MiRNA-155 regulates cumulus cells function, oocyte maturation, and blastocyst formation

2020 ◽  
Vol 103 (3) ◽  
pp. 548-559 ◽  
Author(s):  
Zeinab Dehghan ◽  
Samira Mohammadi-Yeganeh ◽  
Mohammad Salehi
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Target Genes ◽  
Positive Impact ◽  
Polycystic Ovary ◽  
Cumulus Cells ◽  
Cumulus Expansion ◽  
Cytoplasmic Maturation

Abstract Numerous oocytes are retrieved during in vitro fertilization from patients with polycystic ovary syndrome (PCOS). The poor quality of these oocytes leads to lower fertilization and decreases in cleavage and implantation. MiR-155 is one of the microRNA (miRNA) that is increased in serum and granulosa cells of PCOS patients. In this study, we investigate the effects of miR-155 expression and its target genes on oocyte maturation and embryo development. We used the calcium phosphate protocol to transfect vectors that contained miR-155 or miR-off 155 and alone eGFP into cumulus oophorus complex (COCs) of B6D2F1 female mice for in vitro maturation. Cumulus expansion, nuclear, and cytoplasmic maturation, as well as cleavage rates were determined in groups transfected and compared with the control groups. Quantitative real-time polymerase chain reaction was performed to analyze expression levels of miR-155 and the target genes in the cumulus cells, oocytes, and blastocysts. MiR-155 overexpression in COCs suppressed cumulus expansion, oocyte maturation, and inhibition of endogenous miR-155 by miR-off 155 improved cumulus expansion and oocyte maturation by downregulation and expression increase of the Smad2 and Bcl2 genes. On the other hand, overexpression and downregulation of miR-155 in the COCs led to increase and decrease in cleavage rates by changes in expressions of the Mecp2, Jarid2, and Notch1 genes, respectively (P < 0.05). These results suggested that miR-155 overexpression in granulosa cells of PCOS patients can negatively affect nuclear and cytoplasmic maturation, but this miRNA expression has a positive impact on embryo development.

276 FIBROBLAST GROWTH FACTOR 2 PROMOTES BOVINE OOCYTE MEIOTIC MATURATION AND DEVELOPMENTAL COMPETENCE

2011 ◽  
Vol 23 (1) ◽  
pp. 236 ◽  
Author(s):  
K. Zhang ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Relative Abundance ◽  
Cumulus Cells ◽  
Fibroblast Growth Factor 2 ◽  
Fibroblast Growth ◽  
Cumulus Expansion

Oocyte competency is acquired during the course of folliculogenesis and is controlled by various endocrine and paracrine signals. One of these is fibroblast growth factor 2 (FGF2). Its expression is up-regulated in theca and granulosa cells during final maturation of a bovine follicle, and its cognate receptors are expressed in cumulus cells and oocytes throughout the final stages of oocyte maturation. The overall goal of this work was to describe how supplementing FGF2 during oocyte maturation in vitro affects oocyte maturation and subsequent embryo development. Cumulus–oocyte complexes (COC) were collected from bovine ovaries obtained from a local abattoir and cultured in defined TCM-based oocyte maturation medium. Depending on the study, oocytes were examined either during (6 h) or after (21 h) maturation or were fertilized in vitro and examined throughout in vitro embryo development in modified SOFF. Data were analysed with least-squares ANOVA using GLM of SAS. Adding 0.5 to 50 ng mL–1 of FGF2 did not affect cleavage rate or the percentage of 8 to 16 cell embryos at day 3 post-IVF. However, the blastocyst rate at day 7 was greater when oocytes were exposed to 0.5 ng mL–1 of FGF2 during maturation [30.0 ± 1.9% (17/109) v. 16.0 ± 2.6% (23/77) for nontreatment control; 4 replicates; P < 0.05], whereas higher doses of FGF2 did not affect blastocyst rates when compared with controls. Total cell number per blastocyst was not affected by FGF2 addition. The effects of FGF2 on oocyte maturation and cumulus expansion were examined to better understand how FGF2 improves oocyte competency. Adding 0.5 ng mL–1 of FGF2 did not affect the percentage of oocytes containing condensed chromatin after 6 h IVM or metaphase II (MII) rate after 21 h IVM, but 0.5 ng mL–1 of FGF2 treatment increased the cumulus expansion index score after 21 h IVM (P < 0.05). Interestingly, adding 5 ng mL–1 but not 50 ng mL–1 of FGF2 increased MII rate [61.5 ± 4.3% (53/120) for 5 ng mL–1 of FGF2 v. 46.9 ± 5.9% (64/104) for nontreatment controls; 7 replicates; P < 0.05], but neither FGF2 affected rates of chromatin condensation and cumulus expansion. Changes in the relative abundance for several putative oocyte competency markers and maternal genes (CTSB, Sprouty2, EGFR, FSHR, Has2, BMP15, GDF9, JY-1, Follistatin, H2A) were examined at 6 and 21 h after treatment with 0.5 ng mL–1 of FGF2 by quantitative RT-PCR. Relative amounts of 18S RNA was used as an internal control, and 2-ΔΔCT was used to quantify relative gene expression. The relative abundance of most of the transcripts examined was not affected by FGF2, but EGFR mRNA levels were greater after 6 h but not 21 h IVM in cumulus cells isolated from FGF2-supplemented COC (P = 0.057). In summary, improvements in blastocyst development were achieved by FGF2 treatment during oocyte maturation. The reason for the enhanced oocyte competency remains unclear, but it may occur in part because of improvements in cumulus expansion and production of EGFR. This project was supported by NRICGP number 2008-35203-19106 from the USDA-NIFA.

The importance of manganese in the cytoplasmic maturation of cattle oocytes: blastocyst production improvement regardless of cumulus cells presence duringin vitromaturation

Zygote ◽  
2015 ◽  
Vol 24 (1) ◽  
pp. 139-148 ◽  
Author(s):  
Juan Patricio Anchordoquy ◽  
Juan Mateo Anchordoquy ◽  
Matias Angel Sirini ◽  
Juan Alberto Testa ◽  
Pilar Peral-García ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Sod Activity ◽  
Cumulus Expansion ◽  
Production Improvement ◽  
Blastocyst Quality ◽  
Cytoplasmic Maturation

SummaryAdequate dietary intake of manganese (Mn) is required for normal reproductive performance in cattle. This study was carried out to investigate the effect of Mn duringin vitromaturation of bovine cumulus–oocyte complexes (COC) on apoptosis of cumulus cells, cumulus expansion, and superoxide dismutase (SOD) activity in the COC. The role of cumulus cells on Mn transport and subsequent embryo development was also evaluated. Early apoptosis decreased in cumulus cells matured with Mn compared with medium alone. Cumulus expansion did not show differences in COC matured with or without Mn supplementation. SOD activity was higher in COC matured with 6 ng/ml Mn than with 0 ng/ml Mn. Cleavage rates were higher in COC and denuded oocytes co-cultured with cumulus cells, either with or without Mn added toin vitromaturation (IVM) medium. Regardless of the presence of cumulus cells during IVM, the blastocyst rates were higher when 6 ng/ml Mn was supplemented into IVM medium compared with growth in medium alone. Blastocyst quality was enhanced when COC were matured in medium with Mn supplementation. The results of the present study indicated that Mn supplementation to IVM medium enhanced the ‘health’ of COC, and improved subsequent embryo development and embryo quality.

71 MELATONIN IMPROVES PORCINE IN VITRO MATURATION VIA SONIC HEDGEHOG SIGNALLING

2017 ◽  
Vol 29 (1) ◽  
pp. 143
Author(s):  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
G. A. Kim ◽  
B. C. Lee
Keyword(s):  
Embryo Development ◽  
Sonic Hedgehog ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Melatonin Treatment ◽  
Cumulus Expansion ◽  
Hedgehog Signalling

Melatonin (N-acetyl-5-methoxytryptamine) is the hormone synthesised from the mammalian pineal gland, which has an antioxidant property and regulates physiological processes such as cellular metabolism. It is well known that melatonin affects in vitro maturation of oocytes and embryonic development in many species. However, limited information is available on the underlying beneficial effects of melatonin. Sonic Hedgehog (Shh) signalling is important for follicular development, oocyte maturation, and embryo development. To elucidate the relationship between melatonin and Shh signalling, we designed an experiment with the following three groups: (1) control, (2) melatonin, and (3) melatonin with cyclopamine (smoothened inhibitor) during porcine in vitro maturation. Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 28 to 32°C. The contents of follicles 3 to 6 mm in diameter were recovered by aspiration with an 18 G needle. Cumulus–oocyte complexes were pooled and cultured in TCM-199 medium for 44 h. The aim of this study was to evaluate the effects of melatonin (10−9 M) with or without cyclopamine (2 μM) on cumulus cell expansion (a total of 432 cumulus–oocyte complexes were used in 3 replicates), embryo development after parthenogenetic activation (a total of 432 oocytes were used in 4 replicates). Moreover, we detected gene expression related to cumulus expansion, oocyte maturation, and hedgehog signalling in cumulus cells and oocyte. Results indicated that melatonin treatment significantly increased cumulus expansion index (3.75 ± 0.02 v. 3.51 ± 0.03 and 3.59 ± 0.05, respectively; P < 0.05) and blastocyst formation rates (30.4 ± 2.4 v. 21.9 ± 2.2 and 20.0 ± 2.2, respectively; P < 0.05) compared with control and melatonin with cyclopamine. In addition, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, Has2, Ptx-3, and Tnfaip6) and hedgehog signalling-related genes (Shh, Pthc1, Smo, and Gli-1) in cumulus cells were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. Similarly, the expressions of oocyte maturation-related genes (GDF9 and BMP15) in porcine oocytes were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. In conclusion, Shh signalling mediated melatonin to improve porcine cumulus cell expansion, oocyte maturation, and subsequent embryo development. Further studies are needed to evaluate the effect of melatonin on protein levels of Shh signalling. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality and homoscedasticity and then subjected to one-way ANOVA, followed by Duncan’s multiple range test (when the variances were assumed to be equal) or Dunnet’s T3 test (when the variances were assumed to be unequal) to determine differences among experimental groups. All results are expressed as means ± SEM; P-values < 0.05 were considered to be statistically significant. This study was supported by Ministry of Trade, Industry and Energy (#10048948), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (#311011–05–4-SB010, #114059–03–2-SB010), National Research Foundation (2016M3A9B6903410), China Scholarship Council (CSC, No. 2015–3022), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

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.

scholarly journals Investigating the Intrafollicular Factors Affecting Oocyte Developmental Competency

2021 ◽  
Author(s):  
◽  
Zaramasina Clark
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Significant Finding ◽  
High Quality ◽  
Final Maturation

<p>The number of cycles of assisted reproductive technologies (ART) performed increased by ~9.5 % globally between 2008 and 2010. In spite of this, the success rate in terms of delivery was only ~19.0 % (Dyer et al., 2016). This discrepancy between the demand for, and success of, these technologies necessitates the development of tools to improve ART efficiency. To facilitate this, a better understanding of how the microenvironment changes within the developing follicle to culminate in a mature, developmentally-competent oocyte is required. This study employed an in vivo and in vitro ovine model to investigate the relationship between the surrounding microenvironment and oocyte maturation, and in particular, the attainment of oocyte developmental competency and high-quality embryos.  The first objective of this PhD study was to comprehensively investigate the changing microenvironment of in vivo matured, presumptive preovulatory (PPOV) follicles from wild-type (++) and high ovulation rate (OR; I+B+) ewes. The high OR ewes were heterozygous carriers of mutations in BMP15 (I+) and BMPRIB (B+). Functional differences in follicular somatic (granulosa and cumulus) cells between these genotypes, including differential gonadotropin responsiveness of granulosa cells, composition of follicular fluid and gene expression profiles in cumulus cells were evident. These differences emerged as part of a compensatory mechanism by which oocytes from smaller follicles, containing fewer granulosa cells, achieved developmental competency in I+B+ ewes.  The second objective of this PhD study was to develop new approaches for improving current in vitro maturation (IVM) strategies. The first approach utilised in this study focused on developing biomarkers that could be used to improve prediction of developmental competency in oocytes and in vitro produced embryos. This involved interrogating the hypothesis that a combination of molecular and morphokinetic biomarkers would better predict the developmental competency of oocytes and embryos compared to using these biomarkers alone. The second approach utilised in this PhD study tested the effects of modulating IVM conditions to better mimic the follicular microenvironment of a high, compared to a low, OR species on oocyte developmental competency and embryo quality. This involved supplementing IVM media with different ratios of two oocyte-secreted growth factors, i.e. GDF9:BMP15, that were representative of low or high OR species. These approaches demonstrated significant potential and warrant further investigation.  The most significant finding of this study was that despite variances in the surrounding microenvironment during in vivo and in vitro oocyte maturation that culminated in differential gene expression patterns in cumulus cells, and divergent gonadotropin-responsiveness of granulosa cells, the gene expression signatures of developmentally-competent oocytes and the morphokinetics of high-quality embryos were unaltered. This confirms the value of developing such biomarkers for oocyte development competency and embryo quality that remain unaltered despite a changing surrounding environment. Interestingly, simulating the ratio of GDF9:BMP15 that oocytes from high OR species are exposed to during maturation improved developmental competency in oocytes as demonstrated by increased blastocyst rates. Furthermore, this study has demonstrated that combinations of molecular (cumulus cell gene expression) and morphokinetic biomarkers improved the ability to predict developmental competency in oocytes and embryos. Overall, this study revealed novel information regarding the follicular microenvironment during final maturation and identified several novel approaches to improving the efficiency of ART.</p>

172 DETERMINING THE REQUIREMENTS FOR LH AND FSH DURING SHEEP IN VITRO OOCYTE MATURATION

2014 ◽  
Vol 26 (1) ◽  
pp. 200 ◽  
Author(s):  
C. de Frutos ◽  
R. Vicente-Perez ◽  
P. J. Ross
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Mixed Logit ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Grade 3

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

60 EFFECTS OF AY9944 A-7 ON MEIOTIC RESUMPTION OF PORCINE OOCYTES AND CUMULUS CELL EXPANSION

2016 ◽  
Vol 28 (2) ◽  
pp. 160
Author(s):  
S. Lee ◽  
C. Khoirinaya ◽  
J.-X. Jin ◽  
G. A. Kim ◽  
B.-C. Lee
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Cell Expansion ◽  
Cholesterol Biosynthesis ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Mitogen Activated Protein Kinase ◽  
Control Group ◽  
Cumulus Expansion

In vitro studies on mammalian oocytes have shown that follicular fluid-meiosis activating sterol (FF-MAS) can overcome the inhibitory effect of hypoxanthine (Hx) on the resumption of meiosis. FF-MAS, an intermediate in the cholesterol biosynthesis pathway, is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. AY9944 A-7, an inhibitor of Δ14-reductase and Δ7-reductase, induces accumulation of FF-MAS by inhibiting its metabolism. The aim of this study was to evaluate the effects of AY9944 A-7 on meiotic resumption of porcine oocytes, cumulus cell expansion, and gene expression related to M-phase-promoting factor (MPF), mitogen-activated protein kinase (MAPK), and oocyte maturation in oocytes and related to cumulus expansion in cumulus cells. In experiment 1, 1136 cumulus-oocyte complexes (COCs) were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in addition to a meiotic inhibitor (Hx, 4 mM) for 44 h. Oocytes treated with 10 and 20 μM AY9944 A-7 in the presence of Hx had significantly higher GVBD and M2 rates than the control group. However, 40 μM AY9944 A-7 significantly decreased GVBD and M2 rates and increased degeneration of oocytes compared with other groups. In experiment 2, 600 COCs were cultured in IVM media with 4 different concentrations (0, 10, 20, and 40 μM) of AY9944 A-7 in the absence of Hx for 44 h. Cumulus expansion of 40 μM AY9944 A-7 treated group was significantly decreased compared with other groups. In experiment 3, we evaluate the effects of AY9944 A-7 on gene expression, and the experiment was replicated four times. Data on gene expression were analysed using Student’s t-test. Oocytes treated with 10 μM AY9944 A-7 increased expression of genes involved in MPF (Cyclin B and Cdc2), MAPK (C-mos), and oocyte maturation (GDF9 and BMP15). Cumulus cells treated with 10 μM AY9944 A-7 decreased cumulus expansion-related genes (Has2, Tnfaip6, Ptgs2, and Ptx-3). In conclusion, our results suggest that although 10 μM AY9944 A-7 decreased cumulus expansion-related genes, there was no difference in cumulus expansion and it induced meiotic resumption of porcine oocytes with increased MPF, MAPK, and oocyte maturation-related genes. Further studies are needed to evaluate the effect of AY9944 A-7 on porcine embryo development. This study was supported by Ministry Of Trade, Industry & Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

Chlorpyrifos and endosulfan affect buffalo oocyte maturation, fertilization, and embryo development in vitro directly and through cumulus cells

2011 ◽  
Vol 26 (1) ◽  
pp. 57-67 ◽  
Author(s):  
S. Nandi ◽  
P.S.P. Gupta ◽  
S.C. Roy ◽  
S. Selvaraju ◽  
J.P. Ravindra
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Development In Vitro

scholarly journals Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality

2020 ◽  
Vol 21 (15) ◽  
pp. 5340
Author(s):  
Yulia N. Cajas ◽  
Karina Cañón-Beltrán ◽  
Magdalena Ladrón de Guevara ◽  
María G. Millán de la Blanca ◽  
Priscila Ramos-Ibeas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Biological Effects ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Cytoplasmic Maturation

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 μM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 μM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

228 EFFECT OF INSULIN ON BOVINE OOCYTE MATURATION, CUMULUS CELL APOPTOSIS, AND EARLY EMBRYO DEVELOPMENT IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 203
Author(s):  
I. Lindgren ◽  
P. Humblot ◽  
D. Laskowski ◽  
Y. Sjunnesson
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Insulin Treatment ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Early Embryo Development ◽  
Maturation In Vitro

Dairy cow fertility has decreased during the last decades, and much evidence indicates that metabolic disorders are an important part of this decline. Insulin is a key factor in the metabolic challenge during the transition period that coincides with the oocyte maturation and may therefore have an impact on the early embryo development. The aim of this study was to test the effect of insulin during oocyte maturation on early embryo development by adding insulin during the oocyte maturation in vitro. In this study, abattoir-derived bovine ovaries were used and cumulus-oocyte complexes (n = 991) were in vitro matured for 22 h according to standard protocols. Insulin was added during maturation in vitro as follows: H (10 µg mL–1 of insulin), L (0.1 µg mL–1 of insulin), or Z (0 µg mL–1 of insulin). After maturation, oocytes were removed and fixed in paraformaldehyde before staining. Click-it TUNEL assay (Invitrogen, Stockholm, Sweden) was used for apoptotic staining and DRAQ5 (BioNordika, Stockholm, Sweden) for nuclear staining (n = 132). Cumulus-oocyte complexes were evaluated using laser scanning confocal microscope (Zeiss LSM 510, Zeiss, Oberkochen, Germany). Five levels of scans were used to assess oocyte maturation (MII stage) and apoptosis. Because of incomplete penetration of the TUNEL stain (3–5 layers of cumulus cells), only the outer 2 layers of the cumulus complex were investigated regarding apoptosis. Apoptotic index was calculated as apoptotic cells/total cells visualised. Remaining oocytes were fertilized and cultured in vitro until Day 8. Day 7 and Day 8 blastocyst formation was assessed as well as blastocyst stage and grade. Effect of insulin treatment on variables was analysed by ANOVA following arc sin √p transformation. Post-ANOVA comparisons between H+L group v. Z were performed by using the contrast option under GLM (Scheffé test). Results are presented as least squares means ± s.e. P-values ≤ 0.05 were considered as statistically significant. Insulin treatment during oocyte maturation in vitro had no significant effect on oocyte nuclear maturation or apoptotic index of the cumulus cells (Z: 0.052 ± 0.025, L: 0.039 ± 0.016, H: 0.077 ± 0.044, P > 0.05). No effect was seen on cleavage rates (Z: 0.85 ± 0.02, L: 0.85 ± 0.02, H: 0.89 ± 0.03, P > 0.05), but insulin treatment significantly decreased Day 7 rates from fertilized oocytes (Z: 0.19 ± 0.02, L: 0.14 ± 0.02, H: 0.12 ± 0.02, P < 0.05). This study also showed a significantly retarded developmental stage and decreased grade of blastocysts in insulin-treated groups taken together when compared with the control group (P < 0.05). In this study, no effect of insulin supplementation during in vitro maturation was seen on bovine oocyte maturation and apoptosis of cumulus cells, but blastocyst formation and development were negatively affected. Further studies are needed for understanding the relationship between the addition of insulin during maturation in vitro and impaired blastocyst formation. Insulin is a common supplement in the first phase of the first in vitro maturation medium for pig oocytes and is believed to have a beneficial effect on this species.Funding was received from Stiftelsen Nils Lagerlöfs Fond H12–0051-NLA.

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