Studies On Artificially Induced Androgenesis In Mollusks

Androgenesis is defined as all-paternal inheritance. Viable androgenetic diploids can be generated by inhibiting the first cleavage to double paternally derived chromosomes, after fertilization of genetically inactivated eggs with normal sperm. It is a technique that could facilitate the production of completely homozygous isogenic lines, examine sex determination, make genetic analysis, and protect endangered species. Gamma, X-ray and ultraviolet irradiation inactivation are the usual methods used to inactivate egg nuclei. Among them, UV irradiation has been widely used for the convenient facility, safety and relatively low cost. In many fish species, efficient procedures of genetically inactivating of eggs and restoring diploidy have been studied and achievements of successful diploid androgenesis were reported. In contrast, in mollusks, artificial induction of androgenesis has rarely been studied. In this study, optimum inductive parameters in the scallop (Chlamys farreri), and the Pacific oyster (Crassostrea gigas) were tested and cytological processes in induction were observed.Effects of ultraviolet irradiation on genetic inactivation of eggs were examined in these two species. UV irradiation of eggs for 20 s and 30 s respectively at a UV (254 nm) intensity of 2.8 mW- cm"2- s"1 were optimum doses to achieve haploid androgenesis of the scallop and the Pacific oyster on the basis of observations on the fertilization and development rates and chromosome constitutions. The rates of the fertilization and the development of D-shaped larvae decreased with increasing irradiation time, and the development of the genetically inactivated eggs fertilized with the normal sperm terminated before reaching the D-shaped stage. Aneuploids were found in these studies. Because ultraviolet irradiation is known to cause pyrimidine dimerization in the DNA helix, which prevents the replication of genome, the occurrence of these aneuploids is probably attributed to different degree of maternal chromosomal inactivation by UV irradiation and/or DNA repair.Nuclear changes in normal and androgenetic eggs of the above two species were examined under a fluorescence microscope during meiosis, fertilization and earlydevelopment. Cytological processes in induction in the two species were almost the same. Although UV irradiation did not effect either meiotic maturation or the formation of the male and female pronuclei, their developmental progresses were delayed. At metaphase of the first cleavage, the female pronucleus in UV-irradiated eggs inseminated with the normal sperm did not form chromosome, unlike the male pronucleus, but became a dense chromatin body (DCB), which did not participate in the karyokinesis at anaphase of the first cleavage. At completion of cytokinesis of the first cleavage, the DCB was seen either in the cytoplasm of one of the two blastomeres or on the equatorial plate as two partitional parts. Cytological evidence of the induction of androgenesis was demonstrated.The induction of androgenesis diploids by suppression of the first cleavage in the scallop, C.farreri was attemped using the treatment of 6-dimethylaminopurine (6-DMAP). The result showed that it was difficult to induce diploid androgenetic D-shaped larvae. By comparison, treating the zygotes with 6-DMAP (60 ng/ml) at 80 min postinsemination for 15 min at water temperature of 19癈 produced more expected D-shaped larvae. According to Cytological observations, 6-DMAP disrupted the spindle at mitosis and inhibited chromosome movement, resulting in the formation of one diploid male nucleus.