Induction of androgenetic development of the brook charr (Salvelinus fontinalis)× Arctic charr (Salvelinus alpinus) hybrids in eggs derived from the parental species.

Failure of interspecific androgenesis between brook charr (Salvelinus fontinalis, Mitchill 1814) and Arctic charr (Salvelinus alpinus, L.) has been attributed to the conflict between the egg cytoplasm of one species and the sperm nucleus of the other species. To overcome this incompatibility, sperm derived from the brook charr×Arctic charr hybrid male was used to induce androgenetic development in eggs originating from the parental species as well as their hybrids. The eggs were subjected to 420Gy of X-radiation to damage the maternal nuclear DNA and inseminated with untreated sperm. Haploid zygotes were exposed to high hydrostatic pressure shock (7000psi for 4min), which was applied 420min after insemination to inhibit the first cell cleavage and recover the diploid state of the zygote. The androgenetic diploid offspring that hatched from the brook charr, the Arctic charr and the hybrids eggs had survival rates of 4.7±0.6%, 1.2±0.4% and 16.8±0.5%, respectively. Drastic mortality among the hatched androgenetic individuals was observed within the first five months of rearing. Cytogenetic analysis of the androgenetic progenies exhibited residues of the irradiated maternal nuclear genome in the form of radiation-induced chromosome fragments in 47% of the specimens that were examined. Interactions between the egg cytoplasm and the sperm nucleus, the low quality of the gametes, the expression of homozygous paternal lethal alleles and the incomplete inactivation of the maternal chromosomes were identified as factors responsible for the large mortality among androgenetic embryos and hatchlings.

Biochemical and physiological characteristics of semen of sex-reversed female rainbow trout (Oncorhynchus mykiss, Walbaum).

This works studies the biochemical (protein concentration, osmolality, antitrypsin activity, lactate dehydrogenase activity) and physiological characteristics (sperm motility characteristics) of semen of sex-reversed female rainbow trout (n=42) obtained with the application of 11ß-hydroksyandrostendione for sex reversal. All data were arbitrarily divided into three classes depending on the percentage of sperm motility: I XX<25%; II XX 25-50% and III XX>50%. The average percentage of sperm motility was 18±7% n=12 (group I XX); 42±6% n=15 (group II XX) and 65±12% n=15 for group III XX, respectively) to link the values of semen parameters to the maturation stage of semen. Semen from 12 normal males of the same age was used as a reference group. Sperm concentration as well as protein concentration, osmolality, antitrypsin activity, and lactate dehydrogenase activity in seminal plasma of sex-reversed females were higher compared with the values obtained for normal male rainbow trout. The values of these parameters declined with the increasing percentage of sperm motility toward values established for normal males. The fertilization success of semen (3×10(6) spermatozoa/egg) of sex-reversed females was very high (above 90%) for both the percentage of eyed embryos and hatched larvae and was related to sperm motility classes. Correlations between the quality parameters of sex-reversed females semen corresponded to those established previously for the semen of normal male rainbow trout. Antitrypsin activity, lactate dehydrogenase, protein concentration, and osmolality were found to be characteristic of seminal plasma of sex-reversed females. The maturity of sex-reversed female spermatozoa seems to be associated with the decline in the values of those parameters toward the values characteristic for seminal plasma of normal males.

Microcystin-LR induced apoptosis and mRNA expression of p53 and cdkn1a in liver of whitefish (Coregonus lavaretus L.).

There is growing evidence that adverse effects of microcystin-LR (MC-LR) are closely related to oxidative stress processes, free radicals and DNA damage, and involve major gene transcript changes. This study, utilizing gene expression analysis and plasma chemistries was the first to measure the effects of MC-LR in whitefish (Coregonus lavaretus L.), a feasible organism for pollution monitoring in aquatic systems. Fish were injected with different concentrations of MC-LR (0, 10 and 100 microg/kg of body weight) and then sacrificed at either 0, 8, 24, 48 or 72 h later, and their liver tissue were harvested for detailed investigation. Specifically, we were interested whether MC-LR is capable of: (i) modulating expression of two genes, tumor suppressor gene p53 and cdkn1a, p53 direct transcription target, and (ii) inducing apoptosis in whitefish liver. To study these effects, we developed a real-time qPCR assays useful for measuring both p53 and cdkn1a gene transcript levels in liver. To obtain necessary information for the study, either full-length p53 cDNA of whitefish (Wf-p53) was determined, using molecular cloning and rapid amplification of cDNA ends (RACE), or as for Wf-cdkn1a, specific primers were designed based on highly conserved regions of cdkn1a in fish. The Wf-p53 was found to share the same characteristics with a known p53 mRNA sequence of other vertebrates. Whitefish p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. The injection study showed that MC-LR at a higher dose, i.e. 100 microg/kg body weight, up-regulated expression of p53 and cdkn1a genes in whitefish liver, as reflected by the continuous increase in their mRNA levels through the whole experiment. Furthermore, DNA fragmentation was observed in liver cells of whitefish after 24h of exposure to MC-LR (100 microg/kg) that suggests the possibility of apoptosis. Finally, the study confirmed previous observations of severe injury of the liver and loss of normal organ functions as revealed by elevated levels of blood AspAT, AlaAT, and hepatosomatic index (HSI).