Cryopreservation of mature oocytes can result in damage to the metaphase spindle due to the temperature sensitivity of microfilaments and microtubules. Cryopreservation of immature oocytes may circumvent this problem because these structures have not formed yet and the genetic material is enclosed within a nuclear envelope. Because intact oocyte cumulus-oocyte complexes (COC) are essential for normal maturation, we chose to cryopreserve immature intact COC. The aim of this study was to determine if immature COC cryopreserved by slow rate freezing or vitrification would resume meiosis upon thawing. In 2 separate experiments, immature COC (n = 102 and 79) were collected from cross-bred cattle by transvaginal ultrasound-guided aspiration and divided into 2 groups. For both experiments, the first group (n = 64 and 40) was placed directly into maturation medium (TCM 199 supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 2 mM glutamine, and 5 μg mL–1 of FSH) and cultured for 22 h under 5% CO2 in air atmosphere at 39°C. In experiment 1, COC (n = 38) in the second group were cryopreserved by a slow rate freezing protocol. The COC were equilibrated for 5 min in 1.5 M ethylene glycol (EG), and 5 COC were loaded into 0.25-mL straws and placed into the cooling chamber of a Freeze Control unit at –6°C. After 5 min, straws were seeded, then cooled at 0.5°C per min to –35°C before plunging into LN. After storage in LN for 5 days, straws were removed from LN, thawed by placing straws in a 35°C water bath, and COC put into maturation as described above. In experiment 2, COC (n = 39) in the second group were cryopreserved by vitrification using a 3-step procedure. The COC were equilibrated in solutions consisting of 10% glycerol then 10% glycerol and 20% EG in PBS for 5 min each. The COC were then placed in a solution of 24% glycerol and 26% EG, and 1 to 3 COC were placed onto a cryotop device in minimal medium and plunged into LN within 45 s. After storage in LN for 2 days, COC were thawed by placing the cryotop device directly into a warmed dilution solution consisting of 0.5 M galactose in PBS. After 5 min in the dilution solution, COC were put into maturation as described above. For all groups, after 22 h of maturation cumulus cells were stripped from the oocytes by vortexing and oocytes were placed on slides for fixation in methanol acetic acid and stained with 1% orcein to determine the nuclear stage. In experiment 1, oocytes cryopreserved by slow rate freezing matured to MII at the same rate as control oocytes (45 v. 55%, P = 0.44). In experiment 2, oocytes cryopreserved by vitrification matured to MII at a lower rate than controls (49 v. 79%, P = 0.01). These results show that cryopreservation of immature intact COC is a viable alternative to cryopreservation of mature oocytes. Further studies are needed to optimize either slow rate freezing or vitrification of intact COC and determine the developmental competence of cryopreserved oocytes following fertilization.
Synthetic RNA transfection has been an invaluable tool in understanding the mammalian genome because of its ability to deliver exogenous protein without mutagenic effects caused by double-stranded DNA. A common problem associated with the introduction of exogenous mRNA into mammalian cells is the stimulated interferon response. This innate immune response can be avoided with the addition of modified bases during the in vitro transcription process of synthetically derived mRNA. The bases cytidine triphosphate (CTP) and uridine triphosphate (UTP) are replaced with 5-methylcytidine-5′-triphosphate (5-Methyl-CTP) and pseudouridine-5′-triphosphate (Pseudo-UTP) during in vitro transcription. Cellular reprogramming is achieved by the delivery of this mRNA into the cytoplasm. Previous cellular reprogramming experiments lacking modified bases resulted in increased toxicity and a decrease in cellular viability, which lead to the incorporation of modified bases. In the first experiment, bovine fetal fibroblasts were transfected with modified synthetic mRNA encoding green fluorescent protein (GFP) to evaluate the effects on cellular viability and fluorescence. The cellular viability was measured by counting a final number of cells after seeding a constant number of cells in all treatment groups. The control group consisted of bovine fetal fibroblasts cultured in normal growth medium. A no-RNA (NR) group was held under the same conditions with the addition of the transfection reagent, Lipofectamine (Invitrogen, Carlsbad, CA, USA), to account for toxicity resulting from the transfection reagent alone. The cells were transfected every other day for 12 days and were evaluated on days 3, 6, 9, and 12 for viability and fluorescence by flow cytometry. There was no difference in viability of all cells treated with synthetic mRNA encoding GFP when compared to controls (P = 0.9). There was a significant difference in fluorescence on all time points when compared to controls (Day 3, P = 0.004; Day 6, P = 0.004; Day 9, P = 0.007; Day 12, P = 0.04). The second experiment consisted of bovine fetal fibroblasts transfected with modified synthetic mRNA encoding pluripotency factors. The controls were identical to the previous experiment, but treatment groups were transfected with modified synthetic mRNA encoding either three factors (3F: OCT4, SOX2, KLF4) or four factors (4F: OCT4, SOX2, KLF4, c-MYC). The treated cells were transfected every other day and evaluated on Day 24 for cellular viability. There was no difference in cellular viability in all treatment groups when compared to controls (P = 0.2). The introduction of synthetic mRNA containing modified bases maintains cellular viability when compared to controls. The decreased immune response by the inclusion of modified bases may be advantageous in a variety of applications from the introduction of transcription activator-like effector nuclease (TALEN) or zinc finger nucleases for genomic editing to increased efficiency of the development of induced pluripotent stem cells.
In vitro maturation (IVM) is a reproductive technique critical to in vitro embryo production (IVP). Currently, IVP has low efficiency due to an inadequate IVM system where premature meiotic resumption results in low oocyte viability. Meiotic arrest is regulated primarily by 3′,5′-cyclic adenosine monophosphate (cAMP). Some successful methods of improving IVM have utilised cAMP modulators to maintain high intraoocyte cAMP, delaying the onset of nuclear maturation and allowing cytoplasmic maturation to occur. The current experiment is a follow-up to previous work in which an extended 2-step maturation system was examined. In the previous experiments, we found that meiotic resumption was significantly delayed, but the overall maturation rates of extended IVM were about half those of standard IVM, suggesting that the effects of the modulators on the oocytes were not completely reversible. The current experiment compares cAMP concentrations of oocytes in this extended IVM to standard IVM to determine whether high cAMP is the cause of the low maturation rate. Bovine oocytes (n = 686) were obtained from mixed-breed cattle by transvaginal ultrasound-guided aspiration. Oocytes from each cow were divided into 2 groups: standard IVM and extended IVM. Standard IVM consists of a 23-h maturation composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, pen/strep, glutamine, and FSH, and cultured at 39°C in 5% CO2. Extended IVM consists of 2 steps: a pre-IVM phase composed of HEPES-TALP supplemented with 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methylxanthine (IBMX) for 2 h at 39°C, followed by an extended IVM phase composed of standard IVM media supplemented with 20 µM cilostamide for 31 h (39°C, 5% CO2). Additionally, oocytes in the extended IVM treatment group where held in HEPES-TALP media with FSK and IBMX during the 2-h oocyte collection period in order to prevent any decrease in cAMP before the oocytes could be placed in the extended IVM media. Oocytes in standard IVM were sampled at 0, 8, and 23 h of maturation, while oocytes in extended IVM were sampled at 0, 8, 18, and 33 h of maturation. Cumulus cells were removed from all oocytes by vortexing in hyaluronidase solution. Oocytes were stored in groups of 10 at –80°C. A cAMP enzyme immunoassay (GE Healthcare) was performed to determine cAMP concentrations throughout standard and extended IVM. Assay results were analysed using an ANOVA followed by a Tukey's pairwise test (Sigma Stat 3.5) to detect significant differences (P < 0.05). Results indicated significantly higher cAMP levels in extended IVM oocytes during the first 3 h after collection using FSK and IBMX in the holding media (0.505 v. 1.006 fmol/oocyte, P = 0.035) but cAMP levels were not maintained in the cilostamide-only extended IVM medium. This suggests that high cAMP levels were not the cause of low maturation rates in extended IVM, since cAMP concentrations did decrease after 3 h. Possible negative effect of cilostamide on these oocytes may be suggested and need to be analysed.
The efficiency of in vitro production (IVP) has remained low due to an inadequate in vitro maturation (IVM) system. Thus far, the most promising methods of improving IVM utilise cAMP modulators to slow the maturation process by keeping cAMP high. This experiment compares standard IVM to an extended IVM as previously described by Albuz (2010 Hum. Reprod. 25, 2999–3011). The extended IVM consists of a 2-h pre-IVM phase with FSK (an adenylate cyclase activator) and 3-isobutyl-1-methylxanthine [IBMX, a nonspecific phosphodiesterase (PDE) inhibitor], followed by a 31-h IVM phase with cilostamide (a PDE3 inhibitor). 3-Isobutyl-1-methylxanthine inhibits all PDE in both the oocyte and CC, whereas cilostamide inhibits PDE3 only in the oocyte, allowing a gradual reversal of inhibition. Bovine oocytes (n = 363) were obtained by transvaginal ultrasound-guided aspiration of both Brahman and Angus cattle over 4 collection days. Oocytes from each cow were divided into 2 groups. The first group was placed in standard 23-h IVM medium composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, penicillin and streptomycin, glutamine, and FSH, and cultured in 5% CO2 at 39°C. A subset of oocytes was removed from maturation at 8, 13, 18, and 23 h. The second group was placed into a pre-IVM medium of HEPES-TALP supplemented with 100 μM FSK and 500 μM IBMX for 2 h at 39°C, and then moved into standard maturation medium supplemented with 20 μM cilostamide for 31 h (5% CO2, 39°C). Oocytes were sampled at 8, 13, 18, 23, 28, and 33 h. Oocytes were stained with 1% orcein and nuclear status was examined for each sample time (Table 1). Data were analysed using a chi-squared test. At 8 h, there was a significant difference (P < 0.001) between GV stage of IVM and extended IVM (7.1 v. 73.2%), and between metaphase I (MI) stage IVM and extended IVM (76.2 v. 4.9%). At the 23-h time sample, there was a significant difference between metaphase II (MII) IVM and MII extended IVM (78.9 v. 30.6%; P < 0.001). There was also a significant difference between MII oocytes at 23 h IVM and MII oocytes at 33 h extended IVM (78.9 v. 33.3%; P < 0.001). These results are consistent with the hypothesis that cAMP modulators slow the nuclear maturation process. However, results also suggest that the inhibitory effect of the cAMP modulators is not completely reversible. Oocytes appear to arrest at MI by 23-h extended IVM and do not progress to MII at the same rate as standard IVM. Table 1.Nuclear status of bovine oocytes after standard IVM and extended IVM with cAMP modulators
Reduced tolerance to chilling and cryotolerance of oocytes and embryos has been associated with greater cytoplasmic lipids. Previous studies in the cow have demonstrated nutrition-induced modification of follicular components. trans-10, cis-12 conjugated linoleic acid (CLA) was identified as a potent inhibitor of milk fat synthesis in lactating cows, and inclusion of CLA in bovine embryo culture medium reduced embryo lipid content and improved post-thaw embryo survival. Dietary supplementation of cows with CLA could alter oocyte fatty acid metabolism, oocyte lipid composition, and embryo cryotolerance, and responses may be different between Bos indicus and Bos taurus cattle. Therefore, a series of experiments were conducted to evaluate effects of dietary CLA on bovine oocyte lipid content and lipid metabolism and cryosurvival of in vitro-produced embryos from CLA-supplemented oocyte donor cows. Lactating Holstein cows (n = 39) were supplemented 100 g per head/d CLA or Ca salts of palm oil to determine effects of dietary CLA on milk production and milk composition. Nonlactating Holstein (n = 8) and Brahman (n = 17) cows were individually supplemented with 150 g per head/d CLA or no lipid supplement. Cumulus-oocyte complexes (COC) were collected after 41 ± 1 day of CLA supplementation, and mRNA was isolated for qPCR. Relative gene expression in COC from CLA- and control-fed cows was evaluated for genes involved in lipid metabolism (CPT1, FADS2, and PPARα). Crossbred (Angus × Red Angus × Brangus) cows (n = 28) were randomly allotted to a 2 × 2 factorial experiment and fed 150 g per head/d CLA or no lipid supplement. Oocytes were collected (Day 129 and 143 of CLA supplementation), matured, fertilized, and cultured in vitro for 7 days in serum-free culture medium. Embryos were cryopreserved in individual 0.25-mL plastic straws containing 1.5 M ethylene glycol using a slow-cooled method. Post-thaw survival and hatching were evaluated using a 24-h in vitro culture (mSOF with 5% FBS) assay. Milk yield, milk composition, and Nile Red intensity were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). Follicle and oocyte responses were analysed with the Mixed procedure of SAS. Relative gene expression of COC was evaluated using the REST 2009 Software. In vitro embryo production, post-thaw survival, and hatching rates were analysed using Chi Square. Milk fat was depressed (P < 0.001) by 10.1% in lactating Holstein cows fed CLA. Dietary supplementation of Holstien and Brahman cows with CLA did not alter expression of genes (CPT1, FADS2, and PPARα) in COC. Dietary supplementation of crossbred cows with CLA before oocyte collection did not influence cryotolerance of in vitro-produced embryos. Lipid content of oocytes (measured by Nile Red florescence) and follicle, oocyte, and embryo production was not influenced by CLA supplementation of Holstein and Brahman cows. The highly regulated mechanisms involved in fatty acid uptake by ovarian components may help explain the lack of ovarian response to dietary CLA in the current study.
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