RESUMEN
The LEPR gene encodes a leptin hormone receptor, and its mutations are associated with morbid obesity, dysregulation of lipid metabolism, and fertility defects in humans. Spontaneous Lepr mutations have been described in rodents, and Lepr knockout animals have been generated, in particular, using the CRISPR/Cas9 system. Lipid metabolism in rodents significantly differs from that in humans or rabbits, and rabbits are therefore considered as the most relevant model of morbid obesity and lipid metabolism dysregulation in humans. LEPR knockout rabbits have not been reported so far. In this work a LEPR knockout rabbit was generated by introducing a deletion of the region around LEPR exon 10 using the CRISPR/Cas9 system. The body weight of the knockout rabbit was significantly higher than the average body weight of the wild type rabbits. CRISPR/Cas9-mediated generation of LEPR knockout rabbits will allow the development of a model of morbid obesity and endocrine defects due to leptin receptor mutations in humans.
RESUMEN
Reference gene selection in mouse oocytes is an important task required to perform further adequate analysis of target gene expression levels. In the current work we have analyzed expression stability of the seven most commonly used reference genes (Actb, Eef1e1, Gapdh, H2afz, Ppia, Rpl4 and Ubc) in mouse oocytes at the germinal vesicle (GV) stage. We have performed analysis of expression stability of the above-mentioned reference genes with the three most commonly used software tools: geNorm, BestKeeper and NormFinder. Taking into account the results obtained from all of these programmes Gapdh, Rpl4 and H2afz seem to be suitable candidate reference genes in GV oocytes of mouse.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Oocitos/metabolismo , Oogénesis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Ratones , Oocitos/citología , Factores de Elongación de Péptidos/toxicidad , Estándares de Referencia , Proteínas Ribosómicas/genética , Proteínas Supresoras de Tumor/toxicidadRESUMEN
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.
Asunto(s)
Criopreservación , Regulación del Desarrollo de la Expresión Génica/genética , Ovario/metabolismo , Proteínas Ribosómicas/genética , Adulto , Femenino , Perfilación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Humanos , Ovario/crecimiento & desarrollo , Estándares de Referencia , VitrificaciónRESUMEN
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.
Asunto(s)
Criopreservación/métodos , Preservación de la Fertilidad/métodos , Ovario/fisiología , Técnicas de Cultivo de Tejidos/métodos , Femenino , Humanos , Trasplante de Órganos/métodos , Ovario/trasplanteRESUMEN
This review describes the main factors affecting the in vitro development of mouse ovarian follicles under conditions of three-dimensional alginate hydrogel system. The factors discussed include concentration of alginate hydrogel, presence of additives (collagen, fibrin) influencing substrate rigidity; culture conditions; composition of culture media; substances that act like antioxidants (salts of ascorbic acid, glutathione) and contribute to the improvement of lipid metabolism (L-carnitine), hormones and growth factors. The methods for follicle group cultivation in alginate hydrogel and cocultivation of different cell populations with follicles encapsulated in alginate hydrogel are covered in the present article.