RESUMEN
This study aimed to examine the effect of vitrification on the expression of genes that are crucial for porcine early embryo development; cathepsin B (CTSB), growth differentiation factor 9 (GDF9), caudal type homeobox 2 (CDX2), and OCT-4, which play an important role in the maintenance of embryonic cell pluripotency. Their gene expression was investigated in expanded blastocysts (day 6-7) derived from in vitro matured oocytes. The quantitative real-time PCR method was used to assess the amount of relative specific transcripts in 20 vitrified (treatment group) and 32 fresh non-vitrified (control group) blastocysts. Vitrification was performed using 7.5% dimethyl sulfoxide (DMSO) plus 7.5% ethylene glycol (EG), and in the final step, 15% DMSO plus 15% EG and a 0.5 M sucrose solution and cryotop as a vitrification device. The blastocysts were warmed in 1 M, 0.5 M, and 0.25 M sucrose solution and kept in a culture medium for six hours before their fixation and further qPCR analysis. A significant upregulation in the targeted genes CTSB (p<.006), GDF9 (p<.04), and CDX2 (p<.003) was observed in the vitrified embryos compared to the fresh control group. Interestingly, the OCT-4 mRNA expression level was not affected by vitrification and remained comparable to that of the fresh non-vitrified embryos. In summary, the results of this pilot study showed, that vitrification induced substantial alteration in the expression of CTSB, GDF9, and CDX2 genes but did not influence the expression of OCT-4 gene in porcine in vitro derived blastocysts. Our data on the expression of developmentally important genes in vitrified porcine blastocyst may facilitate: (1) future improvements in culture conditions and/or cryopreservation protocol and (2) understanding the mechanism(s) of cryoinjuries inducing compromised post-thaw embryo development followed by the poor pregnancy outcome after blastocyst transfer.
Asunto(s)
Dimetilsulfóxido , Vitrificación , Animales , Blastocisto/fisiología , Criopreservación/métodos , Dimetilsulfóxido/metabolismo , Dimetilsulfóxido/farmacología , Glicol de Etileno/metabolismo , Glicol de Etileno/farmacología , Femenino , Oocitos , Proyectos Piloto , Embarazo , Sacarosa/metabolismo , Sacarosa/farmacología , PorcinosRESUMEN
The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes. The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium. In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.
Asunto(s)
Criopreservación/métodos , Oocitos , Vitrificación , Animales , Calcio/farmacología , Catepsina B/genética , Bovinos , Femenino , Fertilización In Vitro/métodos , Folistatina/genética , Expresión Génica , Técnicas de Maduración In Vitro de los Oocitos , Zona Pelúcida/fisiologíaRESUMEN
Maternal effect genes (MEG) play a crucial role in early embryogenesis. In vitro culture conditions may affect MEG expression in porcine oocytes and embryos. We investigated whether in vitro culture medium supplementation with epidermal growth factor (EGF), IL-1ß or LIF (leukemia inhibitory factor) affects the mRNA level of ZAR-1 (zygote arrest 1), NPM2 (nucleoplasmin 2) and DPPA3 (developmental associated protein 3) in porcine MII oocytes and embryos. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium (control) or in NCSU-37 with EGF 10 ng/ml, IL-1ß 10 ng/ml or LIF 50 ng/ml. After maturation for 44-46 h, MII oocytes were preserved for the analysis of MEG mRNA levels (experiment 1). In experiment 2, COCs were fertilized, and the presumptive zygotes were cultured in the same groups. Then, 2-, 4-, 8-cell embryos, morulae and blastocysts were collected for the analysis of MEG mRNA levels. LIF addition to the maturation medium increased MII oocyte numbers (P < 0.05), while EGF and IL-1ß did not affect oocyte maturation. Medium supplementation with EGF resulted in lower DPPA3 mRNA levels in MII oocytes and in 2- and 4-cell embryos versus control embryos (P < 0.05). LIF treatment increased DPPA3 mRNA levels in morulae and blastocysts (P < 0.05). Culture with EGF and IL-1ß decreased ZAR-1 and NPM2 mRNA levels in 2-cell embryos (P < 0.05). The inclusion of EGF or IL-1ß in the porcine in vitro production system influences ZAR-1, NPM2 and DPPA3 mRNA in MII oocytes and embryos but not beyond the 4-cell stage. LIF stimulates oocyte maturation and affects DPPA3 mRNA in porcine morulae and blastocysts in vitro.
Asunto(s)
Proteínas del Huevo/metabolismo , Embrión de Mamíferos/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Interleucina-1beta/farmacología , Factor Inhibidor de Leucemia/farmacología , Metafase/fisiología , Oocitos/metabolismo , Animales , Proteínas del Huevo/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fertilización In Vitro/veterinaria , Fármacos Gastrointestinales/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Metafase/efectos de los fármacos , Nucleoplasminas/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , PorcinosRESUMEN
The purpose of this retrospective study was to establish a prognosis for implantation, pregnancy and live birth rates in stimulated IVF cycles after transferring embryos derived from: 1/ retrieved immature oocytes that matured overnight in vitro (late mature group: LM); 2/ retrieved immature oocytes that matured overnight in vitro and were added to the embryos derived from retrieved mature oocytes (mixed embryos group: MX); and 3/ retrieved mature oocytes (mature group: M). The obtained implantation, clinical pregnancy and live birth rates for the LM group were: 5.6%, 11.4%, 11.4%; for the MX group were: 4.2%, 14.6%, 11.6%; and for the M group were: 14.6%, 45.2% and 33.3%, respectively. These measurements were significantly lower p<0.05 for the LM and MX groups in comparison to the M group. The number of oocytes retrieved and the number of embryos transferred were the lowest (p<0.001-0.05) for the LM group. It is concluded, that the retrieved immature oocytes are able to mature during overnight culture in vitro, be fertilized and provide developmentally competent embryos with the prognosis of 11% for the successful delivery.
Asunto(s)
Transferencia de Embrión/métodos , Fertilización In Vitro/métodos , Recuperación del Oocito/métodos , Oocitos/crecimiento & desarrollo , Resultado del Embarazo/epidemiología , Análisis de Varianza , Femenino , Humanos , Embarazo , Pronóstico , Estudios RetrospectivosRESUMEN
Controlled ovarian stimulation has become an integral part of infertility treatment. Specific gonadotropin based protocols become the main strategies for controlled stimulation. To avoid the potentially detrimental effect of premature LH surge on oocytes and/or endometrium development, the GnRH analogs have been incorporated into controlled ovarian stimulation strategies. With the availability of recombinant gonadotropins (i.e. recombinant FSH devoided of LH activity) it is necessary to establish precise role of LH in the folliculogenesis and endometrium development. The benefit of exogenous LH may vary with the GnRH-agonists and antagonists regiment used. The optimal amount of LH or ratio FSH to LH used during therapeutically stimulated growth of follicles is still a problem that needs to be solved in the near future.