RESUMO
This study evaluated the effect of adding alpha lipoic acid (ALA) to the vitrification solution of sheep ovarian tissue on 7 days of in vitro culture or 15 days of xenotransplantion. ALA was used at two different concentrations (100 µM: ALA100 and 150 µM: ALA150). Ovarian tissue was evaluated by classical histology (follicular morphology, development, and stromal cell density); immunohistochemistry for forkhead box O3a (FOXO3a); Ki67 (cell proliferation); cluster of differentiation 31 (CD31); and alpha smooth muscle actin (α-SMA). Reactive oxygen species (ROS) levels in ovarian tissue, as well as malondialdehyde (MDA) and nitrite levels in the culture medium, were assessed. Similar percentage of morphologically normal follicles was found in the vitrified ovarian tissue in the presence of ALA100 or ALA150 after in vitro culture or xenotransplantation. Follicular development from all treatments was higher (P < 0.05) than the control group. Moreover, an activation of primordial follicles was observed by FOXO3a. Stromal cell density and immunostaining for Ki67 and CD31 were significantly higher (P < 0.05) in ALA150 vitrified tissue. No difference (P > 0.05) was found in α-SMA between ALA concentrations after in vitro culture or xenograft. ROS levels in the ovarian tissue were similar (P > 0.05) in all treatments, as well as MDA and nitrite levels after 7 days of culture. We concluded that the addition of ALA 150 is able to better preserve the stromal cell density favoring granulosa cell proliferation and neovascularization.
Assuntos
Antioxidantes/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/transplante , Ácido Tióctico/farmacologia , Transplante Heterólogo/métodos , Vitrificação/efeitos dos fármacos , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Folículo Ovariano/fisiologia , Ovário/efeitos dos fármacos , Ovário/fisiologia , Ovário/transplante , OvinosRESUMO
Background: After artificial insemination and multiple ovulation and embryo transfer (MOET), in vitro production of embryos (IVP) represents the third generation of techniques aimed at a better control of animal reproduction. This technique involves four major steps: oocyte collection, oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro development of the resulting embryos (IVD). These different steps are now well established in domestic ruminant species (cattle, sheep and goat) although the variability of the number and quality of the oocytes collected and the low viability of frozen thawed in vitro produced embryos still limit the large-scale use of this promising technology. Beyond the potential use of IVP in breeding schemes, this technique is also required for the establishment of new biotechnologies such as cloning and transgenesis. Additionally, the knowledge of oocyte and embryo physiology acquired through IVP techniques may stimulate the further development of other techniques such as marker assisted and genomic selection of preimplantation embryos and also benefit to assisted procreation in human being. This paper will discuss the possible function of maternal environment in the regulation of early development and the consequences of these functions for IVP, in view to improve IVP embryos viability. Review: Comparisons between in vivo and in vitro produced embryos pointed out several differences in morphology, metabolism and gene expression. IVP embryos have a modified lipid metabolism, resulting in increased triglycerids accumulation, translating into different density. This altered lipid metabolism may account for differences in membrane structure and increased sensitivity to oxidative stress, resulting in lower cryoresistance of these IVP embryos. The identification of modified metabolic pathways leading to these lipidic disorders will provide clues for modification of culture conditions in view to restore normal lipid metabolism through appropriate precursors supplementation of the media. The natural embryo environment from fertilization to blastocyst stage is the oviduct. In vivo, oviduct epithelial cells provide ideal development support by regulating physico-chemical embryo microenvironment. Under in vitro conditions, the lack oviduct support may result in embryo exposure to toxic metabolites and oxidative stress. In addition, in vitro developing embryos may lack oviduct originated embryotrophic factors that regulate and stimulate early development in vivo. The use of co culture systems to mimic natural embryo environment in vitro may allow to improve embryo development, restore normal metabolic parameters, and increase embryo viability and cryoresistance. In addition, such co culture systems involving oviduct epithelial cells will help to identify critical development parameters and to point out potential embryotrophic factors. Conclusion: In vitro embryo production is a promising technique for improvement of selection schemes and diffusion of genetic gain through safe exchanges of embryos. To allow a lager use of this technology, improvements should be obtained in management of oocyte collection and in vitro treatment to improve its quality and in embryo in vitro development systems. A better knowledge of interactions between the developing embryo and maternal environment will allow to improve in vitro systems to produce high viability embryos.