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Biomedical subjects

C Allegrucci

Publications and source records attributed to C Allegrucci.

11 recordsLinked to original sources
PubMed2006

Differences between human embryonic stem cell lines.

The promise of human embryonic stem cell (hESC) lines for treating injuries and degenerative diseases, for understanding early human development, for disease modelling and for drug discovery, has brought much excitement to scientific communities as well as to the public. Although all of the lines derived worldwide share the expression of characteristic pluripotency markers, many differences are emerging between lines that may be more associated with the wide range of culture conditions in current use than the inherent genetic variation of the embryos from which embryonic stem cells were derived. Thus, the validity of many comparisons between lines published thus far is difficult to interpret. This article reviews the evidence for differences between lines, focusing on studies of pluripotency marker molecules, transcriptional profiling, genetic stability and epigenetic stability, for which there is most evidence. Recognition and assessment of environmentally induced differences will be important to facilitate the development of culture systems that maximize stability in culture and provide lines with maximal potential for safety and success in the range of possible applications.

Cell Culture Techniques↗
PubMed2003

Interaction of bovine granulosa and theca cells in a novel serum-free co-culture system.

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles 4-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more androstenedione compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and androstenedione (treatment B). Significantly more androstenedione was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.

Androstenedione↗
PubMed2001

Circulating human antisperm antibodies recognize prostasomes.

PROBLEM: The presence of naturally occurring antisperm antibodies (ASA) is a well-known cause of infertility in men and women, but the antigens for these antibodies are usually poorly characterized. Prostasomes, organelles secreted by human prostatic acinar cells and expelled into the seminal plasma at ejaculation, can adhere to sperm cells. Thus, we have examined whether prostasomes could be an antigen for ASA. METHOD OF STUDY: We have studied the reactivity of chicken antiprostasome antibodies with sperm cells in an agglutination test and conversely the reactivity of serum positive for ASA from 20 infertile patients, with spermatozoa using flow cytometry and with purified prostasomes using enzyme-linked immunosorbent assay. RESULTS: The chicken antiprostasome antibody caused agglutination of sperm cells similarly to the agglutination observed with patients' sera. All of these patients' sera contained IgG antibodies against prostasomes. CONCLUSIONS: The high percentage of patients with antiprostasome antibodies in this study shows that prostasomes could be one of the major targets for ASA.

Animals↗
PubMed2001

Stimulation by n6-cyclopentyladenosine of A1 adenosine receptors, coupled to galphai2 protein subunit, has a capacitative effect on human spermatozoa.

The effects of selective A(1) receptor agonist on human spermatozoa were examined to verify physiological responses and to investigate the signal transduction pathway. N6-Cyclopentyladenosine on uncapacitated spermatozoa did not induce spontaneous acrosome reaction after 5 h capacitation, whereas the number of capacitated spermatozoa, assessed by lysophosphatidylcholine-induced acrosome reaction with Pisum sativum agglutinin staining, was significantly increased. N6-Cyclopentyladenosine was also added to capacitated human spermatozoa to find out whether the agonist could induce the acrosome reaction. Results, although statistically significant, could not be considered biologically significant. A1-Mediated capacitation was followed by the increase of tyrosine phosphorylation of a protein subset ranging between M(r) = 200 000 and 30 000. Stimulation of A1 receptor with the selective agonist elicited an agonist-induced inositol phospholipid hydrolysis leading to a transient rise of inositol triphosphate (IP3). This increase was not induced by A(1) receptor antagonist and was blocked by phospholipase C inhibitor. Coimmunoprecipitation experiments showed that the A(1) receptor is coupled to Galphai2 subunit suggesting that the activation of phospholipase C is mediated by betagamma subunits. In conclusion, the A(1) adenosine receptor in human spermatozoa is coupled to Galphai2, signals via IP3, and affects the capacitative status of ejaculated spermatozoa.

Acrosome Reaction↗
PubMed2000

Molecular and binding characteristics of IP3 receptors in bovine spermatozoa.

We have shown the presence of inositol 1,4,5-triphosphate (IP3) receptors in bovine spermatozoa. These receptors are mainly localized and functionally associated with the acrosome region. Molecular characterization of these bovine IP3 receptors has shown that the functional size of the IP3 binding domain is a protein of 66+/-2 kDa, in agreement with the size of both bovine adrenal cortex and bovine adrenal medullar chromaffin cells IP3 receptors. In contrast, bovine cerebellum IP3 receptor displays molecular weight of 220+/-5 kDa, a value in agreement with data in the literature. Bovine IP3 receptors have a one-affinity state characterized by a low affinity (Kd 750 nM) and a relatively high density (7.5 pmol/mg protein). They are functional and release internal calcium upon the binding of the second messenger. Moreover, the finding that the specific A1 adenosine receptor agonist R-PIA elicits almost the same effect as IP3 might be of some help in understanding the physiological role of these inhibitory adenosine receptors in mammalian spermatozoa.

Acrosome Reaction↗
PubMed2000

Immunolocalization of A1 adenosine receptors in mammalian spermatozoa.

The presence of A1 adenosine receptors (A1AR) in mammalian spermatozoa was previously demonstrated by radiochemical and immunochemical detection. This study was performed to investigate the cellular location of the A1AR to determine whether these receptors were somehow connected with ecto-adenosine deaminase and to evaluate their function in calcium uptake. By immunofluorescence staining we showed that in mammalian spermatozoa A1AR were constantly localized in the acrosomal region. This finding was confirmed by immunogold detection. Confocal analyses with anti-A1 and anti-ADA antibodies showed a high degree of co-localization. Calcium loading assay showed that this association was functional and affected calcium accumulation in mammalian spermatozoa. Therefore, we concluded that the acrosomal localization of A1AR was a constant feature in mammalian sperm. Moreover, these A1 receptors were functionally coupled to ecto-ADA and were able to modulate calcium uptake into an IP3-gated store.(J Histochem Cytochem 48:1163-1171, 2000)

Adenosine Deaminase↗
PubMed1999

CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa.

Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, which showed the localization of CD26 at the postacrosomal region and at the midpiece of the spermatozoa after incubation with vesicles. Moreover, a similar localization of CD26 and ecto-ADA on the spermatozoa was evidenced after fusion. Our results suggest that the interaction CD26/ecto-ADA might be responsible for fusion. Since A(1)ARs are said to be second receptors for ecto-ADA to form ecto-ADA/A(1)AR complexes, and since horse spermatozoa have A(1)ARs at their surface, the interaction CD26/ecto-ADA/A(1)AR during the fusion process cannot be ruled out.

Adenosine Deaminase↗
PubMed1998

Regulation of agonist-receptor binding by G proteins and divalent cations in spermatozoa solubilized A1 adenosine receptors.

Solubilized A1 adenosine receptor (A1AR) was used to investigate the effect of several cations on agonist-binding characteristics and GTP hydrolysis. It was shown by Western blot with G beta-M14 that this preparation contains both G proteins and receptor. The role of the receptor molecule is to facilitate the activation of G proteins as alpha-GTP complex, and GTP hydrolysis has important consequences for the basic deactivation mechanism. Divalent cations, such as Mn2+, Ca2+, and Mg2+, potentiated the agonist-specific binding: Mn2+ had the highest apparent affinity with half-maximal effect at 50 microM. Binding assays, performed in the presence of 100 microM Mn2+, showed an increase in the apparent affinity of the binding sites, whereas, in the presence of 1 mM Mg2+, significant alteration of the apparent affinity, but not of the number of sites, was detected. Concentrations of 1 mM Mg2+ and 100 microM Mn2+ enhanced GTPase activity, whereas 5 mM Ca2+ resulted in the increase of Vmax values without significant alterations of K(m). In the presence of A1-specific agonists, Mn2+ and Mg2+ caused a decrease of Vmax values and an increase of GTP affinity. Other cations, such as Co2+, Cd2+, Cu2+, and Zn2+, inhibited the binding capacity but caused almost no changes in GTP hydrolysis kinetics.

Animals↗
PubMed1997

[3H]-(R)-N6-Phenylisopropyladenosine agonist binding to the solubilized A1 adenosine receptor from bovine epididymal spermatozoa.

The high-affinity agonist radioligand [3H]-(R)-N6-phenylisopropyladenosine ([3H]-R-PIA) was used to investigate agonist A1 adenosine receptors interactions in soluble preparations from bovine spermatozoa. The digitonin-solubilized receptor shows a high-affinity state with a Kd of 5.32+/-1.17 nM and a Bmax of 460+/-33 fmol/mg protein. The binding capacity, higher than that of the membrane-bound form, indicates that the soluble preparation is likely enriched with binding sites. In the presence of guanylyl-5'-imidodiphosphate (Gpp(NH)p), [3H]-R-PIA binds to the soluble receptor with a Kd of 7.97+/-1.44 nM and a Bmax of 400.8+/-27 fmol/mg protein. The radioligand rapidly dissociates with a K(-1) of 0.125 min(-1) although specific [3H]-R-PIA is still found in solubilized A1 receptor. The A1 agonist N6-cyclopentyladenosine differentiates two affinity states, whereas Gpp(NH)p shifts the agonist curve to the right and all the receptors are in the low-affinity state. In the presence of NaCl, the agonist still recognizes two affinity states with a lower affinity than that observed in the absence of NaCl. Analyses of sedimentation profiles show the existence of a population of A1 receptors tightly coupled to Gi, the pertussin toxin-sensitive component of the G protein family.

Animals↗
PubMed1995

Evidence of A1 adenosine receptor on epidydimal bovine spermatozoa.

3H-R-phenylisopropyladenosine (PIA) was used to characterize adenosine receptors on bovine epidydimal spermatozoa membranes. Dypiridamole, an adenosine uptake inhibitor, did not effect the radioligand binding, indicating an external site for the interaction of adenosine with spermatozoa. Steady-state binding was achieved after 45 min at 25 degrees C and lasted for at least 3 h. Scatchard plots were linear with a Kd of 6.98 +/- 1.02 nM and Bmax of 34 +/- 8 fmol/mg protein. N-6-cyclopentyladenosine (CPA), with a Ki of approx. 0.196 nM, was the most potent inhibitor of binding and the agonist order potency series was CPA > R-PIA = N-6-cyclohexyladenosine > N-6-phenyladenosine > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > 2-(p-2-carboxyethyl)phenylamine)-5'-N-ethylcarboxy-amidoa denosine. 1-3-Dipropyl-8-cyclopentylxanthine (DPCPX), an A1 receptor selective antagonist, produced the strongest inhibition with a Ki of 0.46 +/- 0.1 nM. Antagonist order potency series DPCPX > xanthine amine congener > cyclopentyltheophilline = theophylline > caffeine > 1-3-dipropylxanthine > 8-phenyltheophilline was consistent with A1 adenosine receptor (A1AR). Guanylyl-5'-imidodiphosphate did not decrease bound 3-H-R-PIA nor accelerate its dissociation, a behavior consistent with inhibitory receptors only. The incubation of isolated membranes with N-ethylmaleimid followed by a reduction of 57% of the ligand binding further supports the existence of A1AR on bovine epidydimal spermatozoa.

Adenosine↗