phosphorus-radioisotopes and 2--3--dideoxyadenosine-triphosphate

The aims of the present study in ewes were 1) to test the hypothesis that apoptosis in granulosa cells is one of the processes involved in the structural demise of follicles and 2) to define the temporal relationships among the occurrence and degree of apoptosis in granulosa cells, aromatase activity, production of cyclic AMP (cAMP) by granulosa cells in response to FSH or LH, concentrations of estradiol 17 beta (E2) and progesterone in follicular fluid, and the characteristic morphometric changes associated with the process of follicular atresia. To address these aims, ewes were treated with either saline or steroid-free bovine follicular fluid (bFF) at 60 h after estrus, and ovarian follicles > or = 3 mm diameter were recovered at 0, 12, 18, or 24 h later. Apoptotic granulosa cells were identified by the presence of oligonucleosomes after 3'-end labeling of extracted DNA with [32P]alpha dideoxy ATP (ddATP). The degree of oligonucleosome formation, based on the intensity of radiolabeling, was given an apoptosis score (AP) of 0 (nondetectable), 1 (slight), 2 (moderate), or 3 (marked). Moreover, a labeling index (LI) was calculated from the amount of radiolabeled ddATP incorporated into low-molecular weight (< 4.2 kb) DNA fragments. On the basis of morphometric criteria, 73% (141 of 194) of the follicles classified as healthy had apoptotic granulosa cells compared to 86% (18 of 21) of the follicles classified as atretic. In the bFF-but not saline-treated ewes, the concentrations of plasma FSH had declined to basal values at 12 h after treatment. At the beginning of the treatment period, the degree of granulosa cell apoptosis was either undetectable (AP = 0, 47% of follicles) or slight (AP = 1, 44% of follicles) in the majority of follicles. After 12 h from the bFF but not the saline injection, there was a significant increase in the proportion of follicles (> or = 3 mm diameter) per ewe containing apoptotic granulosa cells (p < 0.001) and a significant decrease in the number of follicles per ewe with aromatase activity (p < 0.05) and with follicular fluid E2 > 20 ng/ml (p < 0.05). By 24 h after bFF treatment, apoptosis was evident in all follicles (> or = 3 mm diameter), fewer follicles contained FSH-responsive granulosa cells in terms of cAMP production (p < 0.05), and none were LH-responsive. A significant negative relationship was found between the degree of granulosa cell death as measured by L1 and follicular fluid E2 concentrations. In summary, the p

Topics: Animals; Apoptosis; Aromatase; Cattle; Cyclic AMP; Deoxyadenine Nucleotides; Dideoxynucleotides; DNA; Estradiol; Female; Follicle Stimulating Hormone; Follicular Atresia; Follicular Fluid; Granulosa Cells; In Vitro Techniques; Kinetics; Luteinizing Hormone; Nucleosomes; Ovarian Follicle; Ovulation; Phosphorus Radioisotopes; Progesterone; Sheep

Topics: Cell Line; Deoxyadenine Nucleotides; Dideoxynucleotides; DNA Damage; DNA Repair; DNA Replication; DNA, Neoplasm; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Phosphorus Radioisotopes; Ultraviolet Rays

Dideoxynucleotides have proved to be potent differential inhibitors of DNA polymerases in vitro and in vivo. Used extensively in studies of DNA repair and replication, they have more recently been used as antiviral agents particularly in treating patients for acquired immunodeficiency syndrome (AIDS). Once incorporated, these sugar-modified analogues prevent the further extension of the polynucleotide chain because of the absence of a 3'-hydroxyl group. We demonstrated that, upon injection into Xenopus laevis oocytes, 2',3'-dideoxynucleotides are efficiently removed from plasmid DNA preterminated with these analogues allowing subsequent closure by ligation. The removal process is not sensitive to aphidicolin but is quantitatively inhibited by novobiocin.

Topics: Animals; Autoradiography; Deoxyadenine Nucleotides; Dideoxynucleosides; Dideoxynucleotides; DNA Repair; DNA-Directed DNA Polymerase; Female; Kinetics; Oocytes; Phosphorus Radioisotopes; Plasmids; Protein Biosynthesis; Xenopus laevis