90 research outputs found
Adenosine 3',5'-cyclic monophosphate dependent protein kinase activity in differentiating germ cells of the mouse testis
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RNA synthesis in spermatocytes and spermatids and preservation of meiotic RNA during spermiogenesis in the mouse
No full textThe 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
Particulate and soluble adenylate cyclase activities of mouse male germ cells
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Developmental changes of cyclic adenosine monophosphate-dependent protein kinase activity during spermatogenesis in the mouse
No full textCyclic AMP-dependent protein kinase. activity W8I ItUdied in germ ceIIs at different lUpI of meiosis md spenniogenesis. A constant dec:reue in total protein kinase activity W8I detected in the IOluble fraction of genn ceUs, durin. spennatogalic devdopment. TIUs dec:reue wu not IIIOciated with a redistribution of phosphorylative activity to other compartmentl of the ceDo DEAE-c:eIlulose chromatography of the c:ytosoI of pacbytene spennatocytel md round spermatidl demonstrated the praence of two isoenzyma of protein kinue e1utin. at 35 mM KCI (Peak I) md 170 mM KCI (Peak Il). A selective dec:reue in the amount of Peak I cou1d be observed with the progrcss of spennatid development, md Peak Il W8I the major form praent in e10nptinJ spermaddl. Peak Il isoenzyme W8I me most abundant form of protein kinue aIso in the IOluble fraction of epididymal spermatOZOL A simiIar deve!opmental pattem W8I obtained when cyclic AMP binding activity, rather then phosphorylative activity, wu measured alter ion exchmge chromatography of different germ celllOluble fractiona. The two protein kinue isoenzyma of germ ceUs had characteristic:l similar to those dClcribed in other ceUs of the testis md other tisaua. In particular, Peak I kinase W8I diIIoc:iated md ac:tivated by hip wt concentration md buie proteina, while Peak Il kinue W8I inaensitive to these treatmenti. These distinctive propertia of the two isoenzyma were used to measure me relative concentration of the twO forms direcdy in the crude c:ytosoI preparationa of germ ceUs. Pacbytene spermatocyte kinue activity wu dissociated by hiJh wt to a leve! comparable to that of round spermatidl. The soIuble kinue present in elonpting spermatidl wu, inatead, inaensitive to wt treatment, confmnin. the hypothesis that most of the activity praent in thia celi type hu the cbaracteristic of Peak Il
Electrophoretic pattern of polypeptide synthesis in spermatocytes and spermatids of the mouse
No full textThe 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
Post-meiotic gene activity in spermatogenesis of the mouse
No full textMouse 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
Dna-dependent dna polymerase species in male germ cells of the mouse.
No full textQuasi-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
5',3' Cyclic adenosine monophosphate dependent protein phosphorylation in differentiating male mouse germ cells
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