HPV infection in semen: results from a new molecular approach.

Human papillomavirus (HPV) is the agent of the most common sexually transmitted diseases causing a variety of clinical manifestations ranging from warts to cancer. Oncogenic HPV infection is the major cause of cervical cancer and less frequently of penile cancers. Its presence in semen is widely known, but the effects on fertility are still controversial. We developed a new approach to evaluate virus localisation in the different semen components. We analysed also the specific genotype localisation and viral DNA quantity by qPCR. Results show that HPV DNA can be identified in every fraction of semen: spermatozoa, somatic cells and seminal plasma. Different samples can contain the HPV DNA in different fractions and several HPV genotypes can be found in the same fraction. Additionally, different fractions may contain multiple HPV genotypes in different relative quantity. We analysed the wholeness of HPV DNA in sperm cells by qPCR. In one sample more than half of viral genomes were defective, suggesting a possible recombination event. The new method allows to easily distinguish different sperm infections and to observe the possible effects on semen. The data support the proposed role of HPV in decreased fertility and prompt new possible consequences of the infection in semen.

Sea urchin embryos exposed to cadmium as an experimental model for studying the relationship between autophagy and apoptosis.

The sea urchin embryo is a suitable model that offers an excellent opportunity to investigate different defence strategies activated in stress conditions. We previously showed that cadmium accumulates in a dose- and time-dependent manner into embryonic cells, activating different stress and defence mechanisms, including the synthesis of HSPs and the onset of apoptosis and/or autophagy. In this paper we investigated the functional relationship between autophagy and apoptosis, evaluating apoptosis signals in cadmium-exposed Paracentrotus lividus embryos with inhibited autophagy. We found that the inhibition of autophagy produced the concurrent reduction of apoptosis, suggesting that the two phenomena are functionally related. Considering the catabolic role of autophagy, an energetic hypothesis to explain the relationship was evaluated. Using a substrate for ATP production, we found that apoptosis, assessed by TUNEL and cleaved caspase-3 immunocytochemistry, was substantially restored in cadmium-treated embryos where autophagy was inhibited by 3-Methyladenine. On the basis of these results, we propose that, autophagy could play a crucial role in stress response of this organism because autophagy could energetically contribute to apoptotic execution through its catabolic role.

Manganese overload affects p38 MAPK phosphorylation and metalloproteinase activity during sea urchin embryonic development.

In the marine environment, manganese represents a potential emerging contaminant, resulting from an increased production of manganese-containing compounds. In earlier reports we found that the exposure of Paracentrotus lividus sea urchin embryos to manganese produced phenotypes with no skeleton. In addition, manganese interfered with calcium uptake, perturbed extracellular signal-regulated kinase (ERK) signaling, affected the expression of skeletogenic genes, and caused an increase of the hsc70 and hsc60 protein levels. Here, we extended our studies focusing on the temporal activation of the p38 mitogen-activated protein kinase (p38 MAPK) and the proteolytic activity of metalloproteinases (MMPs). We found that manganese affects the stage-dependent dynamics of p38 MAPK activation and inhibits the total gelatin-auto-cleaving activity of MMPs, with the exclusion of the 90-85 kDa and 68-58 kDa MMPs, whose levels remain high all throughout development. Our findings correlate, for the first time to our knowledge, an altered activation pattern of the p38 MAPK with an aberrant MMP proteolytic activity in the sea urchin embryo.

Localization of mitochondrial Hsp56 chaperonin during sea urchin development.

We have previously demonstrated that Paracentrotus lividus nuclear genome encodes for the heat shock inducible chaperonin homolog Hsp 56 (1) and that the mature protein is localized in the mitochondrial matrix (2). In this paper we report that constitutive Hsp56 is maternally inherited, in fact it is present in the in unfertilized eggs, and that it has a perinuclear specific localization during cleavage. In the later stages both the constitutive and the heat shock inducible chaperonin has a specific territorial distribution. Moreover following heat shock, the Hsp56 appears in the cytoplasm and in the postmitochondrial supernatant beside the mitochondrial fraction.

EGTA treatment causes the synthesis of heat shock proteins in sea urchin embryos.

Paracentrotus lividus embryos, at post-blastular stage, when subjected to a rise in temperature from physiologic (20 degrees C) to 31 degrees C, synthesize a large group of heat shock proteins (hsps), and show a severe inhibition of bulk protein synthesis. We show, by mono- and two-dimensional electrophoresis, that also EGTA (ethylene glycol-bis[beta-aminoethyl ether] tetraacetic acid) treatment induces in sea urchin embryos both marked inhibition of bulk protein synthesis and the synthesis of the entire set of hsps. Furthermore, EGTA-treated sea urchin embryos are able to survive at a temperature otherwise lethal (35 degrees C) becoming thermotolerant. Because incubation with a different calcium-chelator, EDTA (ethylenediaminetetraacetic acid), or in calcium-free medium did not induce hsps synthesis we conclude that the stress response caused by EGTA is not related to its calcium chelator function.

Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin.

Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate-dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution.

Studies on heat shock proteins in sea urchin development.

Work on stress proteins in sea urchin embryos carried out over the last 20 years is reviewed and the following major results are described. Entire sea urchin embryos, if subjected to a rise in temperature at any postblastular stage undergo a wave of heat shock protein (hsp) synthesis and survive. If subjected to the same rise between fertilization and blastula formation, they are not yet able to synthesize hsp and die. Four clones coding for the major hsp, hsp70, have been isolated and sequenced; evidence for the existence of a heat shock factor has been provided, and a mechanism for the developmental regulation of hsp synthesis discussed. Intraembryonic and intracellular hsp location has been described; and a mechanism for achievement of thermotolerance proposed. A chaperonine role for a constitutive mitochondrial hsp56 has been suggested, as well as a role for the constitutive hsp70 in cell division. Heat shock, if preceded by 12-O-tetradecanoylphorbol-12-acetate (TPA) treatment causes apoptosis.

Deciliation: A stressful event for Paracentrotus lividus embryos.

In this report, by using mono- and two-dimensional electrophoretic analysis, we demonstrate that deciliation on sea urchin embryos induces a stress response. Deciliation indeed causes not only the activation of ciliary subroutine, but also a transient decrease of bulk protein synthesis. This decrease is in agreement with our previous results on heat shock response in sea urchin, although deciliation does not induce the expression of the same main hsp set. We were able to characterize one main deciliation-stress protein of 40 kDa whose expression is transiently induced by deciliation and whose localisation is likely to be nuclear.

Apoptosis in sea urchin embryos.

It is demonstrated by DNA electrophoresis analysis, morphological observations and TdT in situ reaction, that Paracentrotus embryos if treated with TPA plus heat undergo an apoptotic reaction. Indication is also obtained that non treated embryos undergo spontaneous apoptosis at the early pluteus stage, especially in the districts of arms and intestine. The possible meaning of this latter observation is discussed.

Sea urchin mitochondrial matrix contains a 56-kDa chaperonine-like protein.

Paracentrotus lividus mitochondrial matrix contains a constitutive hsp of 56-KDa which cross reacts with a serum anti-hsp-60 chaperonine from yeast mitochondria. The localization of hsps preexisting or newly synthesized in different subcellular fractions of gastrula embryos is also analyzed by two-dimensional electrophoresis.

Achievement of thermotolerance through hsps phosphorylation in sea urchin embryos.

TPA treatment of sea urchin embryos is able to induce thermotolerance. Evidence is provided that TPA treatment induces phosphorylation of a constitutive stress protein of 38 KDa.

Two-dimensional electrophoretic analysis of stress proteins in Paracentrotus lividus.

The synthesis of stress proteins in Paracentrotus lividus embryos has been analysed by two-dimensional electrophoresis following either heat shock or ZnSO4 administration. As already shown for the 70-KDa hsp (heat shock proteins) a non responsive period, that is before hatching, exists followed by a responsive period in which up to 16 hsps are synthesized after heat shock, 7 of which are also synthesized after ZnSO4 treatment. Some of them pre-existed the stress, but their synthesis increases following the stress. Only few of them accumulate in amounts detectable by silver staining. The synthesis of all these proteins depends upon RNA synthesis.

Sequence of a sea urchin hsp70 gene and its 5' flanking region.

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.

Stress proteins by zinc ions in sea urchin embryos.

In Paracentrotus lividus embryos, treatment with zinc ions induces the synthesis of the two major stress proteins with the same molecular weight as those induced by heat shock. The developmental stages responsive to zinc ion treatment are the same as those responsive to heat shock. However, zinc treatment induces a longer lasting synthesis of the stress proteins, and, unlike heat shock, does not induce thermotolerance and does not inhibit synthesis of the bulk proteins.

Isolation and characterization of a sea urchin hsp 70 gene segment.

Three clones containing Paracentrotus lividus sea urchin DNA sequences which cross-hybridize to Drosophila heat shock protein (hsp) 70 gene were isolated. The sequence arrangements in the three cloned DNA inserts were compared by restriction and cross-hybridization analysis. The results showed that they contain four different genes related to one Drosophila hsp 70 gene. One of these genes was subcloned, and two of the isolated fragments were shown to hybridize to genomic DNA and to RNA from heat-treated sea urchin embryo.

Acquisition of thermotolerance in sea urchin embryos correlates with the synthesis and age of the heat shock proteins.

Preheating at 31 degrees C induces thermotolerance in Paracentrotus lividus embryos, which therefore become able to withstand 1-h treatment at the otherwise lethal temperature of 35 degrees C, and to develop normally. The acquisition of thermotolerance is positively correlated with the amount of heat shock proteins produced during the 31 degrees C treatment. Evidence is provided that the heat shock proteins, although present in the embryo for long periods after synthesis, lose their effect on thermotolerance within 3 h of the cessation of synthesis.

Response to heat shock of different sea urchin species.

It is demonstrated that sea urchin embryos of the species Sphaerechinus granularis are able to respond to heat shock by producing heat shock proteins at the same stage as embryos of Paracentrotus lividus, i.e. after hatching. Arbacia lixula embryos are able to synthesize heat shock proteins already at the stage of 64-128 blastomeres. Embryonic survival is observed if the embryos are heated at the stages at which they can synthesize the heat shock proteins. The inhibition of the bulk protein synthesis after heating at 31 degrees C is never less than 50%.

Territorial localization of heat shock mRNA production in sea urchin gastrulae.

In situ hybridization experiments with a labeled DNA probe indicate that the ability to respond to heat shock with the production of the mRNA for the 70 kd heat shock protein is segregated into the ectodermal cells already at the gastrula stage or earlier during the embryonic development of Paracentrotus lividus.

Synthesis of heat shock proteins in dissociated sea urchin embryonic cells.

Embryos dissociated into cells retain the stage-specific response to heat shock. The dissociated cells, irrespectively of whether they are reaggregated or not, undergo the developmental changes in their ability to respond to heat shock, at the same time as the entire embryos. The conclusion of the present experiments is that sea urchin embryonic cells become responsive to heat shock and produce heat shock proteins even in the absence of cell interactions.