ventral patterning in the neural tube. In the forebrain of the mutant embryos, severe growth defects and absence of optic vesicles could involve loss of expression of fibroblast growth factor-8, in addition to Shh. Our results also suggest that interaction between gsc and HNF-3beta regulates other signalling molecules required for proper development of the foregut, branchial arches and heart.

Metallothionein levels were determined in the eggs of two sea urchin species, the Mediterranean Sphaerechinus granularis and the Antarctic Sterechinus neumayeri. While appreciable levels of metallothionein were found in S. granularis eggs, a negligible amount was detected in S. neumayeri. Two metallothionein isoforms were purified from S. granularis, and metallothionein cDNAs were obtained by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Two distinct cDNA species were cloned and sequenced. The translated amino acid sequences of these two forms consisted of 67 residues and differed in two amino acid substitutions. Despite the lack of metallothionein in S. neumayeri eggs, a metallothionein cDNA was obtained by RT-PCR amplification and a single amino acid sequence coding for a 63 residues MT was deduced. A comparative analysis of the primary structure of S. granularis and S. neumayeri metallothioneins with those of the other sea urchin metallothioneins has been performed. Sea urchin metallothioneins appear to be less similar to each other than metallothioneins of closely related vertebrates.

X-chromosome inactivation in mammals is regarded as an essentially random process, but the resulting somatic-cell mosaicism creates the opportunity for cell selection. In most people with red-blood-cell glucose-6-phosphate dehydrogenase (G6PD) deficiency, the enzyme-deficient phenotype is only moderately expressed in nucleated cells. However, in a small subset of hemizygous males who suffer from chronic nonspherocytic hemolytic anemia, the underlying mutations (designated class I) cause more-severe G6PD deficiency, and this might provide an opportunity for selection in heterozygous females during development. In order to test this possibility we have analyzed four heterozygotes for class I G6PD mutations: two with G6PD Portici (1178G-->A) and two with G6PD Bari (1187C-->T). We found that in fractionated blood cell types (including erythroid, myeloid, and lymphoid cell lineages) there was a significant excess of G6PD-normal cells. The significant concordance that we have observed in the degree of imbalance in the different blood-cell lineages indicates that a selective mechanism is likely to operate at the level of pluripotent blood stem cells. Thus, it appears that severe G6PD deficiency affects adversely the proliferation or the survival of nucleated blood cells and that this phenotypic characteristic is critical during hematopoiesis.

The effects of follicle-stimulating hormone (FSH) on early meiotic oocytes were studied by cytological, autoradiographic, and photometric techniques. In addition to regulating oogonial proliferation, oogenesis, and folliculogenesis, the hormone influenced germ cell number and the time course of early meiosis. FSH did not affect the timing of DNA replication and amplification and did not change the amount of rDNA accumulated in the nucleus by amplification. A genetic control mechanism for these processes is suggested.

More than 80 variants of glucose-6-phosphate dehydrogenase (G6PD) are associated with chronic nonspherocytic haemolytic anaemia (CNSHA); however, the molecular basis of this association is not fully understood. We have used the polymerase chain reaction and nucleotide sequence analysis to characterize a new G6PD variant, which we designate as G6PD Bari, in a G6PD-deficient boy affected by CNSHA. A single mutation leading to an amino-acid substitution was detected in the G6PD coding region, viz. a C->T transition at position 1187 predicting leucine at residue 396 in the enzyme; proline is invariably present in evolutionary distant G6PD molecules at this position. Inheritance in the patient's family was demonstrated by the polymerase chain reaction followed by diagnostic restriction enzyme analysis. The C->T transition responsible for G6PD Bari maps close to several other mutations previously identified in G6PD variants associated with CNSHA.

In an effort to investigate the subtelomeric region of the X chromosome among Orientals, five DNA sites in the F8C and G6PD genes were analyzed in a sample of 46 chromosomes belonging to the Chinese Li population, an ethnic group characterized by a high prevalence of G6PD deficiency. The DNA sites analyzed, which are highly polymorphic in other populations, have a low degree of heterozygosity in the Li sample and, furthermore, the distribution of the corresponding haplotypes is very different from that previously observed in Italian populations. Interestingly, three unrelated Li G6PD-deficient variants analyzed at the DNA level have the 1376G-->T mutation characteristic of G6PDCanton and they share the same haplotype, including the sites mentioned above, as well as eight DNA polymorphisms in the red/green color vision pigment genes located proximal to G6PD on chromosome X.

We have investigated two unrelated patients with congenital haemolytic anaemia in both of whom we found a combination of hereditary spherocytosis (HS) and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Segregation of the two defects was documented in both families, who had different molecular abnormalities for both HS and G6PD deficiency. In one family the propositus had a reduced level of spectrin and G6PD Seattle (282Asp-->His). In the other family the propositus had a band 3 abnormality and was heterozygous for G6PD Mediterranean (188Ser-->Phe). From a comparison of clinical and haematological findings in family members with either or both abnormalities we conclude that in one case the two defects exhibited a synergistic effect, resulting in a severe chronic haemolytic anaemia; whereas in the other the association was simply additive.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.

The most telomeric region of the human X chromosome within band Xq28 consists of a gene-rich region of about 3 Mb which contains the genes for coagulation factor VIIIc, glucose-6-phosphate dehydrogenase (G6PD), and red/green color vision. We have studied five polymorphic sites from this region, in a sample of normal people from the Cosenza province of Southern Italy. These sites, which span a distance of some 350 kb, are in strong linkage disequilibrium. Of the 32 possible haplotypes only 10 were found, and 4 of these account for 80% of all X chromosomes analyzed. In addition, we found that all G6PD-deficient people with the G6PD Mediterranean mutation belong to only two haplotypes. One of these (Med 1) is found only within a small subregion of the area investigated, west of the Appennine mountain range. Most remarkably, all Med 1 G6PD-deficient individuals also had red/green color blindness. The more frequent haplotype (Med 2) is the same in Calabria and in Sardinia, where it accounts for about 90% of the G6PD Mediterranean mutations, despite the fact that gene flow between the populations of Sardinia and Southern Italy must have been limited. These data do not enable us to determine whether the two types of G6PD Mediterranean have arisen through two separate identical mutational events or through a single mutational event followed by recombination. However, the data indicate relatively little recombination over an extended region of the X chromosome and they suggest that the G6PD Mediterranean mutation is recent by comparison to the other polymorphisms investigated.

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 53 male subjects from Calabria, in southern Italy. Our sequential approach consisted of the following steps: (1) Partial biochemical characterization was used to pinpoint candidate known variants. The identity of these was then verified by restriction-enzyme or allele-specific oligonucleotide hybridization analysis of the appropriate PCR-amplified fragment. (2) On samples for which there was no obvious candidate mutation, we proceeded to amplify the entire coding region in eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis of each fragment. (3) The next step was M13 phage cloning and sequencing of those individual fragments that were found to be abnormal by SSCP. Through this approach we have identified the molecular lesion in 51 of the 53 samples. In these we found a total of nine different G6PD-deficient variants, five of which (G6PD Mediterranean, G6PD A-, G6PD Coimbra, G6PD Seattle, and G6PD Montalbano) were already known, whereas four are new (G6PD Cassano, G6PD Cosenza, G6PD Sibari, and G6PD Maewo). G6PD Mediterranean is the commonest variant, followed by G6PD Seattle. At least seven of the variants are present, at polymorphic frequencies, in the Calabria region, and some have a nonrandom distribution within the region. This study shows that the genetic heterogeneity of G6PD deficiency in Calabria, when analyzed at the DNA level, is even greater than had been anticipated from biochemical characterization. The sequential approach that we have followed is fast and efficient and could be applied to other populations.

More than 80 genetic variants of glucose-6-phosphate dehydrogenase (G6PD) are associated with chronic non-spherocytic haemolytic anaemia (CNSHA). In order to help clarify the molecular basis of this association, we have carried out a detailed biochemical and genetic characterization of two G6PD deficient brothers affected by CNSHA. The G6PD from the two patients has altered electrophoretic mobility, abnormally elevated Michaelis constant (Km) for G6P, and extreme instability in vivo and in vitro. By comparison with published information we found that this is a new G6PD variant which we have designated G6PD Portici. The entire coding region of the gene has been sequenced, and a single point mutation, a G----A transition, was found at position 1178 in exon X, causing a substitution of histidine for arginine at residue 393 in the polypeptide chain. By polymerase chain reaction (PCR) amplification followed by diagnostic restriction enzyme analysis and allele-specific oligonucleotide hybridization we have demonstrated the inheritance of this mutation in the patient's family. Our results support the notion of a causative link between this mutation in the G6PD gene and CNSHA. Our data, in combination with previous data in the literature, suggest that the three-dimensional structure of G6PD is such as to cause interaction in the binding of its two substrates, G6P and NADP.

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.

Glucose-6-phosphate dehydrogenase (G6PD) has been analyzed by gel electrophoresis and by quantitative assay in an unselected sample of 1524 schoolboys from the province of Matera (Lucania) in southern Italy. We have identified 43 subjects with a G6PD variant. Of these, 31 had severe G6PD deficiency, nine had mild to moderate deficiency, and three had a non-deficient electrophoretic variant. The overall rate of G6PD deficiency was 2.6%. The frequency of G6PD deficiency, ranging from 7.2% on the Ionian Coast to zero on the eastern side of the Lucanian Apennines, appears to be inversely related to the distance of each town examined from the Ionian Coast, suggesting that this geographic distribution may reflect, at least in part, gene flow from Greek settlers. Biochemical characterization has shown that most of the G6PD deficiency in this population is accounted for by G6PD Mediterranean. In addition, we have found several examples of two other known polymorphic variants (G6PD Cagliari and G6PD A-); three new polymorphic variants, G6PD Metaponto (class III), G6PD Montalbano (class III), and G6PD Pisticci (class IV); and two sporadic variants, G6PD Tursi (class III) and G6PD Ferrandina (class II). These data provide further evidence for the marked genetic heterogeneity of G6PD deficiency within a relatively narrow geographic area and they prove the presence in the Italian peninsula of a gene (GdA-) regarded as characteristically African.

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.

Study of the germinal epithelium in the adult lizard shows that the germ cells constitute clusters of synchronized cells interconnected by intercellular bridges. Such bridges interconnect oogonia as well as early meiotic prophase oocytes (zygo-pachitene). Besides true intercellular bridges in oocytes, there are plasma membrane interruptions forming large zones that ensure cytoplasmic continuity between adjacent cells. In early diplotene, germ cells are isolated. Later, during auxocytosis, when the polymorphic follicular epithelium around the oocyte starts differentiating, intercellular bridges appear between the follicle cells and oocyte. No relationship is observed between the intercellular bridges found in the germinal epithelium and those found between the follicle cells and oocyte.

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.

Intercellular bridges first appear during lizard oogenesis when follicles are rather small (150 microgram in diameter); at this stage they form connecting links between the oocyte and follicle cells, which have not yet differentiated into pyriform cells. Later on, when the follicles have become larger (1 mm) and the follicular epithelium appears constituted by 3 types of cells (small, intermediate and pyriform cells) they form connecting links between the oocyte and both intermediate and pyriform cells. The establishment of intercellular bridges between pyriform cells and the oocyte precedes the complete differentiation of the former, which excludes the possibility that the fusion between pyriform cells and oocyte occurs only after these cells are completely differentiated. In still larger follicles (up to 2 mm in diameter), during the degeneration of the pyriform cells, the occurrence, inside the bridges, of mitochondria and other cytoplasmic material suggests that these cells at the end of their function transfer their contents into the oocyte.

Detergent treatment of sea urchin eggs at the mid 4-cell stage results in prevention of micromere segregation at the fourth cleavage. In these embryos not only the formation of the primary mesenchyme is suppressed, but synchrony of cell division, which is the rule during the first four cleavage cycles, continues for several cycles after the 16-cell stage while the typical mitotic phase wave that sets in after micromere segregation is abolished. These results support the hypothesis that micromeres act as coordinators of the mitotic activity of the embryo.

The regulation of oocyte growth and of exogenous vitellogenesis by micropinocytosis has been studied in lizard Podarcis sicula kept at 28 degrees during the winter stasis and stimulated by follicle-stimulating hormone (FSH). Under these experimental conditions, oocyte auxocytosis as well as vitellogenesis is stimulated, while the follicular hierarchy is preserved. At the ultrastructural level, the flow of exogenous yolk precursors toward the oocyte increases and the pathway taken by them through intercellular spaces and zona pellucida is the same as that taken by peroxidase (tracer). Yolk precursor endocytosis is found only in oocytes greater than 1500 microns in diameter and takes place through the formation of several coated pits and vesicles. It is suggested that membrane receptors necessary for micropinocytosis are available only in such oocytes. Last, a different permeability of the ovarian follicle to exogenous yolk precursors during the different stages of oocyte growth and endovarian control of vitellogenesis are suggested.

The follicular epithelium of the lizard oocytes undergoes structural and morphological modifications throughout oocyte growth. During this process the number of follicle cells increases and the epithelium acquires a multilayered and polymorphic organization which is characterized by the appearance of large follicle cells (intermediate and pyriform cells). The number of large cells also increases during oocyte growth and this increase parallels that of small cells. However, only the small cells become labelled one hour after [3H-]thymidine administration. Large cells have been found labelled after a longer period of time, i.e. 4--5 months after isotope injection. All these results together indicate that large follicle cells arise from the differentiation of small cells.