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M Mattioli

Publications and source records attributed to M Mattioli.

At least 37 records · Page 2Linked to original sources
PubMed2000

Signal transduction mechanism for LH in the cumulus-oocyte complex.

The paper reviews recently described signalling mechanisms by which cumulus cells exposed to peak levels of gonadotropins, activate oocyte maturation. Cumulus cells react to LH with a prompt Ca raise which diffuses through gap junctions in a few minutes also into the oocyte where a local amplification system spreads the signal all over the cell. Few h later, still as a consequence of LH stimulation, cumulus cells undergo aprogressive depolarisation of their plasma membrane potential. Due to the electric coupling with these cells the oocyte depolarises too and this open specific voltage gated Ca channels responsible for a second wider and more sustained intracellular Ca rise. As a result of changes throughout maturation with a consequent modification of the size and charge of the molecules that can diffuse from one cell compartment to the other. This cell to cell interaction is further modified with cumulus expansion that leads to a progressive uncoupling of outer cumulus cells while the inner cell layer, corona radiata, remains in oocyte maturation by addressing to the oocyte nutrients and instructions in a well-orchestrated sequence. The identification of these mechanisms are a fundamental prerequisite for the development of in vitro systems suitable to produce oocytes matured in vitro with normal developmental competence.

Animals↗
PubMed2000

Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells.

The human monocytic cell line THP-1 differentiates along the macrophage line after phorbol-12-myristate-13-acetate (PMA) supplementation and can be stimulated to secrete tumour necrosis factor alpha (TNF-alpha) by interferon gamma (IFN-gamma) addition. We found that, in the early stage of differentiation (1-48 h), PMA induction elicited an upregulation of intracellular H ferritin and H ferritin binding sites and a downregulation of transferrin receptor. In addition, we found that iron administration to PMA-differentiating cells induced the expression of TNF-alpha mRNA and TNF-alpha secretion to levels even higher than those induced by IFN-gamma alone. The iron chelator desferrioxamine showed the opposite effect and reduced TNF-alpha release. In contrast, preincubation of the cells with iron before PMA induction resulted in a decrease of the TNF-alpha secretion induced by IFN-gamma, whereas the opposite was true after preincubation with desferrioxamine. The data support a co-ordinate interaction between iron and TNF-alpha in monocyte macrophages, with an iron-mediated upregulation of TNF-alpha in the early phase of differentiation and an iron-mediated inhibition at later stages. This complex relationship has to be considered in evaluating the effects of iron on inflammation.

Binding Sites↗
PubMed2000

Vascular endothelial growth factor production in growing pig antral follicles.

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.

Animals↗
PubMed2000

Temperature and impurity transport studies of heated tokamak plasmas by means of a collisional-radiative model of x-ray emission from Mo30+ to Mo39+

This work presents and interprets, by means of detailed atomic calculations, observations of L-shell (n = 3-->n = 2) transitions in highly ionized molybdenum, the main intrinsic heavy impurity in the Frascati tokamak upgrade plasmas. These hot plasmas were obtained by additional electron cyclotron resonance heating (ECRH), at the frequency of 140 Ghz, during the current ramp-up phase of the discharge. Injecting 400 kW on axis and 800 kW slightly off axis, the peak central electron temperature reached 8.0 and 7.0 keV, respectively, for a time much longer than the ionization equilibrium time of the molybdenum ions. X-ray emissions from rarely observed high charge states, Mo30+ to Mo39+, have been studied with moderate spectral resolution (lambda/delta lambda approximately 150) and a time resolution of 5 ms. A sophisticated collisional-radiative model for the study of molybdenum ions in plasmas with electron temperature in the range 4-20 keV is presented. The sensitivity of the x-ray emission to the temperature and to impurity transport processes is discussed. This model has been then used to investigate two different plasma scenarios. In the first regime the ECRH heating occurs on axis during the current ramp up phase, when the magnetic shear is evolving from negative to zero up to the half radius. The spectrum is well reproduced with the molybdenum ions in coronal equilibrium and with a central impurity peaking. In the second regime, at the beginning of the current flat top when magnetic shear is monotonic and sawtoothing activity is appearing, the lowest charge states (Mo33+ to Mo30+), populated off axis, are affected by anomalous transport and the total molybdenum profile is found to be flat up to the half radius. We conclude with the presentation of "synthetic spectra" computed for even higher temperature plasmas that are expected in future experiments with higher ECRH power input.

Journal Article↗
PubMed2000

Can large cell change and high proliferative activity predict hepatocellular carcinoma in patients with hereditary hemochromatosis?

OBJECTIVE: Patients with hereditary hemochromatosis are at high risk of developing hepatocellular carcinoma. This study was undertaken to define whether large cell change and nucleolar organizer regions proliferative index (marker of high proliferative activity) predict hepatocellular carcinoma development in hereditary hemochromatosis. METHODS: Histological staining for large cell change and high proliferative activity were done on baseline liver biopsies of 74 patients with hereditary hemochromatosis (52 with and 22 without cirrhosis), prospectively followed-up for 83 +/- 53 months (range, 1-230 months). RESULTS: Large cell change and high proliferative activity were found only in cirrhotic patients; 16 of 52 patients (31%) had either the large cell change or high proliferative activity. Large cell change was more frequent in patients with hepatitis B surface antigen than in those positive for hepatitis C virus (57% vs 14%, p = 0.04). Hepatocellular carcinoma developed in 7 of 16 (44%) and in 6 of 36 patients (16%) of the patients positive or negative for these morphological variables. The probability of developing hepatocellular carcinoma at 7 yr of follow-up was significantly higher in patients with large cell change or high proliferative activity than in those without. The length of follow-up from baseline histology to hepatocellular carcinoma occurrence was shorter in patients with large cell change or high proliferative activity than in those without these changes (46 +/- 36 and 109 +/- 34 months, p = 0.01). A multivariate analysis indicated that in patients with cirrhosis, large cell change or high proliferative activity (considered as a single variable), and age >55 yr were the only independent variables significantly associated with the risk of developing hepatocellular carcinoma, with a risk ratio of 4.8 (confidence interval 1.2-18.2) and 4.0 (confidence interval 1.1-15.6), respectively. CONCLUSIONS: In hereditary hemochromatosis, the presence of large cell change or high proliferative activity in patients older than 55 yr with cirrhosis should be considered a strong predictor of hepatocellular carcinoma development, especially if hepatitis B virus infection coexists.

Adult↗
PubMed2000

Iron and liver diseases.

A mild to moderate iron excess is found in patients with liver diseases apparently unrelated to genetic hemochromatosis. Iron appears to affect the natural history of hepatitis C virus-related chronic liver diseases, alcoholic liver disease and nonalcoholic steatohepatitis by leading to a more severe fibrosis and thus aiding the evolution to cirrhosis. A higher frequency of mutations of the HFE gene, the gene responsible for hereditary hemochromatosis, is found in patients with liver diseases and increased liver iron than in normal patients. Patients with excess iron are potentially at a higher risk of developing hepatocellular carcinoma. Iron depletion therapy could interfere with fibrosis development and possibly reduce the risk of liver cancer occurrence.

HLA Antigens↗
PubMed2000

Immunohistochemistry of HFE in the duodenum of C282Y homozygotes with antisera for recombinant HFE protein.

BACKGROUND AND OBJECTIVE: HFE is a class-I MHC related protein which carries the C282Y mutation in most patients with hereditary hemochromatosis, an iron overload disease. HFE protein is expected to have a relevant role in the regulation of duodenal iron absorption, and HFE protein was immunohistochemically identified in the crypt cells. The aim of the work was to analyze whether the C282Y mutation affects HFE accumulation in the duodenum. DESIGN AND METHODS: We developed antisera for the extracellular portion of recombinant human HFE protein expressed in E. coli. The antisera were specific for HFE protein and the C282Y mutant in immunoblotting, immunoprecipitation and immunocytochemistry experiments of transfected cells, and they did not cross react with HLA antigens in various analyses. The antisera gave positive results in the staining of paraffin-fixed sections of duodenal slices of subjects with hemochromatosis. RESULTS: The antisera stained evident supranuclear granules in all enterocytes of 7 C282Y homozygous subjects, and a dark area in the same region in 3 other C282Y homozygotes. Granular bodies were absent from the duodenal sections of 8 C282Y negative subjects, from 2 C282Y heterozygotes and 3 C282Y homozygotes, with or without hemochromatosis. INTERPRETATION AND CONCLUSIONS: The detection of HFE-protein in granular bodies in the enterocytes of the large majority (77%) of C282Y homozygotes and not in other subjects suggests that the mutation facilitates protein accumulation in the duodenum.

Amino Acid Substitution↗
PubMed1999

Carbohydrate-deficient transferrin, a sensitive marker of chronic alcohol abuse, is highly influenced by body iron.

Carbohydrate-deficient transferrin (CDT), a microheterogeneous form of serum transferrin (Tf), has been proposed as the most reliable marker of chronic alcohol consumption, although unexplained false-positive and -negative results have been reported. We investigated whether body iron influenced CDT serum levels by studying alcohol abusers with or without iron overload and nonabusers with iron deficiency or iron overload caused by genetic hemochromatosis (GH). In alcohol abusers, CDT was significantly lower in the presence of iron overload than in the absence (24.6 +/- 16.5 U/L vs. 33.3 +/- 11.7 U/L; P <.01), with false-negative results almost exclusively in patients with iron overload. Similarly, in nonabusers with GH, CDT was lower than in normal controls (9.6 +/- 2. 2 U/L vs. 15.7 +/- 3.3 U/L; P <.0001), whereas, patients with iron deficiency anemia had significantly higher levels than controls (28. 1 +/- 5.8 U/L vs. 15.7 +/- 3.3 U/L; P <.0001). In nonabusers, iron supplementation therapy significantly decreased CDT levels in patients with iron deficiency anemia (33.7 +/- 6.6 U/L vs. 21.7 +/- 5.2 U/L; P =.0007), while iron-depletion treatment significantly increased CDT levels in patients with GH (9.7 +/- 2.0 U/L vs. 14.7 +/- 4.0 U/L; P =.001). Alcohol abusers had a significant relationship between liver iron concentration (LIC) and the reciprocal of CDT (r =.65; P <.0001), while in nonabusers, there was a significant correlation between Tf and CDT (r =.72; P <.0001). In conclusion, CDT serum levels are markedly affected by the patient's iron status, with iron overload reducing its sensitivity in alcohol abusers and iron deficiency its specificity in nonabusers. CDT can be considered a reliable marker of alcohol abuse only when iron stores are normal.

Adult↗
PubMed1999

Nerve growth factor production in sheep antral follicles.

Despite their continuous turnover, sheep antral follicles are always regularly innervated. The local production of neurotrophins is probably involved in the control of ovarian innervation. In this context the present investigation was designed to evaluate the ability of sheep antral follicles to produce neurotrophic factors. In the first part of the paper neurotrophic activity was measured in follicular fluid of sheep antral follicles of different size. Using an in vitro model the effect of gonadotrophins on neurotrophin production was then evaluated. The levels of neurotrophic activity in conditioned medium or follicular fluid and the kind of neurotrophin produced were determined by using the chicken embryo dorsal root ganglia test combined with an immunoneutralization step. Follicular fluid from medium-large follicles (>4 mm) contains high levels of NGF (240-250 ng/ml), whereas the factor is nearly undetectable in small follicles (<3 mm) and in early atretic follicles. Experiments in vitro based on the culture of follicle shells for 12 hr confirmed that medium-large follicles can produce NGF. The production is strictly dependent on gonadotrophin stimulation. When gonadotrophins were not added or were added separately, no detectable levels of neurotrophic activity accumulated in medium. By contrast, in the presence of both LH and FSH the production of NGF became apparent showing a clear dose-response behavior. In addition, this production increased progressively with increasing follicle size from 4 to >5 mm up to values of about 60 ng/follicle, whereas follicles with a diameter of less than 3 mm were insensitive to gonadotrophins stimulation and did not produce significant amount of NGF. The data presented demonstrate that sheep follicles produce relevant amounts of NGF as long as the correct hormonal milieu is provided. Under these conditions the production of the NGF increases with increasing follicle size. This may be responsible for the rapid innervation of the wall of growing follicles and/or take part in other non-neural processes that are generally attributed to gonadotrophin stimulation.

Animals↗
PubMed1999

In vitro development of sheep preantral follicles.

Preantral ovarian follicles isolated from prepubertal sheep ovaries were individually cultured for 6 days in the presence of increasing doses of FSH (ranging from 0.01 to 1 microg/ml) and under two different oxygen concentrations, 20% and 5% O2. Follicle development was evaluated on the basis of antral cavity formation as well as the presence of healthy cumulus oocyte complexes. Follicle growth was enhanced by FSH addition to culture medium, while the use of a low oxygen concentration slightly stimulated this process. However, when follicles were cultured in the presence of high doses of FSH (1 microgram/ml) and under low oxygen concentration, a high proportion of them showed the presence of an antral cavity and of a healthy cumulus-oocyte complex. In addition, under this specific culture condition sheep preantral follicles released higher levels of estradiol as compared to those secreted at lower FSH concentrations or under 20% O2. When the meiotic competence of oocytes derived from follicles cultured at 1 microgram/ml FSH was assessed, no significant difference was recorded between the two oxygen groups. These results show that the culture conditions here identified are beneficial to in vitro growth and differentiation of sheep preantral follicles.

Animals↗
PubMed1999

Experimental and simulated neon spectra in the 10-nm wavelength region from tokamak and reversed field pinch plasmas.

Experimental neon spectra (in the 10-nm region), from the tokamak Tore Supra and the reversed field pinch experiment RFX, have been simulated. The spectra include lines from three neon ionization states, namely Ne(7+), Ne(6+), and Ne(5+) ions. Collisional radiative models have been built for these three Ne ions, considering electron collisional excitation and radiative decay as populating processes of the excited states. These models give photon emission coefficients for the emitted lines at electron density and temperature values corresponding to the experimental situations. Impurity modelling is performed using a one-dimensional impurity transport code, calculating the steady-state radial distribution of the Ne ions. The Ne line brightnesses are evaluated in a post-process subroutine and simulated spectra are obtained. The parts of the spectra corresponding to a single ionization state do not depend on the experimental conditions and show good agreement with the simulated single ionization state spectra. On the other hand, the superposition of the three spectra depends on the experimental conditions, as a consequence of the fact that the ion charge distribution depends not only on the radial profiles of the electron density and temperature, but also of the impurity transport coefficients. Simulations of the Ne spectra (including transport) give confidence in the atomic physics calculations; moreover, they allow the determination of the transport coefficients in the plasma region emitting the considered ionization states.

Journal Article↗
PubMed1998

Calcium elevation in sheep cumulus-oocyte complexes after luteinising hormone stimulation.

We investigated Ca2+ levels in intact cumulus-oocyte complexes (COCs) on exposure to peak levels of luteinising hormone (LH). Specific preparations were used where cumulus corona cells were loaded with a membrane-permeant Ca(2+)-sensitive dye (FLUO-3AM), whereas the oocyte was injected directly with the nonpermeant form of the dye (FLUO-3). After exposure to LH, cumulus and corona radiata cells showed distinct rises in intracellular Ca2+ in 50-200 sec. The pattern of Ca2+ response varied in the different cells both for the duration of the transients and for their persistence. Interestingly, Ca2+ elevations were recorded in all the layers of the cumulus mass, including the innermost layer of corona cells, demonstrating the wide diffusion of LH receptors. Following the Ca2+ raise in somatic cells, an intracellular Ca2+ elevation also was recorded within the oocyte with a delay of 100-300 sec. The elevation started at the cortex of the oocyte and then spread all over the ooplasm. The addition of verapamil or manganese chloride did not prevent LH-induced Ca2+ elevation in the COC, whereas mechanical uncoupling of cumulus cells from the oocyte prevented any Ca2+ response within the oocyte. The results indicate that cumulus corona cells are capable of transducing LH message by rising intracellular Ca2+ and show that this signal is rapidly transferred into the oocyte through gap junctions. This may result from the direct diffusion of Ca2+ or its putative releaser IP3 from cumulus cells to the oocyte.

Animals↗
PubMed1998

Expanded cumuli induce acrosome reaction in boar sperm.

The authors investigated acrosomal changes occurring in boar sperm that interact with the expanded cumulus matrix surrounding ovulated pig oocytes. Samples of washed boar sperm obtained from six donors were incubated for 4 hr under capacitating conditions and exposed either to solubilized zonae pellucidae (ZP) or solubilized expanded pig cumuli (SEC) obtained from IVM oocytes. Alternatively, hyaluronic acid, laminin, or fibronectin, components of the extracellular matrix (ECM) were added to capacitated sperm. Acrosomal integrity was evaluated 1 hr later by using FITC-PSA staining. Solubilized cumuli induced acrosome reaction (AR) in a dose-dependent manner with a saturating effect exerted at 2.5 SEC/50 microl. Both 500 nM fibronectin and 500 nM laminin stimulated acrosomal exocytosis, the latter being more effective and inducing saturating levels of AR. By contrast, hyaluronic acid did not affect acrosomal status. Preincubation with anti-laminin antibodies completely prevented the inducing activity of SEC without affecting the activity of solubilized ZP. Consistent with these data, the integrin VLA-6, a receptor with high affinity for laminin, was detected by immunoblotting on the plasma membrane of capacitated boar spermatozoa. In addition, its immunoneutralization, obtained with the preincubation of capacitated sperm with the antibody raised against the alpha chain of VLA-6 integrin, prevented AR upon exposure to laminin or SEC (10.7+/-3.2 and 10.2+/-1.0% respectively), while the samples retained their responsiveness to ZP (29.6+/-1.2%). The results demonstrate that the interaction between laminin, entrapped in the expanded cumuli, and specific integrins present on the sperm membrane can initiate AR, thus taking part in the process of sperm-egg recognition.

Acrosome Reaction↗
PubMed1998

High prevalence of the His63Asp HFE mutation in Italian patients with porphyria cutanea tarda.

Sporadic porphyria cutanea tarda (PCT) is caused by a reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Mild to moderate iron overload is common in PCT, as iron is one of the factors which trigger the clinical manifestations of the disease through the inactivation of URO-D. A role for genetic hemochromatosis in the development of iron overload in sporadic PCT has been hypothesized in the past. The aim of this work was to investigate whether mutations of HFE, which is a candidate gene for hemochromatosis, play the role of genetic susceptibility factors for PCT in Italian patients, who have a high prevalence of acquired triggering factors, such as hepatitis C virus (HCV) chronic infection and alcohol. We determined HFE genotypes of 68 male patients with PCT. Our data do not confirm an association of PCT with the Cys282Tyr HFE mutation, strongly associated with hemochromatosis in Northern European countries. A second mutation of HFE, His63Asp, however, had a significantly increased frequency as it was present in half of the patients. Surprisingly, the presence of the His63Asp mutation was not related to the iron status of patients, suggesting that a subtle abnormality of iron metabolism induced by this mutation could escape detection by the standard parameters of iron status. In PCT patients with liver disease, the presence of the mutation could contribute to the inactivation of URO-D, either directly or through a synergistic action with other factors that cause liver damage.

Adult↗
PubMed1997

Liver iron influences the response to interferon alpha therapy in chronic hepatitis C.

OBJECTIVE: To define whether there is any relation between the iron status of patients with hepatitis C virus (HCV) chronic liver disease and their response to interferon therapy. DESIGN: To evaluate the long-term response to 1 year of interferon therapy with addition of phlebotomies after 3 months of treatment if at that time alanine aminotransferase (ALT) had not normalized in a group of patients with HCV-positive chronic liver disease whose iron status had been characterized. SETTING: A northern Italian hospital. PARTICIPANTS: Fifty-eight anti-HCV-positive patients (four HCV-RNA negative) with biopsy proven chronic hepatitis and no evidence of iron overload as indicated by normal transferrin saturation at the time of enrollment in the study. INTERVENTION: Three times a week intramuscular injection of alpha interferon 3 MU for 1 year with addition of phlebotomies (350 ml/week) till iron depletion if after 3 months of interferon therapy ALT had not normalized. RESULTS: A long-term response was observed in 19 of the 52 patients who completed the treatment, four HCV-RNA negative and 15 positive. The four RNA-negative and seven of the 15 RNA-positive long-term responders had been treated with interferon alone, and the other eight also with phlebotomies. At univariate analysis only HCV genotype, gamma-glutamyltranspeptidase and liver iron concentration were significantly associated with response whereas sinusoidal iron deposition was of borderline significance. No association was found with sex, age, duration of disease, histology, Knodell score, transferrin saturation %, serum ferritin, hepatocytic iron score, and portal iron score. HCV-RNA serum levels, measured in 29 patients, did not correlate with response. At multivariate analysis liver iron concentration was still significant and one unit reduction of liver iron concentration (natural logarithm transformed) was associated with 2.95 odds ratio of response. CONCLUSION: These results indicate that iron in the liver is more closely related to response to interferon than the other variables considered, including HCV characteristics.

Adult↗
PubMed1997

High prevalence of hepatitis C virus type 1b in Italian patients with Porphyria cutanea tarda.

BACKGROUND: A strong association between sporadic porphyria cutanea tarda and chronic hepatitis C virus infection was recently described in Italy, France and Spain. AIMS: To explore whether hepatitis C virus genotype plays a role in porphyria cutanea tarda complicating chronic hepatitis C. PATIENTS: Forty-seven hepatitis C virus-positive porphyria cutanea tarda patients and a control group of 45 patients of similar age with hepatitis C virus-associated chronic liver disease. METHODS: Comparison of frequency of hepatitis C virus genotypes in the two groups and in relation to the age of patients, hepatic histopathology and with the presence of other factors potentially able to trigger porphyria cutanea tarda. RESULTS: A single genotype, hepatitis C virus 1b, was found to be present in nearly 90% of porphyria cutanea tarda-associated chronic liver disease, significantly exceeding the frequency of the same genotype in the control group (p = 0.0001). The presence of hepatitis C virus 1b was not related to the age of patients or disease severity as evaluated by hepatic histopathology. CONCLUSIONS: Hepatitis C virus-associated chronic hepatitis found in the majority of Italian patients with porphyria cutanea tarda is usually sustained by hepatitis C virus genotype 1b. This viral strain might have a direct pathogenic role in inducing porphyria cutanea tarda or could increase the susceptibility of patients of other triggering factors such as iron overload or alcohol abuse.

Adult↗
PubMed1996

Identification of capacitation in boar spermatozoa by chlortetracycline staining.

The functional status of boar spermatozoa undergoing capacitation in vitro was investigated. Two fluorescent stains were used: chlortetracycline (CTC) and a FITC-conjugated lectin (FITC-PSA). The first has been used for the direct identification of the capacitated boar spermatozoa, while the second, based on the identification of capacitated spermatozoa by their ability to undergo zona-induced acrosome reaction (AR), was used to confirm and validate the CTC assay in this species. Spermatozoa obtained from 5 different boars was washed and incubated under capacitating conditions. Aliquots of spermatozoa were collected at 0, 90 and 180 min of incubation and then stained with CTC or FITC-PSA. After CTC staining, 3 different fluorescent patterns were observed: Pattern A with the fluorescence uniformly distributed on the sperm head, Pattern B with the fluorescence concentrated in the post-acrosomial region, and Pattern C with the fluorescence concentrated in the acrosomial region. The percentage of spermatozoa displaying fluorescent Pattern A decreased throughout the incubation while that of spermatozoa with Pattern C showed a concomitant progressive increase. Pattern B fluorescence remained unchanged throughout the maturation period. Exposure to zonae pellucidae (ZP) brought back the levels of Pattern C fluorescence to basal values. Since only the capacitated spermatozoa are believed to react to ZP, this observation together with the rising incidence of Pattern C throughout maturation suggests that fluorescence in the acrosomial region identifies capacitated spermatozoa. The analysis of acrosome integrity carried out with FITC-PSA showed that the proportion of zona-induced AR was nearly the same as that of spermatozoa displaying Pattern C, thus confirming that CTC staining is suitable for the detection of boar sperm capacitation. In the second part of this study, CTC was used to investigate the effects of sperm origin and storage on the capacitation process. Our finding demonstrates that capacitation kinetics show wide variations in sperm samples derived from different boars; moreover, capacitation is also affected by sperm storage. While fresh semen showed a progressive increase in capacitated spermatozoa, ranging from low levels at the beginning of the culture to 46% at the end of incubation, the refrigerated semen had a relatively high percentage of capacitated spermatozoa at the beginning of culture, but this proportion increased only slightly during the following 90 to 180 min of treatment. These data indicate that CTC can be used to identify capacitated boar spermatozoa, and, because of its rapid and easy execution, it can be used routinely to identify the optimal capacitation time for different sperm samples.

Journal Article↗