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R Geremia

Publications and source records attributed to R Geremia.

At least 55 records · Page 3Linked to original sources
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

Cyclic nucleotide phosphodiesterase in developing rat testis. Identification of somatic and germ-cell forms.

Several forms of phosphodiesterase are present in the male gonad, and their relative concentrations vary during testicular development. On the basis of kinetic analysis and chromatography on DEAE cellulose, 3 forms were separated: (a) a high-affinity cGMP phosphodiesterase, regulated by Ca2+ and calmodulin, similar to a form described in different tissues (peak I); (b) a high-affinity cAMP form insensitive to Ca2+, which cannot be readily compared with forms described elsewhere (peak II); and (c) a high-affinity cAMP phosphodiesterase, Ca2+- and calmodulin-insensitive, corresponding to the "hormone-regulated" form described in several systems (peak III). The elution pattern of peak I and calmodulin stimulation were dependent on free calcium concentration during cytosol preparation and chromatography. This datum and rechromatography in the presence or absence of excess calmodulin suggested that the enzyme complexes calmodulin in a Ca2+-dependent manner and is therefore activated. Moreover, whereas peak I was observed in all cell compartments of the testis, peak II was present in germ cells and peak III was found mainly in somatic cells. During development of the testis, a relevant enhancement in the ratio between cAMP and cGMP hydrolytic activity was observed together with an overall increase of phosphodiesterase activity, thus suggesting that the previously described increase in cAMP phosphodiesterase activity during testis maturation should be ascribed to forms present in both germ cells and somatic cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
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↗
PubMed1980

Surface interaction in vitro between Sertoli cells and germ cells at different stages of spermatogenesis.

The presence of surface-recognition mechanisms between somatic and germ cells of the seminiferous epithelium has been studied in the rat by an assay in vitro based on the ability of homogeneous populations of spermatogenic cells, at specific differentiative stages, to adhere to monolayers of cultured Sertoli cells. The results show that germ cells adhere specifically to Sertoli cells and that the adhesion is dependent on the differentiative stage of the germ cells. Pachytene spermatocytes show the highest ability to adhere and form typical junctional specializations with the underlying Sertoli cells, while round spermatids adhere much less to the substrate. The possible regulative role of a somatic cell-germ interaction is discussed.

Animals↗
PubMed1980

Electrophoretic pattern of polypeptide synthesis in spermatocytes and spermatids of the mouse.

The pattern of protein synthesis in different stages of spermatogenesis was examined using two-dimensional polyacrylamide gel electrophoresis followed fluorography. The [3H]leucine labelling was performed by incubating in culture either seminiferous tubules before cell separation or isolated germ cells after fractionation by velocity sedimentation at unit gravity in an albumin gradient. The patterns of soluble polypeptides synthesised in middle-late pachytene spermatocytes, round spermatides (steps 1--8 of spermiogenesis) and intermediate spermatids (steps 9--13) have been compared with each other. Approximately 250 fluorographic spots were detected in middle-late pachytene spermatocytes and round spermatids, but only 100 in intermediate spermatids. From the analysis of fluorograms in the three cell stages examined, 5 categories of labelled polypeptides can be identified: (1) those specific of each cell stage; (2) those present in pachytene spermatocytes and early spermatids but absent in intermediate spermatids; (3) polypeptides labelled during spermiogenesis but unlabelled in meiotic cells; (4) polypeptides showing quantitative differences among the three cell types; and finally (5) polypeptides common to the three cell stages. These results are discussed in relation to differential gene expression during spermatogenesis.

Animals↗
PubMed1978

Post-meiotic gene activity in spermatogenesis of the mouse.

Mouse male germ cells at middle-late pachytene and early spermatid stages obtained by velocity sedimentation at unit gravity in albumin gradients were labelled in culture with [3H]uridine. The newly synthesized RNAs extracted from polysomes of the 2 cell types were studied by sucrose gradient fractionation and poly(U) Sepharose chromatography. The results showed that round spermatids, as well as pachytene spermatocytes, synthesize both ribosomal and polyadenylated RNA molecules. Since these latter are engaged in polysomes they are presumably active messenger RNA molecules.

Animals↗
PubMed1978

DNA-dependent DNA polymerase species in male germ cells of the mouse.

Quasi-homogeneous fractions of male mouse germ cells at definite stages of meiosis and spermiogenesis were obtained by using a separation method based on sedimentation velocity in an albumin gradient. In the various cell types, the total DNA-dependent DNA polymerase activity was determined, and the major enzymatic forms were characterized. The DNA polymerase species present in premeiotic, meiotic and post-meiotic cells were analyzed by glycerol gradient sedimentation. Two types of DNA polymerase were identified in fractions enriched in spermatogonia and preleptotene spermatocytes. One showed a sedimentation coefficient of about 7.5 S and was sensitive to N-ethylmaleimide (NEM); the other exhibited a sedimentation coefficient between 3 and 4 S and was resistant to NEM. On the basis of their sedimentation coefficients, their sensitivity to NEM and their template specificities, these 2 enzymes were identified respectively as alpha and beta DNA polymerases as reported in mammals. The gradient analysis performed on fractions enriched in meiotic and post-meiotic cells revealed the presence of DNA polymerase beta only. A quantitative analysis showed that the activity of the DNA polymerase beta reaches a maximum at middle-late pachytene stage and then drops gradually during spermiogenesis. Although any conclusion as to the biological role of this high level of DNA polymerase activity in pachytene spermatocytes is premature, it is tempting to suggest that this enzyme is involved in meiotic recombination.

Animals↗
PubMed1977

RNA synthesis in spermatocytes and spermatids and preservation of meiotic RNA during spermiogenesis in the mouse.

The rate of RNA synthesis at different stages of spermatogenesis in the mouse, and the preservation of RNA from the diploid to the haploid phase of spermatogenesis were studied in homogeneous germ cell fractions separated by velocity sedimentation at unit gravity. The uridine pool expansion method was used for determining the rate of RNA synthesis: seminiferous tubules were labelled in culture with increasing concentrations of [3H]-uridine and the incorporated radioactivity was estimated in cell fractions separated by velocity sedimentation. The results indicate that before nuclear elongation, round spermatids (steps 1 to 8 of spermiogenesis) synthesize RNA at the same rate per DNA content as middle-late pachytene spermatocytes. The preservation of RNA molecules synthesized in meiosis was investigated by labelling pachytene spermatocytes with T3H]uridine in vivo and collecting samples of germ cells at definite stages of spermatogenesis at various time intervals thereafter. The results show that a considerable proportion the RNA synthesized during the pachytene stage is preserved through spermatid development until late spermiogenesis.

Animals↗
PubMed1977

Histone synthesis and replacement during spermatogenesis in the mouse.

Separation of labelled nuclei by sedimentation velocity at unit gravity (Staput method) was used to study the timing of histone synthesis and replacement by testis-specific basic nuclear protein (TSP) during spermatogenesis in the mouse. Animals were injected (intratesticularly) with 1.25 micronCi per testis 3H-arginine or 2.5 micronCi per testis 3H-lysine, testis nuclei were separated, and the acid extract of each nuclear fraction was analyzed by acrylamide gel electrophoresis. The distribution of labelled histones and TSP in separated nuclei was assessed 2 h after incorporation. Changes in the labelled histone and TSP content of nuclei during subsequent differentiation (1--34 days post-label) was followed in fractions of separated testis cell nuclei and in nuclei of cauda epididymal spermatozoa. Analysis of total histone and (TSP) content indicated quantitative changes during development. Nuclei from primary spermatocytes had relatively larger amounts of histones H1 and H4. Spermatid nuclei showed a relative reduction in histones H1 and H4, coincident with the appearance of TSP in these nuclei. These results suggested that synthesis and/or removal of certain histones must occur in late primary spermatocyte and early spermatid stages of spermatogenesis. Results of labelling experiments indicated several periods of histone synthesis during spermatogenesis: (1) closely associated with the last DNA synthesis(i.e., in early primary spermatocytes), (2) late in meiotic prophase (i.e., in pachytene primary spermatocytes) and (3) simultaneous with TSP synthesis (i.e., in late spermatids). Histone H1 was more heavily labelled toward the end of the primary spermatocyte period. Histone H4 was more heavily labelled in the early primary spermatocyte period, and again at the time of TSP synthesis in spermatids. Histones synthesized before the pachytene primary spermatocyte stage appeared to be replace, but histones synthesized later in spermatogenesis appeared to be at least partially retained in epididymal spermatozoa. These results suggested that repeated specific alterations in the protein complement of the nucleus are an integral part of spermatogenic differentiation in the mouse.

Animals↗
PubMed1976

Kinetics of histone and protamine synthesis during meiosis and spermiogenesis in the mouse.

The separation of mouse spermatogenic cell nuclei by sedimentation velocity at unit gravity has been used to determine the timing of histone and "mouse protamine" synthesis, and the turnover of basic nuclear proteins throughout spermatogenesis. Animals were injected with 3H-arginine or 3H-lysine and at various time intervals (2 hours post-label or from 1 to 30 days post-label) germinal cell nuclei preparations were separated on the staput. Labelled histones and mouse protamine were extracted from staput separated nuclei with hydrocholoric acid and fractionated by polyacrylamide gel electrophoresis. Results indicate that histones are synthesized in association with DNA replication in spermatogonia and preleptotene spermatocytes, in pachytene primary spermatocytes and in spermatids stages 11-16, simultaneously with "mouse protamine". Experiments are reported showing that histones synthesized in pachytene primary spermatocytes and in spermatids stages 11-16 are retained in epididymal spermatozoa, while histones synthesized before meiosis are no longer detectable onto chromatin after meiosis.

Animals↗