scholarly journals Seasonal effects on gene expression, cleavage timing, and developmental competence of bovine preimplantation embryos

Reproduction ◽  
2010 ◽  
Vol 140 (1) ◽  
pp. 73-82 ◽  
Author(s):  
M Gendelman ◽  
A Aroyo ◽  
S Yavin ◽  
Z Roth
Keyword(s):  
Gene Expression ◽  
Cold Season ◽  
Developmental Competence ◽  
Seasonal Effects ◽  
Preimplantation Embryos ◽  
Seasonal Effect ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Hot Season

We examined the association between season and expression of genes involved in early embryonic development with an emphasis on cleavage rate and timing of the first embryonic cleavage. In Exp. 1, oocytes were aspirated during the cold (Dec–Apr) and hot (May–Nov) seasons. Matured oocytes were chemically activated and culturedin vitro. The developmental peak to the two- and four-cell stages occurred earlier, with a higher proportion of first-cleaved embryos, during the cold season relative to the hot season (P<0.01). In Exp. 2, a time-lapse system was employed to characterize the delayed cleavage noted for the hot season. Cleavage to the two-cell stage occurred in two distinct waves: early cleavage occurred between 18 and 25 h post activation, and late cleavage occurred between 27 and 40 h post activation. In Exp. 3, oocytes were aspirated during the cold and hot seasons, maturedin vitro, fertilized, and cultured for 8 days. In each season, early- and late-cleaved two-cell stage embryos were collected. Total RNA was isolated, and semi-quantitative and real-time PCRs were carried out with primers forGDF9,POU5F1, andGAPDHusing18S rRNAas the reference gene. In both seasons, the expression of all examined genes was higher (P<0.05) in early- versus late-cleaved embryos.POU5F1expression was higher (P<0.05) in early-cleaved embryos developed in the cold season versus the hot season counterparts. The findings suggest a deleterious seasonal effect on oocyte developmental competence with delayed cleavage and variation in gene expression.

173 HINDERING OF CLEAVAGE TIMING IN BOVINE PARTHENOTES DURING THE HOT SEASON

2007 ◽  
Vol 19 (1) ◽  
pp. 203 ◽  
Author(s):  
A. Aroyo ◽  
S. Yavin ◽  
Z. Roth ◽  
A. Arav
Keyword(s):  
Thermal Stress ◽  
Embryonic Development ◽  
Elevated Temperatures ◽  
Time Lapse ◽  
Cold Season ◽  
Seasonal Effects ◽  
Cell Stage ◽  
Developmental Potential ◽  
Hot Season

Heat stress is a major contributing factor to low fertility among dairy cattle, as reflected by the dramatic reduction in conception rate during the hot months. The effects of thermal stress on oocyte competence and embryonic development have been well documented. However, timing of embryonic cleavage, which may be considered a parameter for the identification of good-quality embryos, and its association with elevated temperatures have not been studied. Two experiments were performed to examine and characterize seasonal effects (i.e. thermal stress) on cleavage timing of bovine parthenogenetic embryos. Oocytes were aspirated from ovaries collected at the local abattoir in 2 seasons: cold (Dec–Apr) and hot (May–Nov). Matured oocytes were chemically activated (ionomycin followed by 6-DMAP) and cultured in vitro; cleavage timing to the 2- and 4-cell stages was observed and documented. The one-way ANOVA procedure was used for statistical analysis. In the first experiment (n = 5416 oocytes), cleavage was documented at specific time points during development post-activation. The peak in embryonic development to the 2-cell stage was earlier (22 to 27 vs. 27 to 40 h after activation) and the cleavage rate higher (39 vs. 21%; P &lt; 0.0001) during the cold season relative to the hot season, respectively. Similarly, the peak in 4-cell-stage development was also observed earlier (46–52 vs. 52–70 h after activation) and corresponded with a higher proportion of developing embryos (33 vs. 21%; P &lt; 0.0001) during the cold season as compared to the hot season, respectively. These results indicate that embryonic development is delayed and a lower proportion of embryos cleaved during the hot season. To better understand the delay in cleavage timing, a second experiment (n = 308 oocytes) was performed through two consecutive hot seasons. A time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) was employed to collect accurate data on the first cleavage division, known to be indicative of embryo quality. The time-lapse system was pre-programmed to take photos at 1-h intervals such that culture dishes did not need to be removed from the incubator. Similar to the pattern noted for the hot season in the first experiment, a wide distribution of cleavage timing (18-40 h after activation) was observed. Further analysis revealed that embryos cleaved in 2 distinct waves: cleavage timing of the first wave (18 to 25 h after activation) was characterized by a time frame similar to that in the cold season, suggesting good-quality embryos; however, the second wave, from 27 to 40 h after activation, presented a delay in cleavage timing, suggesting that these late-cleaving embryos are of inferior quality. Taken together, the results of the 2 experiments lead to the assumption that oocytes harvested from lactating cows during the hot season are of reduced developmental potential, which may be explained, in part, by the pattern of 2 cleavage waves. Furthermore, cleavage timing appears to be a good indicator of embryo potential and may increase the chances of selecting better in vitro-derived embryos during the hot season for embryo transfer.

scholarly journals Developmental competence and oxidative state of mouse zygotes heat-stressed maternally or in vitro

Reproduction ◽  
2002 ◽  
pp. 683-689 ◽  
Author(s):  
M Ozawa ◽  
M Hirabayashi ◽  
Y Kanai
Keyword(s):  
Hydrogen Peroxide ◽  
Heat Stress ◽  
Mouse Embryos ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Physiological Changes ◽  
Cleavage Rate ◽  
Maternal Environment ◽  
Stressed Group

Mammalian preimplantation embryos are sensitive to maternal and direct heat stress. However, the mechanisms by which heat stress affects early embryonic development in vivo or in vitro are unknown. This study examined whether heat-stress-induced loss of developmental competence in mouse embryos was mediated by physiological changes in the maternal environment or by high temperatures alone. After fertilization, zygotes at the same stage were heat-stressed at 39.5 degrees C for 12 h either maternally (measured by maternal rectal temperature) or directly in culture. Zygotes in each group were cultured at 37.5 degrees C for a further 84 h to assess their developmental ability. Neither type of heat stress affected the first cleavage rate. However, the proportion of embryos that developed to morulae or blastocysts was significantly lower in the maternally heat-stressed group, but not in the directly heat-stressed group. Moreover, maternal heat stress significantly reduced intracellular glutathione concentrations and enhanced hydrogen peroxide concentrations in both zygotes and two-cell embryos that were recovered immediately after heat stress or 12 h later, respectively. In contrast, direct heat stress had little effect on concentrations of glutathione or hydrogen peroxide in cultured early embryos. These results demonstrate that maternal heat stress at the zygote stage reduces the developmental ability of mouse embryos via physiological changes in the maternal environment that lead to an increase in intracellular oxidative stress on the embryo.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 327-335 ◽  
Author(s):  
Hruda Nanda Malik ◽  
Dinesh Kumar Singhal ◽  
Shrabani Saugandhika ◽  
Amit Dubey ◽  
Ayan Mukherjee ◽  
...  
Keyword(s):  
Culture Media ◽  
Combined Treatment ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Quantitative Expression ◽  
Pattern Of Variation ◽  
Better Than

SummaryThe present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

260 BREED INFLUENCES OUTCOME OF IN VITRO PRODUCTION OF EMBRYOS IN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 287 ◽  
Author(s):  
M. C. Abraham ◽  
A. Ruete ◽  
Y. C. B. Brandt
Keyword(s):  
Body Fat ◽  
Age Distribution ◽  
Time Of Day ◽  
Developmental Competence ◽  
Carcass Weight ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Frozen Semen ◽  
Significant Difference

Fertility among cattle breeds can vary. The Swedish Red and White dairy breed (SRB) has been systematically bred for good reproductive traits since 1970 and might therefore have retained a better oocyte quality than other dairy breeds. The aim of this study was to determine if the breed of oocyte donor affects the development of embryos using IVM, IVF, and IVC. Oocyte developmental competence in vitro was compared between the SRB (n = 77 animals), the Swedish Holstein breed (SLB, n = 49), and beef breeds (mixed breeds, n = 97). The oocytes (n = 1380, 18 batches) were aspirated from abattoir-derived ovaries from healthy animals with known identity. Statistical analyses were performed using Student’s t-tests and generalized linear mixed models with random effects. The time of collection in relation to slaughter and time of day, as well as aspiration and the following in vitro procedures, were consistent throughout the experiment. The oocytes were matured, fertilized (frozen semen), and cultured according to conventional protocols without serum. Data are presented as mean ± SEM. The SRB and SLB groups were comparable in age [SRB: 66% cows (over 3 years of age), 27% young cows (calved at least once but not over 3 years of age), and 7% heifers; SLB: 63% cows, 20% young cows, and 17% heifers], carcass classification (scale 1-15, where 15 = highest amount of muscle; SRB: 3.8 ± 0.2, SLB 3.5 ± 0.3), body fat (scale 1-15, where 15 =highest amount of fat; SRB: 8.4 ± 0.4, SLB 8.8 ± 0.5) and kilograms of carcass weight (SRB: 297.3±7.4, SLB: 311.6 ± 9.0). The beef group had a significantly higher mean carcass classification (6.2 ± 0.2) and a different age distribution with a higher proportion of heifers (38% cows, 12% young cows, and 50% heifers), but was comparable in body fat content (8.5 ± 0.4) and kilograms of carcass weight (310.9 ± 7.9). Cleavage rate, number of embryos developed beyond the 2-cell stage by 44 h post-fertilization, and the number of blastocysts developed by Days 7 and 8 were noted. All blastocysts were graded and stained with Hoechst 33 342 and the number of nuclei was determined. Cleavage rate was not different among the breeds (SRB: 71.9 ± 0.03%, SLB: 72.5 ± 0.02%, beef: 73.9 ± 0.03%). The percentage of embryos developed beyond 2-cells (from cleaved) did not differ between the beef and SRB (beef: 65.1 ± 6.1%; SRB: 70.4 ± 4.9%) but SLB was significantly greater than than the other breeds (75.4 ± 4.5%). The percentage of blastocysts developed by Day 8 was significantly higher in the beef (21.1 ± 2.7%) and SRB (23.3 ± 3.5%) breeds compared with the SLB (12.5 ± 2.4%). There was no significant difference in blastocyst grades among breeds (scale 1-4, where 1 = highest grade; SRB: 2.4 ± 0.1, SLB: 2.4 ± 0.2, beef: 2.1 ± 0.2), but the number of nuclei in Day 8 blastocysts was significantly lower in the SLB (SRB: 98.9 ± 7.7, SLB: 79.2 ± 8.7, beef: 101.4 ± 6.9). In conclusion, the breed of origin of the oocytes is an important factors affecting the development during in vitro embryo production in cattle. Funded by Formas.

194 EXPRESSION OF microRNA-15a, 16, AND 21 AND THEIR POSSIBLE ROLES IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 176
Author(s):  
D. X. Zhang ◽  
X. H. Shen ◽  
X. S. Cui ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Copy Number ◽  
Blastocyst Stage ◽  
Early Embryogenesis ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Expression Levels ◽  
Apoptotic Gene

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding RNA molecules that can regulate gene expression by base-pairing with fully or partially sequence-complementary target mRNAs. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. While miRNAs play an important role in animal development, little is known about their biological function during early mammalian development. In order to obtain insight into the role of miRNAs in early embryogenesis, we first determined the expression levels of three apoptosis-related miRNAs, miR-15a, -16, and -21 in mouse preimplantation embryos using TaqMan� MicroRNA Assays. Five embryos of each developmental stage were snap-frozen and amplified by stem-loop RT primer and TaqMan Universal PCR Master Mix (Applied Biosystems Inc., Foster City, CA, USA). The miRNA concentrations (10–X) in embryo samples were calculated by standard curve from synthetic lin-4 miRNA and the absolute copy number per embryo was obtained based on the formula of 6.02 � 10(8–X). All three miRNAs had low expression levels from the zygote to the 8-cell stage and were up-regulated thereafter. In general, among the three miRNAs, miR-15a exhibited the lowest expression in preimplantation embryos, while miR-16 exhibited the highest. Because of the low levels of miRNA-15a, we determined developmental ability and apoptosis of embryos following microinjection of miRNA-15a. The microinjection of miR-15a into zygotes did not affect embryo development up to the blastocyst stage (miR-15a, 90 � 4.5% v. buffer 94.6 � 5.8%); however, it did induce a significant degree of apoptosis (P < 0.05; Tukey's multiple range test). Furthermore, the expression levels of miR-15a and -16 were increased in microinjected blastocysts compared to the control group (copy number per blastocyst, miR-15a, 6991 � 1223 v. 3098 � 592; miR-16, 196216 � 958 v. 133514 � 6059). Real-time RT-PCR data showed that the gene expression levels of the housekeeping gene GAPDH, the anti-apoptotic gene Bcl-xL, and the miRNA pathway-related genes GW182 and Dicer remained unchanged in miR-15a-injected blastocysts compared to the control group. In contrast, the expression of the stem cell-specific transcriptional factor Oct-4 (fold change, 1.451 � 0.12), the pro-apoptotic gene Bax (1.418 � 0.12), and Caspase 3 (1.314 � 0.19) were significantly increased in microinjected blastocysts. In addition, treatment of 2-cell embryos with 600 µm H2O2 induced apoptosis and increased the expression level of miR-16 at the blastocyst stage (P < 0.05). Taken together, the changes in the expression levels of miR-15a, -16, and -21 in various embryonic developmental stages indicate a possible role for them in early embryogenesis. Furthermore, the high expression levels of miR-15a and miR-16 seem to be linked to apoptosis in blastocyst-stage embryos; this may be due to an increase in the expression of pro-apoptotic genes.

scholarly journals Nicotinamide Supplementation during the In Vitro Maturation of Oocytes Improves the Developmental Competence of Preimplantation Embryos: Potential Link to SIRT1/AKT Signaling

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1550 ◽  
Author(s):  
Marwa El Sheikh ◽  
Ahmed Atef Mesalam ◽  
Muhammad Idrees ◽  
Tabinda Sidrat ◽  
Ayman Mesalam ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
In Vitro Maturation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Akt Signaling ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Vitamin B3 ◽  
Cell Stage

Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8–16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.

Comparative expression analysis of embryonic development-related genes at different stages of parthenogenetic and in vitro fertilized embryos in caprine

Zygote ◽  
2013 ◽  
Vol 23 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Renu Singh ◽  
Kuldeep Kumar ◽  
R. Ranjan ◽  
Manish Kumar ◽  
T. Yasotha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Competence ◽  
Cell Stage ◽  
Developmental Abnormalities ◽  
Aberrant Gene Expression ◽  
Comparative Expression Analysis ◽  
Morula Stage ◽  
Aberrant Gene ◽  
Parthenogenetic Embryos

SummaryAberrant gene expression occurs in parthenogenetic embryos due to abnormal epigenetic modifications in the genome that probably diminish viability and enhance developmental abnormalities in these embryos. In the present study, five developmentally important genes (HPRT1, Cx43, Sox2, Mest and IGF2R) were analysed at different stages in parthenotes (haploid and diploid) and compared with similar stages in in vitro fertilized (IVF) embryos. The results indicated that in haploid parthenotes expression of HPRT1 was upregulated (P < 0.05) only at the 2–4-cell stage whereas Cx43 expression was significantly (P < 0.05) downregulated in all stages as compared with the control. However, expression of this gene was upregulated (P < 0.05) in 2–4-cell and morula stages of diploid parthenotes. Expression of Sox2 was significantly (P < 0.05) downregulated in morula stage haploid parthenotes, whereas it was upregulated (P < 0.05) in 8–16-cell stage diploid embryos. The expression of Mest was upregulated (P < 0.05) at the 2–4-cell stage of both haploid and diploid parthenotes, whereas it was downregulated in 8–16-cell stage diploid embryos as compared with control. IGF2R expression was upregulated (P < 0.05) only in morula stage haploid and diploid parthenote as compared with control. These results indicate that parthenogenetic embryos showed aberrant gene expression of developmentally important genes such as HPRT1, Cx43, Sox2, Mest and IGF2R in comparison with IVF embryos, this finding may be one of the major reasons for the poor developmental competence of parthenogenetic embryos.

Spent culture medium analysis from individually cultured bovine embryos demonstrates metabolomic differences

Zygote ◽  
2017 ◽  
Vol 25 (6) ◽  
pp. 662-674 ◽  
Author(s):  
Kayla J. Perkel ◽  
Pavneesh Madan
Keyword(s):  
Culture Medium ◽  
Physiological State ◽  
Proton Nuclear Magnetic Resonance ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Isoleucine Leucine ◽  
H Nmr

SummarySpent culture medium can provide valuable information regarding the physiological state of a bovine preimplantation embryos through non-invasive analysis of the sum/depleted metabolite constituents. Metabolomics has become of great interest as an adjunct technique to morphological and cleavage-rate assessment, but more importantly, in improving our understanding of metabolism. In this study, in vitro produced bovine embryos developing at different rates were evaluated using proton nuclear magnetic resonance (1H NMR). Spent culture medium from individually cultured embryos (2-cell to blastocyst stage) were divided into two groups based on their cleavage rate fast growing (FG) and slow growing (SG; developmentally delayed by 12–24 h), then analyzed by a 600 MHz NMR spectrometer. Sixteen metabolites were detected and investigated for sum/depletion throughout development. Data indicate distinct differences between the 4-cell SG and FG embryos for pyruvate (P < 0.05, n = 9) and at the 16-cell stage for acetate, tryptophan, leucine/isoleucine, valine and histidine. Overall sum/depletion levels of metabolites demonstrated that embryos produced glutamate, but consumed histidine, tyrosine, glycine, methionine, tryptophan, phenylalanine, lysine, arginine, acetate, threonine, alanine, pyruvate, valine, isoleucine/leucine, and lactate with an overall trend of higher consumption of these metabolites by FG groups. Principal component analysis revealed distinct clustering of the plain medium, SG, and FG group, signifying the uniqueness of the metabolomic signatures of each of these groups. This study is the first of its kind to characterize the metabolomic profiles of SG and FG bovine embryos produced in vitro using 1H NMR. Elucidating differences between embryos of varying developmental rates could contribute to a better understanding of embryonic health and physiology.

scholarly journals 240OVINE PREPUBERTAL OOCYTE SHOWS ALTERATE GENE EXPRESSION AND LOW DEVELOPMENTAL COMPETENCE

2004 ◽  
Vol 16 (2) ◽  
pp. 240 ◽  
Author(s):  
G. Leoni ◽  
S. Ledda ◽  
L. Bogliolo ◽  
S. Succu ◽  
I. Rosati ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Transporter ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Quantitative Expression ◽  
Altered Gene Expression ◽  
Altered Gene

The aim of this work was to evaluate developmental competence and gene expression of prepubertal and adult ovine oocytes. GV prepubertal and adult oocytes were matured, fertilized and cultured in vitro until blastocyst stage;; the time (days) needed to reach this stage was recorded. Blastocysts developed on different days were cultured for hatching to evaluate their quality in relation to cleavage rate. Adult and prepubertal GV oocytes and blastocyst-stage embryos produced, respectively, at 6 and 7 days were compared for quantitative expression of poly(A) polymerase (polyA-P), glucose transporter I (Glut-I), desmocollin II (desmoII), plakofilin (plako) and heat shock protein 70.1 (HSP70) genes. Confirming previous results (Ledda et al., 1996 Zygote 4, 343–348), fertilized prepubertal ovine oocytes developed to blastocyst stage at lower rates than the adult ones (19.9 v. 51.3%, respectively, P&lt;0.001) and this stage was delayed 24h in prepubertal compared to adult embryos (P&lt;0.01), reflecting a lower quality (Fenwick et al., 2002 Hum. Reprod. 17, 407–412) of the former. In fact, 44.7, 25.0, 30.3 and 0% of adult blastocysts were obtained after 6, 7, 8 and 9 days, respectively, of postfertilization culture compared to 0, 48.4, 34.3 and 17.2% of prepubertal ones. Faster-developed blastocysts showed higher hatching rate in both prepubertal (54.8%, 7 days of culture) and adult (89.8%, 6 days). Hatching rate dropped to 18.2% when blastocysts were obtained at 8–9 days in prepubertal and to 54.5% and 32.5% at 7 and 8 days, respectively, in adult embryos. Analysis of gene expression showed that HSP70, plako and desmo genes were not expressed in GV oocytes, and Glut-I mRNA was lower in prepubertal GV oocytes than in the adult ones (P&lt;0.01). All genes were expressed in blastocysts;; we found that Glut-I was at lower levels (P&lt;0.01) in prepubertal-derived blastocysts whereas HSP70 was highly expressed (P&lt;0.05) in prepubertal blastocysts than in those derived from adult oocytes. In conclusion this work shows that prepubertal ovine oocytes have a lower developmental competence compared to the adult ones, correlated to an altered gene expression during the growth phase of the oocyte and early embryonic development. Supported by MIUR (cofin).

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