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Biomedical subjects

M Stefanini

Publications and source records attributed to M Stefanini.

At least 127 records · Page 7Linked to original sources
PubMed1984

Catalytic activities of human poly(ADP-ribose) polymerase from normal and mutagenized cells detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

An activity gel procedure is described to identify functional polypeptides of human poly(ADP-ribose) polymerase. Purified or crude enzyme preparations from HeLa cells were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels containing gapped DNA. After renaturation of the peptides in situ, the intact gel was incubated in a poly(ADP-ribose) polymerase reaction mixture containing [32P]NAD. Autoradiograms of the gels consistently exhibited a major activity band at Mr = 116,000-120,000; in many runs, three minor distinct bands at Mr = 125,000, 135,000, and 145,000 were also seen. [32P]NAD appeared to be incorporated into poly(ADP-ribose) since: (i) the activity bands were not detectable when the enzyme-inhibitor 3-aminobenzamide was added to the gel incubation mixture; and (ii) the radioactive polymer, electroeluted from the bands, was completely digested by phosphodiesterase I. Preliminary activity gel analysis of extracts of HeLa cells treated with different DNA-damaging agents revealed that the apparent activity of the Mr = 116,000 form increased by about 10-fold in cells treated with 1 mM dimethyl sulfate and 10-20-fold in cells treated with 10 microM mitomycin C. Only a small increase was obtained in cells treated with 1 mM methyl methanesulfonate, and no change in the activity band pattern was observed after 50 and 100 J/m-2 of UV irradiation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

Study of the interaction between germ cells and Sertoli cells in vitro.

The nature of membrane components involved in the binding between Sertoli cells and pachytene spermatocytes in culture and the metabolic requirements for the binding to occur have been studied. Mild proteolytic digestion of germ cells by trypsin completely inhibited adhesion of germ cells to somatic monolayer. Protein synthesis and glycosylation were required to restore the adhesive properties of trypsin-treated germ cells, showing that surface molecules involved in the binding are glycoproteins. Trypsinization of germ cells after labelling causes a great reduction of several bands which become detectable again after 12 h of recovery from trypsin digestion. Among these, two bands with apparent molecular weight (MW) of 78 000 and 51 000 could be candidate components in cell adhesion.

Animals↗
PubMed1984

Pyruvate and lactate production by cultured Sertoli cells, fibroblasts and muscle satellite cells, and the effect of hormonal and dcAMP stimulation.

Isolated male germ cells survive in culture if medium is supplemented with adequate energy substrates such as lactate or pyruvate. The purpose of the present study was to investigate if cultured Sertoli cells release in the medium lactate or pyruvate and if this production is affected by FSH or dcAMP treatment. We have also studied if the ability to produce lactate and pyruvate is shared by other cell types. The results show that 1) the two metabolites are released from germ-cell-free rat Sertoli cell monolayers, and their release is stimulated by hormone or dcAMP 2) other cell types of mesodermic origin release more lactate and pyruvate than Sertoli cells, but are not stimulated by FSH or dcAMP.

Animals↗
PubMed1983

Changes of Sertoli cell glycoproteins induced by removal of the associated germ cells.

Sertoli cell glycoproteins were studied in culture where these cells were in contact with germ cells (Sertoli cell enriched cultures, SCEC) and in pure Sertoli cell cultures. Sertoli cell only cultures (SCOC) were prepared by a short treatment of SCEC with hypotonic solution or by culturing seminiferous epithelium fragments from prenatally irradiated rats. After metabolic labeling with [3H]fucose. [14C]N-acetylglucosamine or [3H]leucine, SCEC and SCOC particulate fractions (105 000 g pellet) were analyzed by one-dimensional slab gel electrophoresis and fluorography. The comparison of the electrophoretic patterns obtained, demonstrated that a glycoprotein of MW 48 000, undetectable in SCEC, was present in SCOC after labelling with both sugar precursors. The MW 48 000 glycoprotein was also present in the electrophoretic profile of particulate fraction from [3H]fucos-labelled Sertoli cell cultures from prenatally irradiated rat. Such difference was not observed after labelling with [3H]leucine; in this experimental condition a MW 48 000 band was present in the electrophoretic profile of polypeptides from SCEC as well from SCOC. The synthesis of this glycoprotein represented a specific and stable cell response, since it occurred only a few hours after germ cell removal, and it was still detectable 3 days later. FSH stimulation did not influence the synthesis of the MW 48 000 glycoprotein, whereas it increased the synthesis of high MW glycoproteins. The hypothesis is discussed that the appearance of a new glycoprotein when Sertoli cells have lost their contact with germ cells could represent a product of glycosylation of preexisting molecules and their possible location in the Sertoli cell membrane. The results presented here provide additional evidence that Sertoli cell functions may be dependent on the association with the germ cell.

Animals↗
PubMed1983

Intercellular communication in rat seminiferous tubules.

Intercellular electrical coupling in seminiferous tubules from prepubescent and adult Wistar rats has been studied by using conventional techniques. It is found that cells in the seminiferous epithelium are electrically coupled. Experiments performed using "Sertoli cell-enriched" seminiferous tubules indicate the existence of intercellular ionic communication between Sertoli cells. Junctional conductance is independent of the direction of electrical field and it is affected by A23187 Ca ionophore (5 microM) but not by exposure to the neurotransmitter norepinephrine (1-5 X 10(-5) M). Intracellular resistivity (including junctional resistance) is higher in mature as compared to immature germinal epithelium. These findings suggest that cell metabolites or second messenger molecules could be transferred via the low-resistance pathways between epithelium cells to coordinate cellular activity.

Aging↗
PubMed1983

Follicle-stimulating hormone regulates in vivo testicular phosphodiesterase.

The effect of FSH on testicular phosphodiesterase was studied in immature rats in order to verify that the regulation of response to hormone in the gonad involves an increased cyclic AMP catabolism. Hydrolysis of cyclic AMP and cyclic GMP was measured in the homogenates of seminiferous tubules and interstitium of control animals and animals injected i.p. with 50 micrograms ovine FSH twice, 24 h and 12 h before necroscopy. After hormonal treatment, cyclic AMP phosphodiesterase activity in the seminiferous tubules was markedly increased whether results were expressed per testis or per mg protein, while cyclic GMP phosphodiesterase present in the same compartment was apparently unaffected. In the interstitium, cyclic nucleotide hydrolysis was usually decreased after FSH injection. The stimulation of phosphodiesterase, a slow process reaching maximal stimulation after 12 h, was dependent on the dose of FSH injected. In addition, DEAE-cellulose chromatography of cytosol prepared from control and treated seminiferous tubules confirmed that stimulation was restricted to a cyclic AMP hydrolysing enzyme while the activity of the cyclic GMP hydrolysing form was not modified. Thus it is demonstrated that testicular phosphodiesterase is under FSH control. It is proposed that this in vivo regulation is a relevant phenomenon in the modulation of Sertoli cell function and contributes to the refractoriness that follows gonadotropin treatment.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Influence of Sertoli cell products upon the in vitro survival of isolated spermatocytes and spermatids.

We have investigated the effect of Sertoli cell products upon the in vitro survival of isolated pachytene spermatocytes and round spermatids using co-cultures without contact between the somatic and germ cells. 3H-leucine incorporation was measured in such conditions at different culture times and compared with that of isolated germ cells incubated in presence of different energetic substrates. Dependence upon pyruvate and Sertoli cell products with respect to lactate is shown and discussed for both cell types; differences in pyruvate requirement are reported between meiotic and haploid cells.

Animals↗
PubMed1983

Involvement of phosphodiesterase in the refractoriness of the Sertoli cell.

Gonadotropin treatment of the Sertoli cell produces a marked refractory state of the cell to subsequent hormonal stimulation. Because FSH also stimulates the phosphodiesterase activity of these cells, the possible involvement of an altered cAMP catabolism during refractoriness was investigated in an in vitro model. Sertoli cells, after 3 days of culture in a defined medium, were exposed to FSH or isoproterenol for 1-24 h. After this pretreatment, cells were stimulated for 1 h with a maximal FSH dose, and the responsiveness was measured in terms of cAMP accumulation. Sertoli cells previously treated with hormone entered a refractory state, a second exposure being ineffective in elevating intracellular or extracellular cAMP. Addition of the phosphodiesterase inhibitor 3-isobutyl-methylxanthine in the second incubation partially restored the ability of the cell to accumulate cAMP in the presence of hormone. This phosphodiesterase inhibitor also caused an apparent decrease in the potency of FSH to induce the refractory state. Such an impairment of response developed in the intact cell in 4 h, and was accompanied by a partial desensitization of the adenylate cyclase and an increase in phosphodiesterase activity. The stimulation of phosphodiesterase activity, but not the desensitization of adenylate cyclase, was inhibited by cycloheximide. The inhibition of protein synthesis also prevented the onset of the refractory state of the intact Sertoli cell. Pretreatment of the Sertoli cells with either FSH or isoproterenol rendered the cell refractory to a second stimulation with either agonist; in contrast, the adenylate cyclase desensitization in the homogenate was apparent only for the agonist employed in the preincubation. These results indicate that phosphodiesterase regulation is involved in the control of Sertoli cell responsiveness to hormone. Thus, the net decrease in cAMP production of the FSH-treated cells is the result of a decreased adenylate cyclase stimulation and an increased cAMP catabolism mediated by phosphodiesterase. The latter phenomenon appears to be the predominant cause of the partial refractoriness induced by low doses of gonadotropin.

Adenylyl Cyclases↗
PubMed1982

Isolation of Chinese hamster ovary cells with reduced unscheduled DNA synthesis after UV irradiation.

A simple procedure has been worked out to obtain UV-sensitive mutants of Chinese hamster ovary (CHO) cells. In this procedure, conventional mutagenesis is followed by BrdU--light treatment to enrich the population for UV-sensitive cells. Colonies that are allowed to form subsequently are duplicated by replica plating and screened on the master plate for their UV sensitivity and their capacity to carry out UV-induced DNA repair synthesis. Putative mutants are isolated from the replica. With this combination of methods, we succeeded in isolating CHO mutants with an 85-95% reduced level of UV-induced DNA synthesis in combination with an increased UV sensitivity.

Animals↗
PubMed1982

Regulation of follicle-stimulating hormone and dibutyryl adenosine 3',5'-monophosphate of a phosphodiesterase isoenzyme of the Sertoli cell.

The regulatory role of FSH on phosphodiesterase was studied in immature Sertoli cells in culture. Cells were prepared from 15-day-old immature rats, cultured for 3 days in defined medium, and then stimulated for 24 h with gonadotropin or with other factors known to regulate Sertoli cell cAMP. All agents that increased cAMP intracellular levels also had an effect on the total phosphodiesterase activity of cell homogenates, FSH and dibutyryl cAMP being the most potent stimulators. Further, stimulation was more evident when phosphodiesterase was measured at cAMP concentrations below micromolar. With 1 microM cGMP as substrate, no modification of the activity could be detected. Reversal of phosphodiesterase activation was observed at 24 or 40 h when dibutyryl cAMP was removed from the incubation medium. Separation of the isoenzymes present in the soluble fraction of Sertoli cell made it possible to demonstrate a selective stimulation of one isoenzyme. FSH and dibutyryl cAMP increased 10- and 50-fold, respectively, the activity of a high affinity cAMP phosphodiesterase, while the Ca2+-dependent cGMP hydrolyzing enzymes were not apparently affected. The enzyme regulated by FSH and dibutyryl cAMP had an apparent Km for cAMP of 2 microM, was Ca2+ and calmodulin insensitive, and migrated on sucrose density gradients with a sedimentation coefficient of 5.5S. These results indicate that FSH, after stimulation of intracellular cAMP, induces an increase in a high affinity phosphodiesterase and, therefore, an increased cAMP turnover in the Sertoli cell. This, in turn, might be a relevant phenomenon in the control of the responsiveness of this cell to gonadotropin.

1-Methyl-3-isobutylxanthine↗