In the present research we produced triploid, mitogynogenetic (doubled haploid; DH) and meiogynog... more In the present research we produced triploid, mitogynogenetic (doubled haploid; DH) and meiogynogenetic brook trout (Salvelinus fontinalis) to examine efficiency of these technologies and potential susceptibility of chromosome set manipulated individuals for the spinal disorders. Triploidy was induced by shocking (High Hydrostatic Pressure – HHP) of fertilized eggs 30 min. after insemination. In turn, gynogenetic development was induced by activation of eggs with UV-irradiated sperm. Activated eggs were then exposed to HHP shock applied 30 and 420 minutes after insemination to provide meiogynogenotes and gynogenetic DHs, respectively. When compared to non-manipulated diploids, the highest survival rates were observed among triploid brook trout while DHs showed the highest mortality. Malformation rates in the diploid larvae from the control groups did not exceed 7.0% while percentage of malformed triploid individuals equaled 19.1%. Drastically increased number of deformed larvae (>...
In the present research, the eggs from four rainbow trout females were used to provide four group... more In the present research, the eggs from four rainbow trout females were used to provide four groups of gynogenetic doubled haploids (DHs). The quality of the eggs from different clutches was comparable, however, interclutch differences were observed in the gynogenetic variants of the experiment and the survival of DH specimens from different groups varied from 3% to 57% during embryogenesis. Transcriptome analysis of the eggs from different females exhibited inter-individual differences in the maternal genes’ expression. Eggs originating from females whose gynogenetic offspring had the highest survival showed an increased expression of 46 genes when compared to the eggs from three other females. Eggs with the highest survival of gynogenetic embryos showed an up-regulation of genes that are associated with cell survival, migration and differentiation (tyrosine-protein kinase receptor TYRO3-like gene), triglyceride metabolism (carnitine O-palmitoyltransferase 1 gene), biosynthesis of p...
The paper reviews techniques of genome engineering and their potential applications in freshwater... more The paper reviews techniques of genome engineering and their potential applications in freshwater finfish aquaculture. In particular, banking of cryopreserved fish semen combined with androgenesis (both intra- and interspecific) are viewed as the most promising techniques enabling the researcher to protect and restore the gene pools of threatened (wild) and valuable (farmed) fish stocks.
The paper reviews techniques of genome engineering and their potential applications in freshwater... more The paper reviews techniques of genome engineering and their potential applications in freshwater finfish aquaculture. In particular, banking of cryopreserved fish semen combined with androgenesis (both intra- and interspecific) are viewed as the most promising techniques enabling the researcher to protect and restore the gene pools of threatened (wild) and valuable (farmed) fish stocks.
Although brook trout and the Arctic char hybrids are able to reproduce, individuals with decrease... more Although brook trout and the Arctic char hybrids are able to reproduce, individuals with decreased fertility or even fish that are unable to produce any gametes have been also described. Abnormal gonadal development and disturbances in the gamete production in the char hybrid offspring may be triggered by the odd chromosome number and disturbances in their pairing during meiosis. To verify this hypothesis, cytogenetic examination and the gonadal histology analysis of the brook trout x Arctic char hybrids were carried out. Diploid chromosome number in the studied char (F 1 ) hybrids varied from 82 to 84 (FN = 99-102). Among 28 hybrids, 12 males, three females, nine intersex individuals and two sterile specimens were described. In the case of two individuals, gonads were not found. Diploid chromosome numbers in the males and intersex individuals varied from 82 to 84. Chromosome numbers in the females were 82 and 83 chromosomes. Two sterile fish exhibited karyotypes composed of 82 and 84 chromosomes. Predominance of the ovarian component in the intersex gonads and gonadal sex ratio distortion towards the males suggested hybrid females had problems with gonadal differentiation. However, the lack of the clear relationship between chromosome number and gonadal development in the studied hybrids did not support our hypothesis that odd chromosome number may be responsible for such reproductive disturbances in the hybrid individuals. We have presumed that sterility and intersexual development of the gonads may be caused by interactions between brook trout and Arctic char genes on the sex chromosomes and autosomes rather than unpairing of the parental chromosomes.
The development of high resolution methods of chromosome banding helped the finding of homologous... more The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. P...
Cytogenetic or molecular identification of sex chromosomes could help in breeding studies in prod... more Cytogenetic or molecular identification of sex chromosomes could help in breeding studies in producing monosex fish stocks, estimating success of androgenesis, gynogenesis, etc. Among fish species sex chromosomes are recognizable in only a few cases. Some populations of rainbow trout Oncorhynchus mykiss show morphologically differentiated sex chromosomes. A strain from Rutki, Poland, showed a heteromorphic pair of subtelocentric chromosome: presumably of the XY type in the male and XX in the female. Restriction endonuclease and DAPI banding resulted in a characteristic banding pattern enabling identification of the X chromosome.
OCALEWICZ K. 2005. Identification of early and late replicating heterochromatic regions on platyf... more OCALEWICZ K. 2005. Identification of early and late replicating heterochromatic regions on platyfish (Xiphophorus maculatus) chromosomes. Folia biol. (Kraków) 53: 149-153.
A b s t r a c t . The karyotype of bream Abramis brama was analysed by means of C-banding, replic... more A b s t r a c t . The karyotype of bream Abramis brama was analysed by means of C-banding, replication banding, DAPI fluorescent staining and in situ hybridisation with 18S rDNA and telomeric probes. The use of the in vivo 5-bromodeoxyuridine incorporation technique enabled the induction of replication bands of the RBA type in the karyotype. C-bands corresponded to late replicated chromosome regions. 18S rDNA clusters were found on one chromosome pair. Telomeric sequences were observed only on ends of chromatids. The karyotype morphology and NOR phenotype of A. brama are very similar to those found in other species of the leuciscine cyprinids karyotyped so far.
Chromosome distribution of nucleolus organizer regions (NORs) and 5S rDNA loci were investigated ... more Chromosome distribution of nucleolus organizer regions (NORs) and 5S rDNA loci were investigated in platyfish (Xiphophorus maculates ) using conventional and molecular cytogenetic techniques. Chromomycin A 3 (CMA 3 ) staining of GC-rich heterochromatin provided four CMA 3 cytotypes in studied platyfish. As in fish, CMA 3 positive sites are related to NORs, chromosomes sequentially stained by CMA 3 and AgNO 3 showed bright blocks that were transcriptionaly active NORs. Additionally, PRINS with primers enabling amplification of 5S rDNA and CMA 3 staining revealed existence of 5S rDNA sites located close to GC rich regions. The analysis could suggests that minor and major rDNAs may be linked.
Failure of interspecific androgenesis between brook charr (Salvelinus fontinalis, Mitchill 1814) ... more 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.
ABSTRACT Karyotype and cytogenetic characteristics of the invasive round goby (Neogobius melanost... more ABSTRACT Karyotype and cytogenetic characteristics of the invasive round goby (Neogobius melanostomus (Pallas, 1814)) from the population inhabiting the Gulf of Gdansk, Baltic Sea, Poland were investigated using conventional and molecular cytogenetic techniques. The diploid chromosome number of all samples was 2n= 46 and the karyotype consisted of subteloto acrocentric chromosomes that gradually decreased in size. Analysis of chromatin resistance to restriction enzymes (AluI and DdeI) showed pericentromeric and telomeric blocks of heterochromatin. Nucleolus Organizer Regions (NORs) were observed in the pericentromeric regions of two medium-sized chromosomes. Sequential staining (DAPI, CMA3 and AgNO3) showed that the NORs are DAPI-negative and CMA3-positive in round goby chromosomes. Additionally, DAPI negative and CMA3-negative chromatin was identified in the interstitial position of two chromosomes. Fluorescent In Situ Hybridization (FISH) with a telomeric probe did not reveal any Interstitial Telomeric Repeat sequences (ITRs). No morphologically differentiated sex chromosomes were found in the round gobies studied. Considering the increased numbers of bi-armed chromosomes to be a derived condition in some ‘neogobiin’ species, the round goby karyotype of 46 mono-armed chromosomes, which are similar in both female and male cells and have GC-rich NOR-related regions located on the single chromosome pair, fits as an ancestral Neogobius karyotype pattern.
Specimens from two freshwater populations of the ninespine stickleback Pungitius pungitius in Pol... more Specimens from two freshwater populations of the ninespine stickleback Pungitius pungitius in Poland showed morphologically differentiated sex chromosomes. A heteromorphic pair of chromosomes appeared only in male diploid cells. The Y chromosome was the largest chromosome in the P. pungitius karyotype.
In the present research we produced triploid, mitogynogenetic (doubled haploid; DH) and meiogynog... more In the present research we produced triploid, mitogynogenetic (doubled haploid; DH) and meiogynogenetic brook trout (Salvelinus fontinalis) to examine efficiency of these technologies and potential susceptibility of chromosome set manipulated individuals for the spinal disorders. Triploidy was induced by shocking (High Hydrostatic Pressure – HHP) of fertilized eggs 30 min. after insemination. In turn, gynogenetic development was induced by activation of eggs with UV-irradiated sperm. Activated eggs were then exposed to HHP shock applied 30 and 420 minutes after insemination to provide meiogynogenotes and gynogenetic DHs, respectively. When compared to non-manipulated diploids, the highest survival rates were observed among triploid brook trout while DHs showed the highest mortality. Malformation rates in the diploid larvae from the control groups did not exceed 7.0% while percentage of malformed triploid individuals equaled 19.1%. Drastically increased number of deformed larvae (>...
In the present research, the eggs from four rainbow trout females were used to provide four group... more In the present research, the eggs from four rainbow trout females were used to provide four groups of gynogenetic doubled haploids (DHs). The quality of the eggs from different clutches was comparable, however, interclutch differences were observed in the gynogenetic variants of the experiment and the survival of DH specimens from different groups varied from 3% to 57% during embryogenesis. Transcriptome analysis of the eggs from different females exhibited inter-individual differences in the maternal genes’ expression. Eggs originating from females whose gynogenetic offspring had the highest survival showed an increased expression of 46 genes when compared to the eggs from three other females. Eggs with the highest survival of gynogenetic embryos showed an up-regulation of genes that are associated with cell survival, migration and differentiation (tyrosine-protein kinase receptor TYRO3-like gene), triglyceride metabolism (carnitine O-palmitoyltransferase 1 gene), biosynthesis of p...
The paper reviews techniques of genome engineering and their potential applications in freshwater... more The paper reviews techniques of genome engineering and their potential applications in freshwater finfish aquaculture. In particular, banking of cryopreserved fish semen combined with androgenesis (both intra- and interspecific) are viewed as the most promising techniques enabling the researcher to protect and restore the gene pools of threatened (wild) and valuable (farmed) fish stocks.
The paper reviews techniques of genome engineering and their potential applications in freshwater... more The paper reviews techniques of genome engineering and their potential applications in freshwater finfish aquaculture. In particular, banking of cryopreserved fish semen combined with androgenesis (both intra- and interspecific) are viewed as the most promising techniques enabling the researcher to protect and restore the gene pools of threatened (wild) and valuable (farmed) fish stocks.
Although brook trout and the Arctic char hybrids are able to reproduce, individuals with decrease... more Although brook trout and the Arctic char hybrids are able to reproduce, individuals with decreased fertility or even fish that are unable to produce any gametes have been also described. Abnormal gonadal development and disturbances in the gamete production in the char hybrid offspring may be triggered by the odd chromosome number and disturbances in their pairing during meiosis. To verify this hypothesis, cytogenetic examination and the gonadal histology analysis of the brook trout x Arctic char hybrids were carried out. Diploid chromosome number in the studied char (F 1 ) hybrids varied from 82 to 84 (FN = 99-102). Among 28 hybrids, 12 males, three females, nine intersex individuals and two sterile specimens were described. In the case of two individuals, gonads were not found. Diploid chromosome numbers in the males and intersex individuals varied from 82 to 84. Chromosome numbers in the females were 82 and 83 chromosomes. Two sterile fish exhibited karyotypes composed of 82 and 84 chromosomes. Predominance of the ovarian component in the intersex gonads and gonadal sex ratio distortion towards the males suggested hybrid females had problems with gonadal differentiation. However, the lack of the clear relationship between chromosome number and gonadal development in the studied hybrids did not support our hypothesis that odd chromosome number may be responsible for such reproductive disturbances in the hybrid individuals. We have presumed that sterility and intersexual development of the gonads may be caused by interactions between brook trout and Arctic char genes on the sex chromosomes and autosomes rather than unpairing of the parental chromosomes.
The development of high resolution methods of chromosome banding helped the finding of homologous... more The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. P...
Cytogenetic or molecular identification of sex chromosomes could help in breeding studies in prod... more Cytogenetic or molecular identification of sex chromosomes could help in breeding studies in producing monosex fish stocks, estimating success of androgenesis, gynogenesis, etc. Among fish species sex chromosomes are recognizable in only a few cases. Some populations of rainbow trout Oncorhynchus mykiss show morphologically differentiated sex chromosomes. A strain from Rutki, Poland, showed a heteromorphic pair of subtelocentric chromosome: presumably of the XY type in the male and XX in the female. Restriction endonuclease and DAPI banding resulted in a characteristic banding pattern enabling identification of the X chromosome.
OCALEWICZ K. 2005. Identification of early and late replicating heterochromatic regions on platyf... more OCALEWICZ K. 2005. Identification of early and late replicating heterochromatic regions on platyfish (Xiphophorus maculatus) chromosomes. Folia biol. (Kraków) 53: 149-153.
A b s t r a c t . The karyotype of bream Abramis brama was analysed by means of C-banding, replic... more A b s t r a c t . The karyotype of bream Abramis brama was analysed by means of C-banding, replication banding, DAPI fluorescent staining and in situ hybridisation with 18S rDNA and telomeric probes. The use of the in vivo 5-bromodeoxyuridine incorporation technique enabled the induction of replication bands of the RBA type in the karyotype. C-bands corresponded to late replicated chromosome regions. 18S rDNA clusters were found on one chromosome pair. Telomeric sequences were observed only on ends of chromatids. The karyotype morphology and NOR phenotype of A. brama are very similar to those found in other species of the leuciscine cyprinids karyotyped so far.
Chromosome distribution of nucleolus organizer regions (NORs) and 5S rDNA loci were investigated ... more Chromosome distribution of nucleolus organizer regions (NORs) and 5S rDNA loci were investigated in platyfish (Xiphophorus maculates ) using conventional and molecular cytogenetic techniques. Chromomycin A 3 (CMA 3 ) staining of GC-rich heterochromatin provided four CMA 3 cytotypes in studied platyfish. As in fish, CMA 3 positive sites are related to NORs, chromosomes sequentially stained by CMA 3 and AgNO 3 showed bright blocks that were transcriptionaly active NORs. Additionally, PRINS with primers enabling amplification of 5S rDNA and CMA 3 staining revealed existence of 5S rDNA sites located close to GC rich regions. The analysis could suggests that minor and major rDNAs may be linked.
Failure of interspecific androgenesis between brook charr (Salvelinus fontinalis, Mitchill 1814) ... more 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.
ABSTRACT Karyotype and cytogenetic characteristics of the invasive round goby (Neogobius melanost... more ABSTRACT Karyotype and cytogenetic characteristics of the invasive round goby (Neogobius melanostomus (Pallas, 1814)) from the population inhabiting the Gulf of Gdansk, Baltic Sea, Poland were investigated using conventional and molecular cytogenetic techniques. The diploid chromosome number of all samples was 2n= 46 and the karyotype consisted of subteloto acrocentric chromosomes that gradually decreased in size. Analysis of chromatin resistance to restriction enzymes (AluI and DdeI) showed pericentromeric and telomeric blocks of heterochromatin. Nucleolus Organizer Regions (NORs) were observed in the pericentromeric regions of two medium-sized chromosomes. Sequential staining (DAPI, CMA3 and AgNO3) showed that the NORs are DAPI-negative and CMA3-positive in round goby chromosomes. Additionally, DAPI negative and CMA3-negative chromatin was identified in the interstitial position of two chromosomes. Fluorescent In Situ Hybridization (FISH) with a telomeric probe did not reveal any Interstitial Telomeric Repeat sequences (ITRs). No morphologically differentiated sex chromosomes were found in the round gobies studied. Considering the increased numbers of bi-armed chromosomes to be a derived condition in some ‘neogobiin’ species, the round goby karyotype of 46 mono-armed chromosomes, which are similar in both female and male cells and have GC-rich NOR-related regions located on the single chromosome pair, fits as an ancestral Neogobius karyotype pattern.
Specimens from two freshwater populations of the ninespine stickleback Pungitius pungitius in Pol... more Specimens from two freshwater populations of the ninespine stickleback Pungitius pungitius in Poland showed morphologically differentiated sex chromosomes. A heteromorphic pair of chromosomes appeared only in male diploid cells. The Y chromosome was the largest chromosome in the P. pungitius karyotype.
Uploads
Papers by Konrad Ocalewicz