Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of differentiated cells as the donor karyoplast. Blastocyst production and development to term of cloned embryos has been hypothesized to differ between population doublings of the same cell line as a consequence of changes in the levels of DNA methyltransferase 1 (DNMT1) and methylated DNA during in vitro culture. The objective of this study was to determine embryo production, developmental potential, and gene expression patterns of prehatched and posthatched embryos generated using donor cells with different levels of DNMT1 transcript. Day 7 embryos generated using donor cells with high and low levels of DNMT1 mRNA were transferred to recipient cows. Embryos recovered on Day 13 were morphologically characterized or used for gene expression analysis of DNMT, INFT, and MHC1. A higher proportion of 8- to 16-cell embryos developed to the blastocyst stage when cells with low levels of DNMT1 mRNA were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of DNMT1 mRNA and capable of developing beyond the 8- to 16-cell stage produced a larger number of apparently developing embryos, larger conceptuses, and a higher expression of DNMT3A transcript than NT embryos reconstructed using cells with high levels of DNMT1 mRNA. However, abnormal gene expression of DNMT, INFT, and MHC1 was noted in the majority of cloned embryos, indicating inefficient nuclear reprogramming and retarded embryo development. Furthermore, aberrant DNMT1 expression may partially contribute to the inefficient nuclear reprogramming observed in cloned embryos.
Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.
Bos indicus embryos have a lower survival rate compared with Bos taurus after cryopreservation. It has been hypothesized that the lower survival rate is due to higher intracellular lipid content. The objective of this study was to determine if there is a difference in intracellular lipid content of oocytes from mature purebred Brahman and Angus cows. Donor females used in the study were maintained on pasture prior to the onset of the experiment and on a grain-supplemented hay ration during the study. Oocytes were collected from cows at 30-day intervals (3 aspirations/donor) by transvaginal ultrasound-guided oocyte aspirations (TUGA). Mature oocytes were evaluated using a sucrose step gradient procedure and Nile Red staining. FSH (Folltropin-V®; Bioniche Animal Health, Beltsville, Ontario, Canada) administration began on Day 4 of the estrous cycle (estrus = Day 0) twice daily for 3 days in decreasing doses (Brahman 232 mg and Angus 280 mg total), and on Day 8 oocytes were recovered. The mean number of follicles aspirated/donor and oocytes recovered/donor were 20 and 16.61 oocytes for the Brahman donors (n = 6) and 12 follicles and 7.06 oocytes/donor for the Angus donors (n = 10). Oocytes (individual donor basis) were then incubated in TCM-199 supplemented with 10% fetal bovine serum + bLH and bFSH (0.01 U mL−1) at 38.5°C. After 20 h, mature oocytes were denuded by vortexing for 3 min in HEPS + BSA (4 mg mL−1). Buoyancy was tested for individual mature oocytes using a sucrose step density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% sucrose (indicating high lipids) to 35% sucrose (indicating lower lipids). Oocytes recovered from the sucrose were fixed for 24 h in paraformaldehyde for evaluation with Nile Red stain. Oocytes were stained for 24 h, and then placed in Prolong® Gold (Invitrogen, Carlsbad, CA, USA) and evaluated under fluorescence. Oocytes images were evaluated using a Scion Image camera (Scion Corp., Frederick, MD, USA) to calculate mean (± SE) Nile Red units (NRU) (higher NRU = higher lipid content). Treatment groups were analyzed using one-way ANOVA. In summary, Brahman M-II oocytes had significantly lower (P ≤ 0.05) buoyant density, with a significantly higher mean NRU score, when compared with oocytes harvested from Angus donors (Table 1). Based on these results, Brahman oocytes have a higher intracellular lipid content then Angus oocytes. Table 1.Percent sucrose levels and Nile Red units for bovine oocytes from three replicates per donor
Intracellular lipid content has been found to be higher in Bos indicus oocytes compared with Bos taurus oocytes (Ballard et al. 2007 Reprod. Fertil. Dev. 19, 170). The objective of this study was to further determine whether there was a correlation between oocyte lipid content and circulating cholesterol (CHL) and triglyceride (TRG) levels of mature purebred Brahman (BR; n = 11) and English breed (EN; n = 13) beef cows. All donor cows were maintained on the same ration 30 days prior to and during the oocyte collection interval. Oocytes were collected from donors at 30-day intervals (3 replicates/female) by transvaginal ultrasound-guided aspirations. Blood samples were collected to evaluate total circulating CHL and TRG levels just prior to CIDR insertion and again prior to oocyte collection. Follicle-stimulating hormone (Folltropin-V; Bioniche, Belleville, Ontario, Canada) was given twice daily for 3 days in decreasing doses beginning on Day 4 after CIDR insertion, with oocyte recovery on Day 8. The mean number of follicles aspirated/donor and oocytes recovered/donor were 12 and 11 for the BR and 18 and 7 for the EN donors, respectively. Oocytes were matured in TCM-199 with 10% fetal bovine serum + bovine LH and bovine FSH (0.01 U mL–1). After 20 h, mature oocytes with a visible polar body (BR = 242/391 or 61%, and EN = 170/258 or 65%) were denuded by vortexing in Tyrodes HEPES medium + BSA (0.03 mg mL–1). Visual color scores (1 = light, 2 = medium, 3 = dark) were then assigned to the oocytes. Buoyancy of mature oocytes was then measured by using a sucrose step-density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% (high lipids) to 35% (lower lipids). The oocytes were stained with Nile Red and evaluated by fluorescent microscopy. Oocyte images were evaluated by using Scion Image software to calculate the Nile Red units (NRU; higher NRU = higher lipid content). Serum was analyzed (Texas Veterinary Medical Diagnostic Laboratory) for CHL and TRG concentrations. Data were analyzed by one-way ANOVA. There was no difference between visual color scores for oocytes from BR and EN donors. However, BR oocytes had significantly (P < 0.05) lower buoyant density and significantly higher mean NRU scores (32% and 173) compared with oocytes harvested from EN donors (33% and 152), respectively. Serum CHL and TRG levels were both significantly higher (P < 0.05) in the BR donors (150 and 33.2) compared with the EN donors (110 and 25.5), respectively. These results suggest that serum cholesterol and triglyceride levels may be predictive of donor oocyte lipid content.
Numerous studies have reported aberrant gene expression levels in embryos attributed to suboptimal culture conditions. This study investigated the effects of different culture systems and protein sources on the development of IVP embryos as measured by cleavage and blastocyst rates, cell number, and relative abundance levels of Oct-4, Connexin 43, Nanog, and glucose transporter-1 (Glut-1) when compared with in vivo embryos. Experiment (Exp) 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium (KSOMaa) supplemented with amino acids. Exp 2 compared the same two culture systems with and without the addition of calf serum (CS). Oocytes were matured for 22 h, fertilized in vitro, and then cultured in the appropriate treatment medium. RNA was extracted from pools of blastocysts, reverse transcribed to cDNA, and primer-specific amplified via Q-PCR. Exp 1 analyzed 10 pools per treatment, and either 10 (in vivo) or 11 pools were analyzed per treatment in Exp 2. One-way ANOVA followed by multiple pair-wise comparisons using Tukey's test was used to detect differences between treatments and in vivo embryos (P < 0.05). Results from both experiments indicated that, despite similar cleavage and blastocyst rates among treatments, significant differences were detected at the mRNA level and in cell numbers between treatments. In Exp 1, Oct-4 was found to have a mean abundance mRNA level significantly greater in KSOMaa-cultured blastocysts than in either SOFaa-cultured blastocysts or in vivo embryos. The same pattern of upregulation of Oct-4 in KSOMaa or KSOMaa with CS-cultured blastocysts was detected in Exp 2. In contrast to that reported by others, Connexin 43 was not expressed at detectable levels in the in vivo embryos analyzed in our studies. Connexin 43 was not detected in IVP blastocysts used in Exp 1. Conversely, Connexin 43 was detected in KSOMaa, SOFaa, and SOFaa with CS-cultured blastocysts in Exp 2. There was no significant difference in expression of the ICM-specific transcript Nanog in either experiment. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of Glut-1 when compared with other treatments and in vivo embryos. Overall, the transcript levels of the majority of the genes analyzed were altered by the in vitro culture condition. Differences continue to be observed between in vitro-cultured and in vivo embryos, and until these differences are minimized, aberrations in in vitro development will continue to arise. Further research will possibly modify the current culture conditions, allowing for the production of in vitro embryos of higher developmental potential similar to that observed in in vivo-derived embryos.
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