Baicalin improves IVM of pig oocytes and subsequent preimplantation embryo development by inhibiting apoptosis

2019 ◽  
Vol 31 (5) ◽  
pp. 983 ◽  
Author(s):  
Qing Guo ◽  
Mei-Fu Xuan ◽  
Zhao-Bo Luo ◽  
Jun-Xia Wang ◽  
Song-Shan Jin ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cell ◽  
Subsequent Development ◽  
Control Group ◽  
Preimplantation Embryo Development ◽  
Nuclear Maturation ◽  
Expression Of Genes ◽  
Pig Oocyte ◽  
Treatment Control Group

Baicalin, a monomer of flavonoids extracted from dried roots of Scutellaria baicalensis, is used to treat female infertility. However, the effect of baicalin on oocyte maturation is unknown. In this study we investigated the effects of baicalin on the IVM of pig oocytes and subsequent embryo development following parthenogenetic activation (PA). We found that 0.1µgmL−1 baicalin significantly (P<0.05) increased the IVM rate of oocytes compared with the non-treatment (control) group by reducing levels of reactive oxygen species (ROS). In addition, the mRNA expression of genes related to nuclear maturation and cumulus cell expansion, mitochondrial membrane potential and ATP content was significantly (P<0.05) higher in baicalin-treated than control oocytes. To determine whether baicalin treatment during IVM of pig oocytes improves subsequent development of PA embryos, we measured the cleavage and blastocyst formation rates, as well as the number of cells per blastocyst. All these parameters were significantly (P<0.05) higher in the baicalin-treated than control group. In conclusion, this study demonstrates that baicalin improves pig oocyte maturation and subsequent embryo development invitro by inhibiting production of ROS and reducing apoptosis in oocytes.

329 SUPPRESSION OF BOVINE OOCYTE IVM BY SERUM AND FSH DEPRIVATION OR BY α-AMANITIN: EFFECT ON FERTILIZATION AND EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 272
Author(s):  
K. Kananen-Anttila ◽  
M. Eronen ◽  
J. Matilainen ◽  
M. Kallio ◽  
J. Peippo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryo Cleavage ◽  
Bovine Oocytes ◽  
Sperm Aster

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 249 ◽  
Author(s):  
N. Maedomari ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
A. Takizawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Control Group ◽  
Nuclear Maturation ◽  
First Polar Body

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

scholarly journals X-Linked Huwe1 Is Essential for Oocyte Maturation and Preimplantation Embryo Development

iScience ◽  
2020 ◽  
Vol 23 (9) ◽  
pp. 101523
Author(s):  
Alaa A. Eisa ◽  
Scott Bang ◽  
Katherine J. Crawford ◽  
Emily M. Murphy ◽  
William W. Feng ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Preimplantation Embryo ◽  
Preimplantation Embryo Development

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Dionne ◽  
A J Watson ◽  
D H Betts ◽  
B A Rafea
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Embryo Culture ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Control Group ◽  
Blastocyst Development ◽  
Preimplantation Embryo Development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA & 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p &lt; 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p &lt; 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

Erratum to: 264 EXOGENOUS LINOLENIC ACID IN OOCYTE MATURATION MEDIA PROMOTES NUCLEAR MATURATION AND PARTHENOGENETIC PREIMPLANTATION EMBRYONIC DEVELOPMENT IN THE GOAT

2013 ◽  
Vol 25 (1) ◽  
pp. 280
Author(s):  
A. Veshkini ◽  
A.-A. Khadem ◽  
M. Soleimani ◽  
R. Jahanbin ◽  
M. Salehi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Linolenic Acid ◽  
Oocyte Maturation ◽  
Mass Spectrometry Analysis ◽  
Control Group ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Wide Range ◽  
And Control

Dietary intakes of polyunsaturated fatty acids are thought to mediate a wide range of actions in reproductive tissues. This includes the effects on ovarian follicle and corpus luteum functions via improved energy efficiency as well as providing precursors for the synthesis of signalling molecules such as steroids and prostaglandins. An appropriate level of α-linolenic acid (ALA) in the oocyte maturation medium has been shown to induce molecular changes associated with oocyte maturation and embryo developmental competence. In that light, we hypothesised that supplementation of exogenous ALA to maturation media could enhance nuclear maturation and embryonic development in the goat. A preliminary experiment was executed to measure the level of ALA in antral follicles by gas chromatography/mass spectrometry analysis. Our results revealed that the concentration of ALA in follicular fluids ranged from 0.006 to 0.02 mg mL–1 (21.5 to 71.8 µM, with a mean of ~50 µM). To test the effect of ALA on the competence of goat oocytes to complete meiotic maturation to metaphase II and sustain embryonic development, ovaries were obtained from a local abattoir. Cumulus–oocyte complexes were recovered by the slicing method followed by selection of oocytes with a homogenous cytoplasm and at least three layers of compact cumulus cells. The cumulus–oocyte complexes were placed in maturation media supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, several oocytes from the treatment (n = 170) and control (n = 166) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. Other groups of oocytes from both the treatment (n = 70) and control (n = 61) groups were subjected to parthenogenetic activation by applying 1 min of exposure to 2.5 µM ionomycin followed by 2 mM 6-DMAP treatment for 3 h. After activation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated above. Four replications were performed. Differences in developmental rates were analysed for significance by one-way ANOVA using SAS version 8.0 (SAS Institute Inc., Cary, NC, USA), considering P < 0.05 to be significant. As a result, supplementation of the maturation media with ALA did not appear to affect cumulus expansion. In contrast, IVM of goat oocytes in the presence of ALA resulted in a significantly higher maturation rate compared with maturation without ALA supplementation (66.4% v. 57.9%). Likewise, addition of ALA to the IVM medium significantly increased the rate of cleavage (60.1% v. 52.4%) and blastocyst formation (22.6% v. 14.9%), calculated from the activated oocytes. Collectively, the results of our study show that supplementation of IVM media with 50 µM ALA promotes nuclear maturation, increases cleavage rate, and results in higher blastocyst rate in goat oocytes after parthenogenetic activation. Thus, providing appropriate levels of ALA in maturation media could have beneficial effects on embryo development and reproductive efficiency in the goat.

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.

scholarly journals 311EFFECT OF OOCYTE MATURATION MEDIA ON THE SPEED OF MEIOTIC PROGRESSION AND BLASTOCYST DEVELOPMENT OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 275
Author(s):  
D. Fischer ◽  
J. Bordignon ◽  
C. Robert ◽  
D. Betts
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Immunofluorescence Microscopy ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Meiotic Progression

Environment is crucial for in vitro development of gametes and embryos. The recent progression of culture media towards defined conditions brought to surface the impact of different medium supplements on oocyte and embryo development. In this work we evaluate the effect of various oocyte culture media on bovine oocyte maturation and subsequent embryo development. Bovine cumulus-oocyte complexes were recovered from slaughterhouse ovaries and matured in vitro in either TCM-199 (Gibco) or SOF (Synthetic Oviduct Fluid) media supplemented with BSA (fatty acid-free) or serum (fetal bovine serum). Oocytes from each treatment group were denuded and fixed at 18, 20, 22, 24, 26 and 28h post-maturation (p.m.). Oocyte meiotic progression was monitored in each of the groups (n=28–40 oocytes/group) by immunofluorescence microscopy of chromatin. Oocytes matured in SOF showed a slower rate of meiotic progression when compared to the other groups, with the highest percentage of oocytes reaching the MII stage by 28h p.m. (60.71% SOF-BSA, 71.43% SOF-Serum). The fastest developmental rate was observed in oocytes matured in TCM-serum (77.15% at 24h p.m.) followed by oocytes matured in TCM-BSA (74.29% at 26h p.m.). In order to evaluate the effect of nuclear maturation on chromosome segregation, chromosomal organization of MII oocytes was evaluated by immunofluorescence microscopy within each media group (n=26–31 oocytes/group) at 18, 22 and 26h p.m.. No chromosomal abnormalities were found at 18h p.m.. Both media supplemented with BSA induced lower frequencies of chromosomal abnormalities (0 to 3.23%) and (3.57 to 7.69%) for SOF and TCM, respectively, when compared to their serum-supplemented counterparts (7.14 to 11.54%) and (10 to 10.71%) for SOF and TCM, respectively at 22 and 26h p.m.. Remarkably, the maturation medium and its supplements influenced the speed of blastocyst development. For this experiment, oocytes were matured in TCM-BSA, TCM-Serum, SOF-BSA or SOF-serum, fertilized in vitro in a TALP-base media supplemented with BSA and cultured in SOF-BSA. Blastocyst development was assessed at 7, 8 and 9 days of culture. Cleavage rates were similar between the groups (84–90%), whereas development rates to blastocyst stage varied among treatment groups. Maturation in SOF-BSA induced a delay in blastocyst formation that reached its highest percentage only on day 9 of culture (30.8%); moreover, blastocyst development was carried over until Day 12. When oocytes were matured in the presence of serum, the number of blastocysts did not increase after Day 8 of culture (26.6%, TCM-serum). These results provide evidence of a severe impact of oocyte culture media on the nuclear maturation of oocytes and their subsequent embryonic development after IVF. Moreover, the difference in the rate of oocyte maturation and blastocyst formation emphasizes the necessity for reviewing and adapting current protocols to new systems such as SOF-BSA. [Research funded by NSERC and OMAF of Canada.]

Sign in / Sign up

Export Citation Format

Share Document