178 TRANSCRIPTIONAL DIFFERENCES IN GENES RELATED TO GLUCOSE METABOLISM BETWEEN COMPETENT AND INCOMPETENT BOVINE IMMATURE CUMULUS-OOCYTE COMPLEXES SELECTED BY BCB

2017 ◽  
Vol 29 (1) ◽  
pp. 197
Author(s):  
I. Lamas-Toranzo ◽  
D. A. Martínez-Corona ◽  
E. Pericuesta ◽  
P. Bermejo-Álvarez
Keyword(s):  
Glucose Metabolism ◽  
Growth Phase ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Transcriptional Analysis ◽  
Glutathione Metabolism ◽  
Anaerobic Glycolysis ◽  
G6pdh Activity ◽  
Morphological Criteria

In vitro maturation is a key step of in vitro embryo production, being the main factor responsible for the low blastocyst yield. In vitro maturation requires the selection of competent immature cumulus-oocytes complexes (COC), which is usually accomplished based on morphological criteria and follicle size. Competent immature COC have finished their growth phase and show decreased G6PDH activity. Brilliant cresol blue (BCB) is a dye that is degraded by G6PDH and, therefore, can be used to distinguish COC that have finished their growth phase (BCB+) from those that are still growing and are less competent (BCB-). The objective of this study was to determine the metabolic differences between BCB- and BCB+ COC by performing a transcriptional analysis of genes related to glucose metabolism. The COC obtained from slaughterhouse ovaries were selected based on BCB staining. The BCB+ and BCB- COC were fertilized and cultured in vitro to determine the differences in developmental ability. For gene expression analysis, BCB+ and BCB- COC were denuded by vortexing and 5 groups of 10 oocytes; their corresponding cumulus cells per group were snap-frozen until analysis. Messenger RNA was extracted by Dynabeads (Dynal Biotech, Lake Success, NY, USA) and relative mRNA abundance was analysed by quantitative PCR using PPIA1 as housekeeping. Statistical differences were determined based on ANOVA (P < 0.05). The genes analysed were G6PDH, its positive regulator SIRT2, 2 glucose transporters SLC2A1 and SLC2A5, 2 genes involved in anaerobic glycolysis GAPDH and LDHA, 2 genes related with Krebs cycle CS and ATP5A1, and 1 gene related to glutathione metabolism GPX1. As expected, the BCB+ group showed a higher cleavage rate (85.6 ± 1.8 v. 74.2 ± 1.3%, BCB+ v. BCB-; P < 0.05) and blastocyst yield (Day 9: 33.3 ± 3.8 v. 16.1 ± 1.4%, BCB+ v. BCB-; P < 0.05) compared with BCB-. Genes SIRT2, GAPDH, and LDHA were significantly up-regulated in BCB- cumulus cells (SIRT2: 1 ± 0.04 v. 1.45 ± 0.21; GAPDH: 1 ± 0.17 v. 1.46 ± 0.15; LDHA: 1 ± 0.22 v. 1.65 ± 0.12; BCB+ v. BCB-; P < 0.05), whereas no significant differences were found in the other genes and in oocytes. In conclusion, the differences in G6PDH activity between BCB+ and BCB- COC are not controlled by G6PDH transcript abundance, but seem to be mediated by SIRT2 regulation of G6PDH activity. The BCB- cumulus cells showed an up-regulation of GAPDH and LDHA, suggesting a higher activity of anaerobic glycolysis in BCB- COC compared with BCB+.

Glucose Metabolism Characterization During Mouse In Vitro Maturation Identifies Alterations In Cumulus Cells†

2021 ◽  
Author(s):  
Nazli Akin ◽  
Lucia von Mengden ◽  
Anamaria-Cristina Herta ◽  
Katy Billooye ◽  
Julia Leersum ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
In Vitro Maturation ◽  
Culture Media ◽  
Cumulus Cells ◽  
Routine Practice ◽  
Glycolytic Activity ◽  
Direct Measurements ◽  
Final Maturation

Abstract In vitro maturation (IVM) is an assisted reproduction technique with reduced hormone-related side effects. Several attempts to implement IVM in routine practice have failed, primarily due to its relatively low efficiency compared to conventional in vitro fertilization (IVF). Recently, capacitation (CAPA)-IVM, a novel two-step IVM method, has improved the embryology outcomes through synchronizing the oocyte nuclear and cytoplasmic maturation. However, the efficiency gap between CAPA-IVM and conventional IVF is still noticeable especially in the numerical production of good quality embryos. Considering the importance of glucose for oocyte competence, its metabolization is studied within both in vivo and CAPA-IVM matured mouse cumulus-oocyte-complexes (COCs) through direct measurements in both cellular compartments, from transcriptional and translational perspectives, to reveal metabolic shortcomings within the CAPA-IVM COCs. These results confirmed that within in vivo COC, cumulus cells are highly glycolytic, whereas oocytes, with low glycolytic activity, are deviating their glucose towards pentose phosphate pathway. No significant differences were observed in the CAPA-IVM oocytes compared to their in vivo counterparts. However, their cumulus cells exhibited a precocious increase of glycolytic activity during the pre-maturation culture step and activity was decreased during the IVM step. Here, specific alterations in mouse COC glucose metabolism due to CAPA-IVM culture were characterized using direct measurements for the first time. Present data show that, while CAPA-IVM cumulus cells are able to utilize glucose, their ability to support oocytes during final maturation is impaired. Future CAPA-IVM optimization strategies could focus on adjusting culture media energy substrate concentrations and/or implementing co-culture strategies.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

Influence of cumulus cells on in vitro maturation and fertilization of equine oocytes

1996 ◽  
Vol 45 (1) ◽  
pp. 263 ◽  
Author(s):  
M.V. Marcos ◽  
A.R. Spell ◽  
M.D. Butine ◽  
M.J. Arns
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells

Use of pregnancy‐associated plasma protein‐A during oocyte in vitro maturation increases IGF‐1 and affects the transcriptional profile of cumulus cells and embryos from Nelore cows

2019 ◽  
Vol 86 (11) ◽  
pp. 1694-1704
Author(s):  
Alan B. Giroto ◽  
Patrícia K. Fontes ◽  
Fernanda F. Franchi ◽  
Priscila H. dos Santos ◽  
Eduardo M. Razza ◽  
...  
Keyword(s):  
Plasma Protein ◽  
In Vitro Maturation ◽  
Protein A ◽  
Cumulus Cells ◽  
Transcriptional Profile

Lysophosphatidic acid increases in vitro maturation efficiency via uPA-uPAR signaling pathway in cumulus cells

2018 ◽  
Vol 113 ◽  
pp. 197-207 ◽  
Author(s):  
Seon-Ung Hwang ◽  
Kyu-Jun Kim ◽  
Eunhye Kim ◽  
Junchul David Yoon ◽  
Kyu Mi Park ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Lysophosphatidic Acid ◽  
In Vitro Maturation ◽  
Cumulus Cells

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
R. Oishi ◽  
Y. Isaji ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
In Vitro Maturation ◽  
Basal Medium ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Medium ◽  
Dibutyryl Camp ◽  
Mouse Oocytes ◽  
Junctional Communication

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P < 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P > 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P < 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

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