IGF-I Medium Supplementation Improves Singly Cultured Cat Oocyte Maturation and Embryo Development In Vitro.

Embryo production is a routine procedure in several species. However, in felids, the effectiveness of this approach is far behind that in the majority of laboratory species. The development of a suitable environment starts with the proper composition of culture media. Therefore, for the improvement of assisted reproduction techniques and their outcome in cats, this is an urgent task. As the addition of insulin-like growth factors (IGF-I, IGF-II) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was beneficial in other mammalian species, this study aims to check whether these components, combined with other factors (such as type of fertilisation or type of culture) can provide a benefit in the felid culture system in current use. Thus, these supplements, in different concentrations and combinations, were merged with the use of two fertilisation techniques and randomly assigned to single or group culturing. The results showed that the addition of IGF-I and/or GM-CSF produced an increase in morula and blastocyst rate in a single culture system. In particular, the supplementation with 20 ng/mL of IGF-I incremented the maturation rate by 10% and significantly increased the morula and blastocyst rates in single culturing. This result is especially remarkable for wild felids, where only a few oocytes and/or embryos are available.

Deuterated stearic acid uptake and accumulation in lipid droplets of cat oocytes.

Fatty acid uptake and accumulation in lipid droplets are essential processes of lipid metabolism. Oocyte in vitro culture in media enriched with fatty acid is used to modify the lipid content and composition, aiming to study the consequences of obesity and enhance cell cryotolerance. We applied Raman spectroscopy and deuterium labeling approach to quantify stearic acid uptake and investigate its incorporation within oocytes. Our data suggest that deuterium labeling does not affect oocyte maturation rates. The efficiency of deuterated stearic acid (dSA) uptake was shown to decrease with the increase of its concentration in culture medium and the duration of in vitro culture. The molar ratio between dSA and bovine serum albumin has no significant effect on the dSA uptake for 200 µM but modifies concentration dependence of the lipid uptake. dSA accumulates in all the lipid droplets inside oocytes. Different lipid droplets within the same oocyte exhibit different concentrations of dSA. The scatter in the dSA concentration in lipid droplets decreases with the culture time. Using dSA as an example, we provide a comprehensive description of how fatty acid concentration, its molar ratio versus bovine serum albumin, and culture time affect the uptake of the fatty acids in oocytes. Raman microspectroscopy of deuterium-labeled fatty acids is a nondestructive tool providing information about fatty acid uptake and heterogeneity of their accumulation between lipid droplets within the single oocyte.

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis.

BACKGROUND: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. METHODS: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. RESULTS: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. CONCLUSIONS: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis

BACKGROUND: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. METHODS: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. RESULTS: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. CONCLUSIONS: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Dynamic changes in mitochondrial DNA, distribution and activity within cat oocytes during folliculogenesis.

Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial (n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral (n = 44), early antral (n = 66), small antral (n = 59), antral (n = 41) and advanced antral (n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mtDNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes.