scholarly journals Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential

2017 ◽  
Vol 29 (5) ◽  
pp. 876 ◽  
Author(s):  
Denise Laskowski ◽  
Ylva Sjunnesson ◽  
Patrice Humblot ◽  
Marc-André Sirard ◽  
Göran Andersson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Quantitative Polymerase Chain Reaction ◽  
Blastocyst Stage ◽  
Specific Gene ◽  
Bovine Oocyte ◽  
Developmental Potential ◽  
Metabolic Imbalance ◽  
Negative Effect

Metabolic imbalance impairs fertility, because changes in concentrations of metabolites and hormones in the blood and follicular fluid create an unfavourable environment for early embryonic development. Insulin is a key metabolic hormone known for its effects on fertility: insulin concentrations are increased during energy balance disturbances in diabetes or metabolic syndrome. Still, insulin is frequently used at supraphysiological concentrations for embryo in vitro culture with unknown consequences for the developmental potential of the offspring. In the present study we investigated the effects of insulin exposure during in vitro bovine oocyte maturation on developmental rates, embryo quality and gene expression. Supplementation of the maturation media with insulin at 10 or 0.1 µg mL–1 decreased blastocyst rates compared with an insulin-free control (19.8 ± 1.3% and 20.4 ± 1.3% vs 23.8 ± 1.3%, respectively; P < 0.05) and led to increased cell numbers (nearly 10% more cells on Day 8 compared with control; P < 0.05). Transcriptome analysis revealed significant upregulation of genes involved in lipid metabolism, nuclear factor (erythroid-derived 2)-like 2 (NRF2) stress response and cell differentiation, validated by quantitative polymerase chain reaction. To conclude, the results of the present study demonstrate that insulin exposure during in vitro oocyte maturation has a lasting effect on the embryo until the blastocyst stage, with a potential negative effect in the form of specific gene expression perturbations.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

94 EFFICIENT BOVINE EMBRYO SPLITTING FOR GENE EXPRESSION AND IN VIVO DEVELOPMENT STUDIES

2014 ◽  
Vol 26 (1) ◽  
pp. 161
Author(s):  
A. Velasquez ◽  
D. Veraguas ◽  
F. O. Castro ◽  
J. F. Cox ◽  
L. l. Rodriguez-Alvarez
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Gene Markers ◽  
Developmental Potential

It is known that embryos produced in vitro are less competent than their in vivo-derived counterparts. When embryos are produced or manipulated in vitro, their developmental potential decreases significantly, which impinges upon the production of viable offspring. In bovines, embryos that will be transferred to a surrogate mother are selected at the blastocysts stage using noninvasive methods, such as their morphological features. However, many of those embryos are not able to implant or to maintain a normal pregnancy because embryo morphology does not reflect its developmental potential and a correct gene expression pattern that support a normal development. It seems that the ideal method for embryo selection would be based on the screening of gene markers that correlate with successful pregnancy after embryo transfer. In that sense, we have proposed an approach to characterise gene expression pattern of early (Day 7) bovine blastocysts and to correlate this gene expression with further developmental potential in vivo, i.e. upon elongation until Day 17. For that, it was established an efficient method to produce identical and viable hemi-embryos by splitting IVF bovine blastocysts in order to set the expression profile of certain genes in one hemi-embryo at blastocyst stage, while the counterpart embryo elongates in vivo for 10 days. A total of 129 blastocysts were split. Six groups of blastocysts were used for splitting and the results compared: 1) Day-7 early blastocysts (n = 20); 2) Day-7 expanded blastocysts (n = 25); 3) Day-7 hatched blastocysts (n = 17); 4) Day-8 early blastocysts (n = 10); 5) Day-8 expanded blastocysts (n = 12); and 6) Day-8 hatched blastocysts (n = 45). Hemi-embryos derived from day-8 grade I and well expanded blastocysts had the greatest survival rate, in vitro re-expansion (67.7%; P < 0.05) and both hemi-embryos conserved a normal morphology with a total cell number over 80 after 6 h in culture. Also both hemi-embryos at blastocyst stage showed homogeneous expression pattern of the genes OCT4, SOX2, NANOG, CDX2, ACTB, and GAPDH (P < 0.05). Finally, the in vivo survival of hemi-embryos was assessed and compared with nonsplit embryos (control) by transferring to recipient cow and collecting at Day 17 of development. For this, hemi-embryos derived from Day-8 hatched blastocyst were used. From 14 transferred hemi-embryos, 5 (35.7%) were collected, and 9 elongated from 17 controls were recovered (52.9%). Also the elongation rate was significantly lower in hemi-embryos than in control; the length of hemi-embryos had a range between 1 and 5 cm, whereas 60% of the control embryos were longer than 10 cm. Our results provide an initial approach to study the correlation among the gene expression characteristics of early bovine embryos with their further development. However, it seems that embryo splitting hampers their elongation in vivo. It might be possible that the development of split embryos is retarded because of manipulation. This work was partially supported by Fondecyt grant no. 11100082 from the Ministry of Education of Chile.

scholarly journals Elevation of MPF and MAPK gene expression, GSH content and mitochondrial distribution quality induced by melatonin promotes porcine oocyte maturation and development in vitro

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9913
Author(s):  
Zimo Zhao ◽  
Ling Yang ◽  
Dan Zhang ◽  
Zi Zheng ◽  
Ning Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Developmental Potential ◽  
Expression Levels ◽  
Maturation In Vitro ◽  
Porcine Oocyte ◽  
Maturation Rate ◽  
Mitochondrial Distribution ◽  
Mapk Gene

The MPF and MAPK genes play crucial roles during oocyte maturation processes. However, the pattern of MPF and MAPK gene expression induced by melatonin (MT) and its correlation to oocyte maturation quality during the process of porcine oocyte maturation in vitro remains unexplored. To unravel it, in this study, we cultured the porcine oocytes in maturation medium supplemented with 0, 10−6, 10−9, and 10−12 mol/L melatonin. Later, we analyzed the MPF and MAPK gene expression levels by RT-PCR and determined the maturation index (survival and maturation rate of oocytes). The GSH content in the single oocyte, and cytoplasmic mitochondrial maturation distribution after porcine oocyte maturation in vitro was also evaluated. We also assessed the effects of these changes on parthenogenetic embryonic developmental potential. The oocytes cultured with 10−9mol/L melatonin concentration showed higher oocyte maturation rate, and MPF and MAPK genes expression levels along with better mitochondrial distribution than the 0, 10−6, and 10−12 mol/L melatonin concentrations (p < 0.05). No significant difference was observed in the survival rates when the oocytes were cultured with different melatonin concentrations. The expression of the MPF gene in the oocytes cultured with 10−6 mol/L melatonin was higher than with 10−12 and 0 mol/L melatonin, and the expression of the MAPK gene in 10−6 and 10−12 group was higher than the control (p < 0.05). As far as the embryonic developmental potential is concerned, the cleavage and blastocyst rate of oocytes cultured with 10−6 and 10−9 mol/L melatonin was significantly higher than the 10−12 mol/L melatonin and control. In conclusion, 10−9–10−6 mol/L melatonin significantly induced the MPF and MAPK gene expression; besides, it could also be correlated with GSH content of single oocyte, mitochondrial maturation distribution, and the first polar body expulsion. These changes were also found to be associated with parthenogenetic embryo developmental potential in vitro.

The expression level of SOX2 at the blastocyst stage regulates the developmental capacity of bovine embryos up to day-13 of in vitro culture

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 398-404 ◽  
Author(s):  
A.E. Velásquez ◽  
D. Veraguas ◽  
J. Cabezas ◽  
J. Manríquez ◽  
F.O. Castro ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
The Other ◽  
Embryo Survival ◽  
Gene Markers ◽  
Developmental Potential ◽  
Pluripotency Markers ◽  
Negative Effect ◽  
Developmental Capacity

SummaryQuality of in vitro-produced embryos is influenced by changes in gene expression in response to adverse conditions. Gene markers for predicting ‘good embryos’ do not exist at present. We propose that the expression of pluripotency markers OCT4–SOX2–NANOG in D9 (day 9) bovine demi-embryos correlated with development at D13 (day 13). Day 8 in vitro-produced blastocysts were split in two cloned halves, one half (D9) was subjected to analysis of pluripotency markers and the other was kept in culture until D13 of development. Embryo development was scored and correlated with its own status at D9 and assigned to one of two categories: G1, arrested/dead; or G2, development up to D13. SOX2 and NANOG expression levels were significantly higher in embryos from G1 and there was also negative correlation between SOX2 and embryo survival to D13 (G3; r = −0.37; P = 0.03). We observed a significant reduction in the expression of the three studied genes from D9 to D13. Furthermore, there was a correlation between the expression of pluripotency markers at D9 and embryo diameter and the expression of trophoblastic markers at D13 (TP1–EOMES–FGF4–CDX2–TKDP1). Finally, the quotient between the relative expression of SOX2 and OCT4 in the D9 blastocysts from G1 and G2 showed that embryos that were considered as competent (G2) had a quotient close to one, while the other group had a quotient of 2.3 due to a higher expression of SOX2. These results might indicate that overexpression of SOX2 at the blastocyst stage had a negative effect on the control of embryonic developmental potential.

265 DEVELOPMENT AND DYNAMICS OF GENE EXPRESSION OF BOVINE EMBRYOS CULTURED IN VITRO IN THREE DIFFERENT MEDIA

2006 ◽  
Vol 18 (2) ◽  
pp. 240
Author(s):  
H. Sagirkaya ◽  
M. Misirlioglu ◽  
A. Kaya ◽  
H. Odaman ◽  
N. First ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Apoptotic Cells ◽  
Glucose Transporter 1 ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Glut 1

Dramatic reprogramming of gene expression occurs during embryonic genome activation (EGA), an essential event initiating as early as the 1-cell zygotic stage in the bovine and increasing gradually as embryonic development advances. It is this reprogramming of gene expression that sets the stage for later development. Expression of embryonic genes is altered in different culture conditions and this may influence developmental potential both during pre-implantation and during fetal development. The objective of this study was to define some most commonly used embryo culture media (KSOMaa, CR1aa, and SOFaa) based on their ability to support embryonic development to the blastocyst stage, mean cell number, percentages of apoptotic cells, and the expression patterns of a panel of developmentally important genes. Oocytes with several layers of cumulus cells obtained from an abattoir were matured in TCM 199 (supplemented with 0.25 mM pyruvate, 0.5 μg/mL FSH, 5 μg/mL LH, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FCS) for 24 h and in vitro-fertilized (Day 0) using frozen bull semen. Presumptive zygotes were transferred into three different media (KSOMaa, CR1aa, and SOFaa) 16–18 h post-insemination, supplemented with 10% FCS on Day 4, and cultured until Day 8 at which time they were fixed or frozen for further analysis. Mean cell numbers and percentages of apoptotic cells in blastocysts were determined using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Real-time quantitative PCR was performed to assess gene transcripts of glucose transporter-1 (Glut-1), heat shock protein 70.1 (Hsp70), interferon-tau (IF-tau), insulin-like growth factor II receptor (Igf-2r), desmosomal glycoprotein desmocollin III (DcIII), and DNA methyltransferase 3a (Dnmt3a). Gene expression data were analyzed relative to transcripts of housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (Gapdh). In three separate trials, a total of 538, 518, and 503 oocytes were used for KSOMaa, CR1aa, and SOFaa groups, respectively. Cleavage rates were 79.2%, 77.5%, and 80.2%; and rates of development to the blastocyst stage were 22.2%, 23.4%, and 32.9% for KSOMaa, CR1aa, and SOFaa groups, respectively. The blastocyst rate of the SOFaa group was significantly higher than those of the KSOMaa and CR1aa groups (P < 0.05). Mean cell numbers were 109.3, 101.0, and 114.0; and the percentages of apoptotic cell numbers per blastocyst were 1.25, 1.91, and 1.87 for KSOMaa, CR1aa, and SOFaa groups, respectively. There was no difference among groups in terms of mean cell numbers and percentages of apoptotic cells per blastocyst. The expressions of Glut-1 and DcIII genes did not differ among the groups. However, expressions of Hsp70, IF-tau, and Dnmt3a genes were all significantly up-regulated in the CR1aa group as compared to the SOFaa and KSOMaa groups (P < 0.05). In conclusion, SOFaa supports higher development to the blastocyst stage than KSOMaa and CR1aa, and culture conditions influence gene expression.

scholarly journals Exocannabinoids effect on in vitro bovine oocyte maturation via activation of AKT and ERK1/2

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. 603-612 ◽  
Author(s):  
A P López-Cardona ◽  
M J Sánchez-Calabuig ◽  
P Beltran-Breña ◽  
N Agirregoitia ◽  
D Rizos ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Cannabinoid Receptors ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Oocyte ◽  
Rt Pcr ◽  
Interferon Tau ◽  
Reproductive Events ◽  
Bovine Oocytes

Endocannabinoids are known to mediate practically all reproductive events in mammals; however, little is known about their role in oocyte maturation. Through RT-PCR and immunocytochemistry, this study confirms the presence of CB1 and CB2 cannabinoid receptors in bovine oocytes and shows how exposure to the exogenous cannabinoids HU-210 and THC during their in vitro maturation (IVM) activates the phosphorylation of AKT and ERK1/2 proteins associated with the resumption of meiosis. Although supplementation with HU-210 or THC during IVM did not increase blastocyst yields, the expression of interferon tau (IFNτ) and gap junction alpha-1 protein (GJA1) was enhanced at the blastocyst stage. Our data suggest that cannabinoid agonists may be useful IVM supplements as their presence during oocyte maturation upregulates the expression in blastocysts of key genes for embryo quality.

164 EPIGENETIC ANALYSIS OF GENOMIC DNA IN PREPUBERAL AND ADULT BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 184
Author(s):  
M. Diederich ◽  
J. Heinzmann ◽  
W. Kues ◽  
U. Baulain ◽  
T. Haaf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Methylation Status ◽  
Control Group ◽  
Specific Gene ◽  
In Vitro Production ◽  
Adult Cattle ◽  
Developmental Potential ◽  
Bovine Oocytes

The use of oocytes obtained from prepuberal cattle shortens the generation interval by producing descendants of genetically valuable animals before achieving actual cultivation maturity. However, several studies proved that oocytes derived from prepuberal animals differ significantly from oocytes of adult animals with regard to their developmental capability and therefore reproductive potential. Epigenetic events are taken into consideration as a possible reason for this phenomenon. Particularly DNA methylation, allele specific gene expression in a parent-of-origin-specific manner (imprinting), and certain histone modifications, like acetylations, carboxylations, and phosphorylations, play an important role. This project aims to gain knowledge about the mechanisms involved in attaining of the full developmental potential of bovine oocytes. Using immature and in vitro matured oocytes of prepuberal and adult cattle, a comparative study was conducted by measuring mRNA expression of 4 developmentally relevant, but non-imprinted genes (GDF9, GLUT1, PRDX1, and ZAR1) as well as the general DNA methylation status, performed by bisulfite sequencing of 2 satellite sequences [bovine testis satellite I DNA segment 2 (BTSS2) and Bos taurus α satellite I DNA (BTS)]. After various pretreatments, immature bovine oocytes were collected from prepuberal calves [6–9 months, either left untreated (Ca1) or treated with FSH (Ca2) or FSH+IGF1 (Ca3) or FSH+IGFK (Ca4)] and adult animals [≥2nd lactation, either left untreated (Ad1) or treated with FSH (Ad2)] using the Ovum-pick-up (OPU) technique. The Ad1 group was considered the control group. First results of the qPCR analyses of immature oocytes show differences between treatment groups for GLUT1, PRDX1, and ZAR1 transcripts. Compared with Ad1, GLUT1 expression increased in Ad2 [fold change (FC) 2.2], Ca1 (FC 2.0), Ca2 (FC 1.8), and Ca3 (FC 1.4). The genes PRDX1 and ZAR1 were reduced in all groups by 0.02 to 0.07 in comparison with Ad1. The GDF9 showed generally a very low expression. The methylation analysis shows for BTSS2 and BTS significant differences before and after in vitro maturation in the groups Ad1 (BTSS2: 49.6 v. 64.9%), Ad2 (BTS: 76.7 v. 52.5%), Ca1 (BTSS2: 74.6 v. 53.3%), Ca2 (BTS: 72.8 v. 57.8%) and Ca3 (BTSS2: 60.6 v. 71.7%). Currently, the first experiment and statistical analysis are under way. The preliminary data confirm differences in gene expression between prepuberal and adult animals, and demonstrates the dependence of the methylation pattern on age and maturation status. These results contribute to a better understanding of the developmental potential of prepuberal oocytes in order to optimize their use for in vitro production of embryos. This work was supported by the H. Wilhelm Schaumann Foundation, Hamburg.

scholarly journals The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

2021 ◽  
Author(s):  
Er-Meng Gao ◽  
Bongkoch Turathum ◽  
Ling Wang ◽  
Di Zhang ◽  
Yu-Bing Liu ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Cumulus Cells ◽  
Cholesterol Transport ◽  
Vitro Fertilization ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Morphological Differences

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

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