RESUMO
PURPOSE: To select reference genes with stable messenger RNA (mRNA) expression for quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis of vitrified/thawed human ovarian tissue and to evaluate in human ovarian tissue the levels of key proteins which are commonly used as reference proteins. METHODS: Pieces of ovarian tissue were obtained during laparoscopy from patients (n = 10, 24-36 years old) who suffered from types of cancer that does not affect reproductive system. Tissue strips from the intact group were immediately placed into liquid nitrogen. Tissue strips from the second group were successively placed into solutions with cryoprotective agents. Then, these strips were rapidly placed into liquid nitrogen. After thawing, ovarian tissue strips were cultured during 2 h in complete growth medium. Gene expression levels were measured using quantitative RT-PCR. Also, protein levels of three key reference genes were measured using Western blot. Statistical analysis of obtained data was performed by BestKeeper, NormFinder, and geNorm software utilities; correlation coefficients were also calculated. RESULTS: The most suitable reference genes for qRT-PCR analysis of human cortical ovarian tissue after cryopreservation by vitrification are genes of ribosomal proteins RPL4, RPLP0, RPS18, and heat shock protein HSP90AB1. The protein levels of three commonly used reference genes (ACTB, GAPDH, and HSP90) were measured in two groups of samples of human ovarian tissue: intact and vitrified/thawed. The levels of ACTB, GAPDH, and HSP90 proteins were similar in native and vitrified/thawed samples. CONCLUSION: Selection of suitable reference genes is the first aim of any research dedicated to the investigation of gene expression, because the interpretation of obtained results largely depends on selection of appropriate reference genes. Nowadays, there are many mathematical approaches allowing to select not only single reference gene but also a group of the most stably expressed reference genes. The use of mathematical models which take into account multiple reference genes will allow to obtain more accurate data on the expression of target genes.
Assuntos
Criopreservação , Regulação da Expressão Gênica no Desenvolvimento/genética , Ovário/metabolismo , Proteínas Ribossômicas/genética , Adulto , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Humanos , Ovário/crescimento & desenvolvimento , Padrões de Referência , VitrificaçãoRESUMO
The results of a complex morphofunctional study with markers of proliferation (PCNA and Ki-67), angiogenesis (CD31 and CD34), and structural integrity of mesenchymal cells (vimentin) suggest that the pool of primordial follicles was well preserved in vitrified ovarian tissue and that viability of its cellular components can be rapidly restored during incubation at 37°C within 4 h after thawing.
Assuntos
Criopreservação/métodos , Folículo Ovariano/citologia , Ovário/citologia , Vitrificação , Adulto , Antígenos CD34/genética , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Sobrevivência Celular , Crioprotetores/farmacologia , Culdoscopia , Feminino , Expressão Gênica , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Pessoa de Meia-Idade , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Técnicas de Cultura de Tecidos , Vimentina/genética , Vimentina/metabolismoRESUMO
In the present review, the main strategies of female fertility preservation are covered. Procedures of fertility preservation are necessary for women who suffer from diseases whose treatment requires the use of aggressive therapies, such as chemotherapy and radiotherapy. These kinds of therapy negatively influence the health of gametes and their progenitors. The most commonly used method of female fertility preservation is ovarian tissue cryopreservation, followed by the retransplantation of thawed tissue. Another approach to female fertility preservation that has been actively developed lately is the ovarian tissue in vitro culture. The principal methods, advantages and drawbacks of these two strategies are discussed in this article.
Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Ovário/fisiologia , Técnicas de Cultura de Tecidos/métodos , Feminino , Humanos , Transplante de Órgãos/métodos , Ovário/transplanteRESUMO
No neutron dose rate effects in the wide range of 10(-3) Gy/s to 10(6) Gy/s were observed in yeast diploid cells for induction of mitotic segregation and crossing-over. The RBE values for these effects were determined as doses ratio (Dgamma/D(n)) at maximum effects. The RBE were 2.2-1.9 for neutrons of the reactor BR-10 (E = = 0.85 MeV) and the pulse reactor BARS-6 (E = 1.44 MeV). The RBE values for genetic effects were 1.0 at the equal survival level for neutrons and gamma-rays 60Co.
Assuntos
Nêutrons , Saccharomyces cerevisiae/efeitos da radiação , Segregação de Cromossomos/efeitos da radiação , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/efeitos da radiação , Troca Genética/efeitos da radiação , Diploide , Relação Dose-Resposta à Radiação , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
We report here a comparative analysis of RBE for lethality of a single pulse (duration 65 micros) of fast neutron with ultra high dose rates (up to 6 x 10(6) Gy/s) and continuous neutron radiation (3.6 x 10(3) s) of the pulse reactor BARS-6. Three diploid strains, one haploid strain and three diploid repair-deficient strains (rad52-1/rad52-1; rad54/rad54; rad2/rad2) were used. The RBE values (D(0gamma)/1D(0n)) of a single pulse and continuous neutron irradiation were equal (1.7-1.8) with maximum RBE (4.1-3.1) in region of low doses (shoulder region). Haploid cells were found to be more (3 times) sensitive to both gamma-rays and neutrons than the wild type. There was no obvious decrease in the RBE of 1.9 in highly sensitive haploid cells as compared with highly resistant diploid cells. The repair-deficient strains (rad52-1/rad52-1; rad54/rad54) were more (up to 10 fold) sensitive to both neutrons and gamma-rays as compared with their parent line. The RBE values of 1.5-1.7 of neutrons for these mutants (independent by of the mode of irradiation) were found. The repair-deficient mutant rad2/rad2 had similar sensitivity as a wild type and a RBE value was 2.0. We have concluded that biological effectiveness of the neutrons of pulse reactor BARS-6 was independent of the dose-rate, differing up to 10(8) fold. The RBE didn't vary significantly with the capacity of cells to repair DNA damages.
Assuntos
Reparo do DNA/genética , Nêutrons Rápidos , Tolerância a Radiação/genética , Saccharomyces/efeitos da radiação , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Radiação Ionizante , Saccharomyces/genéticaRESUMO
The relative contribution of photo- and non-photoreactivable damages to the lethal effect of far-(250 nm) and mid-(313 nm) wave UV in isogenic bacterial cells Escherichia coli WP2 (wild type, uvrA and recA mutants) was estimated. It has been demonstrated that the value of non-photoreactivable damages increases with lambda of UV (250----313 nm) and depends on the genotype (uvrA and recA).
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Genes Bacterianos/efeitos da radiação , Mutação , Recombinases Rec A/genética , Raios Ultravioleta , Proteínas de Bactérias/efeitos da radiação , Escherichia coli/efeitos da radiação , Genótipo , Recombinases Rec A/efeitos da radiaçãoRESUMO
A relative contribution of photoreactivated (modified by visible light) and non-photoreactivated (modified by temperature) damages to UV-irradiated (250-334 nm) E. coli B cells was estimated. The contribution of damages modified by temperature to a lethal effect of UV-radiation was invariable within the range from 250 to 334 nm. The photoreactivation of E. coli B cells was also independent of lambda-inactivating UV-light within 250-313 nm, and its value exceeded that of the wild-type E. coli WP2 which did not vary by the mode of UV-damages repair. Moreover, in contrast to E. coli B. cells, the value of the photoreactivation of E. coli WP2 decreased, as lambda-inactivating UV-light increased from 250 to 313 nm.
