Cytochemical and Biochemical Studies on Progesterone-induced Maturation in Amphibian Oocytes: 2. DNA synthesis

Abstract

During the maturation of amphibian oocytes, Feulgen positive bodies originating from the nucleoli (nucleolar organizers, or ‘cores’) appear in the cytoplasm. The possibility that their deoxyribonucleicacid (DNA) might be replicated in the cytoplasm (rDNA) has been studied, following three different lines of approach: effects of inhibitors of DNA synthesis; cyto-photometric analysis of the DNA content of the Feulgen stained cytoplasmic bodies; characterization, in CsCI gradients, of the DNA which is synthesized after injection of 3H-thymidine into Xenopus laevis oocytes. The results can be summarized as follows: (1) None of the inhibitors used(hydroxyurea, 2′-deoxyadenosine(dA), cytosine arabinoside (Ara-C), bromodeoxyuridine, dimethylbenzylrifampicine, ethidium bromide, X-rays) inhibits progesterone-induced maturation in Rana pipiens and X. laevis oocytes. Only dA (10−2M) and Ara-C (10−2M) slowed down or arrested maturation; but this is probably due to the acidity of the drugs rather than to an inhibition of DNA synthesis. Experiments in which the inhibitors were injected into the oocytes show that the appearance of Feulgen-positive bodies in the cytoplasm is not influenced by agents which block DNA synthesis. (2) Quantitative cytophotometric measurements of the DNA content of the Feulgen positive cytoplasmic bodies rule out the possibility of a large rDN A synthesis during maturation. (3) There is a small DNA synthesis during maturation in x. laevis oocytes. However, this synthesis is merely the continuation of the slow DNA synthesis during oogenesis: it is not increased by progesterone-treatment. No radioactive peak is detectable at the density of rDNA. Enucleation experiments and autoradiography observations show that DNA synthesis takes place in the cytoplasm of the oocytes. Experiments on homogenates and characterization of the radioactive DNA strongly suggest that the neo-synthesized DNA is of mitochondrial origin. It is concluded that there is no other DNA synthesis, during maturation, than the continuation of the mitochondrial DNA synthesis which is already taking place during oogenesis, and that DNA synthesis is not required for successful maturation.