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E Moretti

Publications and source records attributed to E Moretti.

At least 37 records · Page 2Linked to original sources
PubMed2001

FISH assessment of aneuploidy frequencies in mature and immature human spermatozoa classified by the absence or presence of cytoplasmic retention.

Previously, a relationship has been found between diminished cellular maturity of human spermatozoa and low-level expression of the testis-specific chaperone protein, HspA2. Because HspA2 is a component of the synaptonemal complex in rodents, and assuming that this is also the case in men, it was postulated that the frequency of chromosomal aneuploidies would be higher in immature versus mature spermatozoa. This question was examined in spermatozoa from semen and from 80% Percoll pellets (enriched for mature spermatozoa) of the same ejaculate in 10 oligozoospermic men. Immature spermatozoa with retained cytoplasm, which signifies spermiogenetic arrest, were identified by immunocytochemistry. Using fluorescence in-situ hybridization (FISH), approximately 7000 sperm nuclei were evaluated in each of the 20 fractions (142 086 spermatozoa in all) using centromeric probes for the X, Y and 17 chromosomes. The proportions of immature spermatozoa were 45.4 +/- 3.4 versus 26.6 +/- 2.2% in the two semen versus the Percoll groups (medians: 48.2 versus 25%, P < 0.001, n = 300 spermatozoa per fraction, total 6000 spermatozoa). There was also a concomitant decline in total disomy, total diploidy and total aneuploidy frequencies in the 80% Percoll versus semen fractions (0.17 versus 0.54%, 0.14 versus 0.26% and 0.31 versus 0.81% respectively, P < 0.001 in all comparisons). The mean decline of aneuploidies was 2.7-fold. With regard to the hypothesis that aneuploidies are related to sperm immaturity, there was a close correlation between the incidence of immature spermatozoa and disomies (r = 0.7, P < 0.001) but no correlation with diploidies (r = 0.03), indicating that disomies originate primarily in immature spermatozoa. It is suggested that the common factor underlying sperm immaturity and aneuploidies is the diminished expression of HspA2. In addition, the lack of this chaperone may also cause diminished cellular transport of proteins, such as DNA-repair enzymes or of the retention of cytoplasm that is extruded from normally maturing spermatozoa during spermiogenesis.

Aneuploidy↗
PubMed2001

Genetic sperm defects and consanguinity.

BACKGROUND: The existence of a genetic component to human infertility has been suggested, although neither the specific abnormalities involved, nor their genetic mechanism of transmission, are currently defined. We have examined, by transmission electron microscopy (TEM), ejaculate from 1600 males with fertility problems. Among the subjects studied, we focused on a group of patients whose family histories revealed different degrees of consanguinity, in order to evaluate the relationship between consanguinity and particular sperm alterations. METHODS AND RESULTS: A total of 64 consanguineous individuals were identified. In this group, excluding two azoospermic patients, 17 patients (27%) were found to have well recognized genetic ultrastructural defects affecting their entire sperm population: eight subjects had spermatozoa with "stunted tails", four "detached tail" spermatozoa, two "Kartagener's syndrome", two "miniacrosome" and one "round headed" spermatozoa. Since these alterations affect the total sperm population and do not respond to medical treatment, they are suspected of having a genetic origin. The remaining group of 1506 non-consanguineous patients suffered from the same genetic defects in only 15 cases (<1%). CONCLUSIONS: From the data presented, it appears that some very peculiar and rare sperm defects may have a genetic basis since they occur more frequently in consanguineous patients, and are related to different degrees of consanguinity. Since the ejaculate of the remaining patients, both consanguineous and not, showed diverse types of ultrastructural sperm anomalies that did not affect the entire sperm population, they might represent pathologies lacking a genetic basis.

Acrosome↗
PubMed2001

Antibodies to an epitope from the Cha human autoantigen are markers of Chagas' disease.

Chagas' disease is a prevalent disease in South America that is thought to have an autoimmune etiology. We previously identified human Cha as a new autoantigen recognized by chagasic sera. Those sera recognized an epitope spanning amino acids 120 to 129 of Cha, named R3. In the present study we have used the synthetic R3 peptide for the detection of serum immunoglobulin G antibodies from patients at different stages of Chagas' disease, including a therapeutically treated group. The immunoreactivity with R3 by enzyme-linked immunosorbent assay (ELISA) showed 92.4% sensitivity and 100% specificity for Chagas' disease sera. This sensitivity and specificity were higher than for any other autoantigen described to date. No anti-R3 antibodies were detected in sera from Leishmania-infected or idiopathic dilated cardiomyopathy patients or healthy controls from the same areas. Moreover, anti-R3 antibody reactivity detected by ELISA correlated with conventional serological tests as indirect immunofluorescence and ELISA assays with Trypanosoma cruzi extracts and other diagnostic tests as indirect hemagglutination. The levels of anti-R3 antibodies increased with progression and symptomatology of Chagas' disease. More interestingly, a statistically significant fall in anti-R3 antibody titer was observed in patients treated with antiparasitic drugs. Those results suggest that the presence of anti-R3 antibodies is a highly specific marker of Chagas' disease and that R3 ELISA could be helpful in the diagnosis and monitoring of this disease.

Amino Acid Sequence↗
PubMed1999

A quick molecular method for the simultaneous detection in spermatozoa of nuclear, acrosomal and axonemal structure by fluorescent microscopy.

Ejaculated spermatozoa from infertile men presenting to our laboratory for semen analysis were processed with a new molecular method which reveals simultaneously, in the same sperm cell, the status of the acrosome, by testing the hyaluronidase content, the texture of the nucleus, by checking the DNA strands breaks, and the structure of the axoneme, revealing the tubulin content. The presence of hyaluronidase and tubulin is essential for the sperm function, and the analysis of the DNA status reveals the eventual apoptotic process. Using this method in normal spermatozoa, the staining of the acrosomal hyaluronidase reveals, by yellow-green fluorescence, the shape of the acrosomal complex and its texture. At the same time, in the same sperm cell, the staining of the axonemal tubulin demonstrates, by a red labeling, the presence of the protein and therefore the consistence of the axonemal structure. Simultaneously, at the head level, the absence of red labeling from nuclear DNA indicates that the apoptotic process is not present. This protocol allows quantification of the frequency of the presence of normal or abnormal spermatozoa, by an easy scoring and calculation of the apoptotic sperm or of the sperm with generic defects at acrosomal or flagellar level. The percentage of normal spermatozoa evaluated by the triple staining method has been compared with the results of the PAP staining and of the ultrastructural analysis, statistically elaborated. Triple staining results more severe than the PAP method, but TEM analysis is the finest technique to detect sperm abnormality because it considers the entire panel of sperm defects.

Acrosome↗
PubMed1998

New insights into the interaction between the gp120 and the HIV receptor in human sperm (human.sperm/gp120/galactoglycerolipid/antigalactosylceramide/seminolip id/spermatogonia).

The human immunodeficiency virus (HIV) can infect some cell types which lack CD4. Galactosylceramide, a glycolipid present in the nervous system and colonic epithelial cells, has been implicated in the virus entry in these cells. Our data demonstrate that the HIV surface glycoprotein gp120 binds to the galactosyl-alkyl-acylglycerol (GalAAG), a glycolipid structurally related to galactosylceramide present on the surface membrane of the spermatozoa. In this paper, we review our previous data and further confirm the specificity of the interaction between this galactoglycerolipid and the gp120. Consistent with the structural similarity to galactosylceramide, the sperm GalAAG is capable of specifically binding the gp120. The specificity of the binding of antibodies antigalactosylceramide and the gp120 to the sperm extract and to the purified GalAAG fraction prepared from the same extract has been demonstrated utilizing an ELISA assay which favors sensitivity and specificity. Immunofluorescence and immunoelectron microscopy data show a different localization for the GalAAG and its sulfated form the seminolipid (SGalAAG). The GalAAG is preferentially localized in the equatorial segment and the middle piece of the sperm tail, while the seminolipid is widely distributed on the membrane of the spermatozoa. These data indicate that human sperm express on their surface membrane a glycolipid similar in structure to galactosylceramide, the receptor for HIV identified in the CD4 cells, that could function as a HIV receptor and possibly be implicated in its transmission.

Adult↗
PubMed1998

Localization of human follicle-stimulating hormone in the testis.

Localization of the follicle-stimulating hormone (FSH) molecule and its receptor (FSHR), as well as the role of FSH in Sertoli cell mitosis and maturation, has been demonstrated by several investigators in human and murine testis by detecting the localization of anti-FSH antibodies or [(131)I]-labeled FSH and by detecting FSH receptor (FSHR) mRNA by in situ hybridization, or FSHR by anti-FSHR antibodies. The presence of FSH in germinal cells is controversial or, in humans, excluded. We have investigated the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions, at the submicroscopical level, by using a better antigenicity conservative procedure. Thus, the distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has now been clarified. In germinal cells, our observations demonstrate the presence of FSH and the FSHR mRNA: the first on the plasma membrane and in endocytotic vesicles, and the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia to spermatocytes, including round spermatids. Penetration was by the endocytosis via membrane vesicles in which the FSHR is present, whereas its messenger is largely present in the cytoplasm and is responsible for the binding and subsequent internalization of the FSH molecule. As a control, human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The FSH molecule has been localized by monoclonal antibodies on plasma membranes and vesicles, and the FSHR mRNA was found scattered in the cytoplasm after in situ hybridization. We can now conclude that FSH is present in Sertoli cells and in round germinal cells, both expressing the FSHR. FSH penetrates in a similar way in both kinds of cells via endocytosis, and is therefore subsequently localized in the same membranous organelles.

Animals↗
PubMed1998

Identification, separation and characterisation of two forms of cytosolic 5'-nucleotidase/nucleoside phosphotransferase in calf thymus.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, endowed with phosphotransferase activity, is a widespread enzyme responsible for the regulation of intracellular IMP and GMP concentrations and the phosphorylation of purine nucleoside pro-drugs. The enzyme activity is stimulated by ATP, ADP and 2,3-bisphosphoglycerate (BPG), and is inhibited by phosphate. Calf thymus possesses two active proteins with a different electrophoretic mobility. In this report we show that the two forms can be separated by ADP-agarose affinity chromatography. Whereas form A binds weakly to the column, form B is tightly bound and is released by the addition of ADP into the elution buffer. The two enzyme forms differ in terms of electrophoretic, chromatographic behaviour and regulatory characteristics. Form B, as already described for the enzyme purified from the same source (Pesi et al., 1996, Biochim Biophys Acta 294, 191-194), exhibits three different sites for the three activators with a synergistic effect between ADP and BPG. Form A has a high affinity regulatory site for BPG, while ADP and ATP appear to share the same low affinity site and no synergistic effect is observed.

5'-Nucleotidase↗
PubMed1998

Localization of lamins in mammalian spermatozoa.

Little information is available concerning lamins in the nucleus of germinal cells. In this paper we briefly describe and compare the organization of A- and B-type lamins in several mammalian spermatozoa. Nuclear lamin B is localized primarily in the postacrosomal sheath of human, bull and rabbit spermatozoa; lamin A/C is a major component of the equatorial segment in most of mammalian sperm, with the exception of rodents. In mouse sperm, devoid of equatorial segment, only lamin B appears to be expressed. The same happens in human pathological spermatozoa in which the equatorial segment is altered or absent.

Animals↗
PubMed1997

G protein-coupled receptor kinase GRK4. Molecular analysis of the four isoforms and ultrastructural localization in spermatozoa and germinal cells.

G protein-coupled receptor kinase 4 (GRK4) presents some peculiar characteristics that make it a unique member within the GRK multigene family. For example, this is the only GRK for which four splice variants (GRK4alpha, -beta, -gamma, -delta) have been identified. We developed a simple assay to study kinase activity, and we found that GRK4alpha, but not GRK4beta, -gamma, and -delta, was able to phosphorylate rhodopsin in an agonist-dependent manner. GRK4alpha kinase activity was inhibited by Ca2+/calmodulin (CaM) (IC50 = 80 nM), and a direct interaction between GRK4alpha and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. The other three GRK4 isoforms did not interact with CaM in parallel experiments. The present investigation also aimed to define cellular and ultrastructural localization of GRK4. A substantial expression of GRK4 mRNA was only found in testis and in the spermatogonia cell line GC-1 spg. Specific GRK4 immunoreactivity was only found on sperm membranes, and immunochemical and ultrastructural analyses showed that it is associated to the acrosomal membranes and to the outer mitochondrial membranes. GRK4gamma was the only detectable isoform in human sperm. We concluded that: i) only GRK4alpha can phosphorylate rhodopsin and that this activity is inhibited by CaM; ii) the other three isoforms do not phosphorylate rhodopsin and do not interact with CaM; and iii) the association of GRK4 with highly specialized sperm organelles, which are essential for fertilization, strongly indicates that this kinase is involved in this process.

Animals↗
PubMed1997

The effect of follicle stimulating hormone therapy on human sperm structure (Notulae seminologicae 11).

The effects of follicle stimulating hormone (FSH) treatment on the quality of human spermatozoa were assessed by examining the ultrastructure and the function of infertile human spermatozoa using a previously-defined formula. Using the spermatozoa as an andrological monitor shows that the therapeutic effect of FSH depends on the type of sperm defect. The response to FSH is, in many cases, positive and can be evaluated by examining the state of the ejaculated spermatozoa. From an initial group of 81 patients, 15 were placebo-treated controls, and 19 were non-responders (mainly with microbially infected semen). Out of 47 responders, after therapy nine achieved improved sperm quality which approached the natural fertility threshold. These responders all had spermatozoa affected by immaturity or apoptosis (n = 27). The 20 microbially-infected responders also had immature spermatozoa and never achieved the quality level of natural fertility. Thus, a natural fertility level was only achieved by nine responders out of 27 (three with immature spermatozoa, and six with apoptotic spermatozoa). Using our method of sperm analysis, these patients' spermatozoa were clearly categorized before treatment as either immature or apoptotic. In consequence, the success of the therapy was predictable. The response of individual organelles to therapy was examined. Certain qualities of the acrosome, the chromatin, the mitochondria, and the axoneme appear to be sensitive to FSH. Most of the previous conflicting results reported in the literature may be due to a lack of relevant discrimination between the different defects present in the spermatozoa of the patients, without assessing the likelihood of their response.

Acrosome↗
PubMed1997

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 3. Partial zona dissection (PZD) (Notulae seminologicae 12).

This paper belongs to a series of application of the Baccetti's et al. (1995) formula to the submicroscopical mathematical examination of the human spermatozoa used for assisted reproduction. The present experiment concerns partial zona dissection, a technique requiring a careful evaluation of sperm quality in order to predict the success of the program. Our results demonstrate that the sperm submicroscopic characters introduced in the formula are clearly correlated with the result of PZD. In fact the two numbers concerning the sperm ultrastructural quality (percentage and total number of spermatozoa free from defects in the ejaculate) obtained in successful and unsuccessful PZD groups, showed a large difference (P < 0.01). The most important characteristics seem to be the quality of the acrosomal complex, the condition of the chromatin and the mitochondrial assembly. All these characteristics are expressed with largely different means in successful and unsuccessful ejaculates (from P < 0.05 to P < 0.01). A comparison with the results previously obtained in ICSI (Strehler et al., 1995) and IVF (Piomboni et al., 1996) shows that sperm quality is significantly more implicated in the success of IVF than of PZD or ICSI.

Female↗
PubMed1997

[Chagas disease. Serologic response to crude and semipurified T. cruzi antigens in different clinical groups of patients].

The immune response of people infected by the protozoan parasite Trypanosoma cruzi varies in indeterminate or chronic periods of infection, according to the course of the disease. Apparently, the antibody pattern is different in asymptomatic patients than in patients with cardio-myopathy. The study of those differences for clinical purposes depends on the availability of adequate antigenic preparations. We studied the behavior of crude and partially purified antigens of T. cruzi in acute and chronically infected patients. The level of total antibodies was measured by conventional serology (indirect immunofluorescence, IF, and indirect hemagglutination, HA) and the level of antibodies against different fractions of antigens obtained by analytical chroma-tofocusing, by using enzymatic immunoassay (ELISA). Chronic patients were classified according to Kuschnir et al. in: Group 0 (G0) or Group 1 (G1) according to the absence or presence of electrocardiographic alterations. In acute patients, conventional serology was negative in most cases. However, the ELISA performed with the soluble fraction of the lysate of parasites, showed positive results in 60% of patients. By classical serology, the behavior of chronic patients sera was different in the two groups when assayed by IF; G1 showing more elevated titles than G0. On the other hand, by HA the results were similar in both groups. When the sera were studied by ELISA using the different fractions, the fraction collected at pH 4-4.5, named F IV, detected distinct reactivity in some of, but not in all, the patients of G1. In that subgroup, the highest ELISA indes (optical density of sera vs OD of negative controls) was observed. By associating IF plus FIV-ELISA the results became more apparent: 22% of sera showed titles by IF equal or more than 1:128 and ELISA index equal or more than 3.5, whereas only 2% of G0 showed this characteristic and all the sera with index equal or more than 4 belonged to G1. With lesser antibody titles it was not possible to differentiate between the two groups. It can be concluded that the study of the pattern of antibodies by using different antigens and serological methodologies can offer useful information in the different periods of Chagas disease, by adding the higher sensitivity of methods using crude antigens with the more specific and discriminative results obtained by the use of purified or synthetic antigens.

Adolescent↗
PubMed1996

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 2. In vitro fertilization. (Notulae seminologicae. 7)

This paper belongs to a series of applications of the Baccetti et al. formula (1) to the submicroscopical mathematical examination of human spermatozoa used for assisted reproduction. The present experiment concerns IVF, a technique requiring careful evaluation of sperm quality to predict the success of the program. Our results demonstrate that the sperm submicroscopic characters introduced in the formula are clearly correlated with the result of IVF. In fact the two numbers concerning sperm quality (percentage of spermatozoa free from structural defects and total number in the ejaculate of spermatozoa free from defects) obtained in successful and unsuccessful IVF groups, showed a large difference. The t distribution in both cases reached a significance of 0.005. The synthetic parameters obtained are therefore a good tool in the prediction of sperm power in in vitro insemination techniques. The most important characteristics seem to be the quality of the acrosomal complex, the status of the chromatin, the shape of mitochondria, the axonemal pattern, and the membrane integrity. All these characteristics are expressed with largely different means in successful and unsuccessful ejaculates (t distribution significant at 0.005). All these data confirm that submicroscopic mathematical diagnosis offers a convincing evaluation of sperm structure and function, involving all organelles, including acrosome function and cell motility. It is also demonstrated that sperm quality is a major factor in the success of IVF and that it is clearly revealed by the integrity of the majority of the sperm organelles.

Acrosome↗
PubMed1995

Localisation of two classes of acetylcholine receptor-like molecules in sperms of different animal species.

The distribution of different classes of acetylcholine (ACh) receptor-like molecules in sperms of different invertebrate and vertebrate species is described. ACh receptor molecules belong to one of two classes: muscarinic receptors (mAChRs), associated with signal transduction mechanisms in the inner domain of the cell, and nicotinic receptors (nAChRs), capable of opening Na+ channels when activated by the ligand. Molecules immunologically related to mAChRs and to ACh can be identified by specific antibodies, and revealed by immunofluorescent or immunogold staining; the nicotinic receptor-like molecules are localised as curare-sensitive affinity sites for alpha-bungarotoxin. In all species studied, both classes of receptors were found, with a similar distribution. Muscarinic-like molecules were found mainly in the sperm head regions of most species; such a localisation may be correlated to a function in sperm-egg interaction, for instance in the regulation of the block to polyspermy. Nicotinic-like molecules are present mainly in the tail and in the post-acrosomal region of most animals, thus confirming their function in the regulation of sperm propulsion, but are also present at the acrosomal region of most species. The distribution patterns of the different classes of molecules indicate that both may be involved in sperm-egg interactions, in addition to their known function in the regulation of sperm propulsion.

Acrosome↗
PubMed1995

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. I. Intracytoplasmic sperm injection. (Notulae seminologicae 6).

After a large introduction concerning the influence of sperm quality in the success of in vitro fertilization, the debated problem of the importance of the sperm quality in intracytoplasmic sperm injection is investigated. The spermatozoa are studied by electron microscopy, and the results evaluated by the formula of Baccetti et al. (1995). The quality of spermatozoa has been correlated with the success of ICSI. The first conclusion has been that ejaculates which produced embryos contained higher percentages and overall higher total number of 'healthy' spermatozoa than those which obtained fertilizations arrested at the two pronuclei stage, or showed no fecundation at all. A second conclusion was that the quality of sperm organelles is mainly involved in the oocyte activation, but after the 2 pronuclei stage, segmentation usually proceeds with few influences from sperm quality. The highest involvement of spermatozoa is therefore the initiation of oocyte activation. A third conclusion confirmed the need of ultrastructural evaluation of sperm cell organization, deeply investigating the inner organelles. Moreover, we observed that the head organelles are more involved that the tail ones. Three acrosomal characteristics are concerned: the shape, the dimensions and the content. All of them are significantly better in the oocyte activating than in non activating spermatozoa. Also the nuclear shape and the status of the chromatin (frequently strictly interdependent) are significantly concerned with the oocyte activation and with the embryo segmentation. In the tail, the mitochondrial shape is significantly different in activating and not activating spermatozoa, and also in those producing embryo segmentation. Moreover, the absence of dynein arms seems to be determinant in impeding the oocyte activation.

Fertilization in Vitro↗
PubMed1994

Focal cerebral ischemia and patent cardiac foramen ovale.

By means of transthoracic contrast echocardiography, the prevalence of a patent foramen ovale (PFO) was studied, in a continuous series of 48 patients aged less than 50 years with a recent episode of acute cerebral ischemia. A PFO was found in 11 subjects (23%). In the subgroup of younger patients (aged less than 30 years), the prevalence was much higher than in those aged 30 or more (58% against 11%, p = 0.0022). In the 19 patients with clear evidence of extracardiac causal factors of cerebral ischemia, there was no PFO; of the remaining 29 subjects, a PFO was present in 11 (38%) (p = 0.0015). In conclusion, the possible presence of a PFO must be carefully investigated in subjects with cerebral ischemia aged less than 30, as well as in subjects aged between 30 and 50 in whom there is no acceptable explanation for their cerebral ischemic episode.

Adult↗