172 Expression of proliferation and apoptosis markers in cumulus cells surrounding matured and aged oocytes exposed to luteotropic factors during the second phase of in vitro maturation

2019 ◽  
Vol 31 (1) ◽  
pp. 210
Author(s):  
I. Lebedeva ◽  
O. Mityashova ◽  
A. Smekalova ◽  
E. Montvila ◽  
G. Singina ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Caspase 3 ◽  
Cumulus Cells ◽  
Treated Group ◽  
Second Phase ◽  
Proliferation And Apoptosis ◽  
Oocyte Aging ◽  
Apoptosis Markers

The quality and developmental capacity of mammalian oocytes depends on cooperation with surrounding cumulus cells. The functional state and activity of cumulus cells changes with oocyte maturation, especially during the oocyte transition from metaphase I (MI) to metaphase II (MII) stage. In the present work, effects of 3 luteotropic factors, progesterone (P4), prolactin (PRL), and LH, during the second phase of in vitro maturation (IVM) on the subsequent expression of proliferation and apoptosis markers in bovine cumulus cells surrounding matured and aged oocytes were studied. A total of 1532 cumulus-oocyte complexes (COC) were cultured for 12h in TCM-199 containing 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, COC were transferred to the following IVM systems: (1) TCM-199 containing 10% FCS (Control 1) and (2) a monolayer of granulosa cells (GC) precultured for 12h in TCM-199 containing 10% FCS (Control 2). In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (50ng mL−1) or ovine LH (5μg mL−1); then, the COC were matured for next 12h. Half of the COC matured for 12h in both systems were cultured for an additional 24h in fresh TCM-199 containing 10% FCS to test long-term hormonal effects during oocyte aging. After culture, the cumulus expression of the proliferation marker proliferating cell nuclear antigen (PCNA) and the pro-apoptotic markers caspase-3 and Bax was assessed by the immunocytochemical method. The data from 4 to 5 replicates using 84 to 106 COC per treatment were analysed by ANOVA. After IVM in System 1, the rate of PCNA-positive cumulus cells was higher (P<0.05) in the PRL-treated group (41.3±1.6%) than in the control (34.6±2.3%) or LH-treated group (29.9±2.9%), but did not differ from that in the P4-treated group (38.2±4.8%). In the presence of GC (System 2), the respective rates did not change but were more variable. Aging of COC matured in both systems led to a 1.4- to 1.9-fold reduction in the proportion of the cells containing the proliferation marker PCNA (P<0.05). Meanwhile, none of the hormones tested had any long-term effect on the proliferative activity of senescent cumulus cells. The rate of cumulus cells expressing caspase-3 in different groups varied from 48.5±4.9 to 53.8±5.8% and did not depend on the hormones, IVM system, or oocyte aging. The proportion of the Bax-positive cells was also unaffected by luteotropic factors but increased 1.4 to 1.6 times (P<0.01) following 24h of COC aging. Our findings indicate that PRL can exert a short-term stimulatory action on the proliferative activity of bovine cumulus cells in the course of the second phase of IVM. Meanwhile, the cumulus expression of pro-apoptotic markers caspase-3 and Bax is not responsive to P4, PRL, or LH during the second step of IVM. The study was supported by the Russian Science Foundation (project 16-16-10069).

55 The Role of Poly (ADP-Ribose) Polymerases in Porcine Cumulus - Oocyte Complex During In Vitro Maturation and Embryonic Development

2018 ◽  
Vol 30 (1) ◽  
pp. 166
Author(s):  
D. H. Kim ◽  
S. T. Shin ◽  
H. T. Lee
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Parp Inhibitor ◽  
Development Program ◽  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Mrna Levels

Poly(ADP-ribosyl)ation (PARylation) is related to DNA repair, chromatin modification, and apoptosis and is catalyzed by PARylation polymerases (PARP). Previous studies have shown that PARylation regulates pre-implantation development and participates autophagy mechanism in mouse and pig. However, the involvement of PARylation and pro-survival autophagy in pre-implantation development from cumulus–oocyte complexes (COC) to the blastocyst stage has not yet been documented. Thus, we investigated the role of PARylation during in vitro maturation (IVM) of porcine COC and their embryonic development. To study the effect of PARylation, COC were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during IVM. Nuclear maturation rates of oocytes were showed no significant differences between 2 groups in all stages (from GV to MII). However, the expansion rates of cumulus cells were significantly decreased in 3-ABA–treated COC compared with control (11.05 ± 1.09 v. 48.40 ± 0.67%). When we analysed mRNA levels of maturation- and expansion-related genes in cumulus cells, levels of PTX3, CX43, and COX-2 were increased but levels of HAS2 and TNFAIP6 were decreased in treatment group. In addition, expression levels of GDF9 and BMP15 in oocytes were up-regulated in treated group. Then, we examined the development of IVF embryos from IVM oocytes in the presence and absence of 3-ABA and their quality at the blastocyst stage. We found that the developmental rates of embryos were significantly decreased in 3-ABA-treated group. In particular, the proportion of expanded blastocysts was lower in the treated embryos (2.65 ± 1.53) compared with control embryos (12.27 ± 3.05). Furthermore, the transcript levels of autophagy-related genes (ATG5, BECLIN1, and LC3) in 3-ABA-treated embryos were lowered in all stages. In addition, we found a higher rate of apoptosis in treated blastocysts compared with the control (total apoptosis index; 15.65 ± 2.73 v. 4.89 ± 0.67). Finally, SQSTM1/p62 aggregate increased in 3-ABA-treated blastocysts, indicating that the inhibition of PARylation regulates selective autophagy pathways to utilise SQSTM1/p62. Therefore, these results indicate that PARylation by PARPs during IVM of COC is deeply involved in the pro-survival autophagy and influences the development and quality of porcine embryos. This research was supported by a Grant from the Bio & Medical Technology Development Program (2015M3A9C7030091) of the National Research Foundation (NRF) funded by the Korean government.

scholarly journals Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

Reproduction ◽  
2003 ◽  
pp. 369-376 ◽  
Author(s):  
S Ikeda ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Dna Fragmentation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Treated Group ◽  
Developmental Competence ◽  
Pcr Analysis ◽  
Control Group ◽  
Dependent Manner

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.

scholarly journals Expression of the apoptosis regulatory gene family in the long-term in vitro cultured human cumulus cells

2021 ◽  
Vol 9 (1) ◽  
pp. 8-13
Author(s):  
Rafał Sibiak ◽  
Rut Bryl ◽  
Katarzyna Stefańska ◽  
Błażej Chermuła ◽  
Wojciech Pieńkowski ◽  
...  
Keyword(s):  
Ovarian Follicle ◽  
Regulatory Gene ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Oocyte Aging ◽  
Follicle Maturation ◽  
Insight Into ◽  
Junction Proteins

Abstract Human cumulus cells (CCs) play a key role in the regulation of ovarian follicle maturation and oocyte fertilization. They influence the oocyte development by transferring the various molecules via the specific gap junction proteins, also known as the connexins, which provide a direct transmembrane connection between the oocyte and CCs. The human CCs were obtained in the patients diagnosed with infertility, who underwent the procedure of the controlled ovarian stimulation, and the following in vitro fertilization to elucidate the possible involvement of the CCs in the regulation of the fertilization and oocyte aging. Collected samples were long-term cultured and harvested after 7, 15, and 30 days of cultivation. Afterward, we assessed the relative expression of the following apoptosis regulatory genes - BAX, CASP9, and TP53 - using the RT-qPCR method. We noted a decrease in the expression of all above-mentioned genes in the samples harvested after 15 and 30 days, in reference to 7 days in vitro cultured CCs. In summary, our results provide precious insight into the dynamics of changes and confirm the continuous expression of the proapoptotic genes – BAX, CASP9, and TP53 in the long-term cultured CCs. Running title: Apoptotic gene expression in the human cumulus cells

130 THE SYNERGIC EFFECT OF NERVE GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR ON IN VITRO MATURATION AND DEVELOPMENTAL COMPETENCE IN BOVINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 169
Author(s):  
B. Kim ◽  
I. M. Saadeldin ◽  
B. Lee ◽  
G. Jang
Keyword(s):  
Growth Factor ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Treatment Groups ◽  
Endothelial Growth Factor

Nerve growth factor (NGF) has been reported to increase the mRNA expression of vascular endothelial growth factor (VEGF) in granulose cells of human, rat via TrkA signaling; VEGF has been shown to exert beneficial effects during bovine in vitro maturation (IVM) as well as early embryonic development. The aims of this study were 1) to investigate not only the direct effect of NGF but also the collaborative effect of NGF and VEGF during bovine in vitro maturation (IVM), in vitro culture (IVC), or both; and 2) to validate the correlation among transcript abundance of 7 genes (VEGF164, VEGF120, Flt-1, Flk-1, TrkA, PTGS2, and CYP11A1) in bovine cumulus cells and the results of IVM or IVC among the differently treated groups. In Experiment 1, concentrations of 0, 10, and 100 ng mL–1 NGF were added to our established IVM medium without serum, and in Experiment 2, control and treatment groups (concentration of 0, 10, and 100 ng mL–1 NGF with VEGF 100 ng mL–1) were added into chemically defined media. The oocytes of each group in Experiments 1 and 2 were determined by the proportion of MII oocytes after 24 h, and embryos were assessed after parthenogenetic activation. Cumulus cells from the differently treated matured cumulus cell–oocyte complexes (COC) were separated and synthesised into cDNA for RT-PCR and real-time PCR in order to measure relative abundance of 7 genes in a dose-dependent manner or a time-dependent manner. In Experiment 1, the concentration of 10 ng mL–1 (57.40%) and 100 ng mL–1 (62.75%) NGF treatment groups did not significantly increase the proportion of MII oocytes compared with the control group (55.06%). In Experiment 2, both the NGF 10 ng mL–1 with VEGF 100 ng mL–1 treated group (67.69%; P ≤ 0.01) and the NGF 100 ng mL–1 with VEGF 100 ng mL–1 treated group (72.24%; P ≤ 0.001) had a significantly higher percentage of polar body extrusion than control group (51.77%) and the group which was treated with VEGF 100 ng mL–1 (56.39%). The NGF treatment group with VEGF increased transcriptional level of VEGF164 and VEGF120 compared with the control group and only NGF- or VEGF-treated groups. In addition, either the NGF-treated group or NGF plus VEGF showed significantly increased mRNA abundance in VEGF164, VEGF120, Flt-1, Flk-1, and TrkA genes, whereas the NGF plus VEGF-treated group indicated the up-regulation of VEGF164, VEGF120, CYP11A1, and PTGS2 genes. In conclusion, NGF and exogenous VEGF have a synergic effect during bovine IVM and the early stage of embryo development; the elevated VEGF mRNA abundance in cumulus cells might contribute to the viability of bovine oocytes and early embryonic development. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program.

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 >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.

scholarly journals Effects of Silver Nanoparticles on Proliferation and Apoptosis in Granulosa Cells of Chicken Preovulatory Follicles: An In Vitro Study

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1652
Author(s):  
Dorota Katarzyńska-Banasik ◽  
Anna Kozubek ◽  
Małgorzata Grzesiak ◽  
Andrzej Sechman
Keyword(s):  
Silver Nanoparticles ◽  
Granulosa Cells ◽  
Caspase 3 ◽  
In Vitro Study ◽  
Poultry Production ◽  
Cell Death Pathways ◽  
Preovulatory Follicles ◽  
Proliferation And Apoptosis ◽  
Apoptosis Process

The continuous development of poultry production related to the growing demand for eggs and chicken meat makes it necessary to use modern technologies. An answer to this demand may be the use of nanotechnology in poultry farming. One of the promising nanomaterials in this field are silver nanoparticles (AgNPs), which are used as disinfectants, reducing microbial pollution and the amounts of greenhouse gases released. This study aimed to evaluate the effect of AgNPs on the proliferation and apoptosis process in the granulosa cells of chicken preovulatory follicles. The in vitro culture experiment revealed that both 13 nm and 50 nm AgNPs inhibited the proliferation of the granulosa cells. However, a faster action was observed in 50 nm AgNPs than in 13 nm ones. A size-dependent effect of AgNP was also demonstrated for the caspase-3 activity. AgNPs 13 nm in size increased the caspase-3 activity in granulosa cells, while 50 nm AgNPs did not exert an effect, which may indicate the induction of distinct cell death pathways by AgNPs. In conclusion, our study reveals that AgNPs in vitro inhibit granulosa cell proliferation and stimulate their apoptosis. These results suggest that AgNPs may disrupt the final stage of preovulatory follicle maturation and ovulation.

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