321 OOCYTE DIAMETER INFLUENCES THE MEIOTIC RESUMPTION AND PROGRESSION INDUCED BY OKADAIC ACID IN DOG

2006 ◽  
Vol 18 (2) ◽  
pp. 268
Author(s):  
F. Ariu ◽  
L. Bogliolo ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Control Group ◽  
Oocyte Diameter ◽  
Treatment Groups ◽  
Different Diameters

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: <110 �m, 110-120 �m, and >120 �m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 �M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5�C, 5% CO2 in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 �m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes <110 �m (16/108, 14.8%; P < 0.05) and 110-120 �m (70/130, 53.8%; P < 0.01) as compared to that of oocytes in the <110 �m and 110-120 �m control groups (2/58, 3.4%; 24/82, 29.3%). The percentage of oocytes in the 110-120 �m OA group that underwent in vitro maturation to metaphase II (MII) was significantly higher than in the 110-120 �m control group (18/130, 13.8% vs. 4/82, 4.9%, respectively; P < 0.05). In contrast, smaller oocytes (<110 �m) did not develop to MII with or whitout OA. Meiotic resumption rate of >120 �m OA group (64/78, 82.0%) was similar to the >120 �m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P < 0.01). Low rates of meiotic resumption were observed in denuded >120-�m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 �m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.

245 EFFECTS OF MELATONIN ON THE IN VITRO OOCYTE MATURATION OF PORCINE CUMULUS - OOCYTE COMPLEXES

2008 ◽  
Vol 20 (1) ◽  
pp. 202
Author(s):  
J. K. Kang ◽  
O. J. Koo ◽  
D. K. Kwon ◽  
G. Jang ◽  
S. K. Kang ◽  
...  
Keyword(s):  
Beneficial Effect ◽  
Granulosa Cells ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Polar Body ◽  
Cumulus Cells ◽  
Control Group ◽  
Radical Scavenger ◽  
Melatonin Receptor

In mammalian species, melatonin is a multifunctional hormone that mediates several circadian and seasonal processes, including reproduction. In addition to its hormonal actions, melatonin has a strong antioxidant effect and acts as a powerful free radical scavenger. Accordingly, the objective of this study was to investigate the local expression of the melatonin-receptor I (Mel-RI) gene and the effect of melatonin as an antioxidant on the in vitro maturation of porcine cumulus–oocyte complexes. Oocytes were obtained from aspiration of slaughterhouse ovaries and then matured in tissue culture medium 199 (TCM199) supplemented with 0.1% polyvinyl alcohol (PVA), 20 ng mL–1 epidermal growth factor (EGF), and 4 IU mL–1 pregnant mare serum gonadotropin (PMSG)/hCG. Expression of Mel-RI gene in cumulus cells, granulosa cells, or oocytes was evaluated by RT-PCR (Exp 1). The effect of various concentrations (0, 10, 50, and 100 ng mL–1) of melatonin on the in vitro maturation in terms of nuclear maturation (polar body extrusion) (Exp 2) and measurement of reactive oxygen species (ROS) by dichlorohydrofluorescein diacetate (Yang et al. 1998 Hum. Reprod. 13, 998–1002) (Exp 3) were investigated. Each experiment was replicated at least four times, and each replication included more than thirty oocytes. For statistical analysis, one-way anova in the Prizm software program (GraphPad, San Diego, CA, USA) was used. The Mel-RI gene was locally expressed in cumulus and granulosa cells but not in oocytes. Melatonin at 10 ng mL–1 had a beneficial effect on in vitro maturation (84.6%) compared to 0 ng mL–1 (75.6%), 50 ng mL–1 (80.9%), and 100 ng mL–1 (76.5%). Levels of ROS were also significantly decreased by melatonin treatment at 10 (14.07), 50 (14.42), and 100 ng mL–1 (13.03) of melatonin (P < 0.001) compared to that of the control group (28.21). The presence of melatonin receptor in cumulus cells and granulosa cells indicates a potentially important role for this hormone in regulating porcine ovarian and reproduction function. In conclusion, the present study demonstrated that melatonin had a beneficial effect on in vitro maturation of porcine cumulus–oocyte complexes, probably through binding a receptor and decreased ROS production. Further studies are required to elucidate the relationship between the appearance of melatonin receptor and mechanisms of melatonin function in the embryo.

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 &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.

scholarly journals LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

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.

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

2010 ◽  
Vol 22 (1) ◽  
pp. 322
Author(s):  
D. D. Bücher ◽  
M. A. Castro ◽  
M. E. Silva ◽  
M. A. Berland ◽  
I. I. Concha ◽  
...  
Keyword(s):  
Cell Viability ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Number ◽  
Control Group ◽  
Cumulus Expansion ◽  
Gm Csf ◽  
Nuclear Stage

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

312 EFFECTS OF TRANSFORMING GROWTH FACTOR ALPHA AND INSULIN-LIKE GROWTH FACTOR 1 SUPPLEMENTATION ON IN VITRO MATURATION OF CANINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245
Author(s):  
A. Sato ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
A. Hara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Synergistic Effect ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Germinal Vesicle ◽  
Control Group ◽  
Insulin Like Growth Factor ◽  
Treatment Groups

Although in vitro maturation (IVM) of oocytes has been successfully established for many species, the efficiency of IVM in canine oocytes is still very low. As growth factors have been shown to promote oocyte maturation in some species, we investigated whether use of transforming growth factor α (TGF-a) and insulin-like growth factor 1 (IGF-1) might overcome the difficulties of achieving meiotic maturation in cultured canine cumulus-oocyte complexes (COC). Ovaries were obtained from bitches at 6 months to 7 years of age by ovariohysterectomy and were sliced repeatedly to release COC. In the first experiment, the COC were cultured at 38.8°C for 48 h in 5% CO2 in air in medium 199 supplemented with either TGF-a (0, 1, 10, or 100 ng mL–1) or IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). In the second experiment, the synergistic effect of TGF-a and IGF-1 was investigated by culturing COC in medium 199 supplemented with both TGF-a (0, 1, 10, or 100 ng mL–1) and IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). At the end of the culture period, the oocytes were denuded of cumulus cells by pipetting with a fine bore glass pipette; the denuded oocytes were then fixed in Carnoy's solution and stained with Hoechst 33342. The nuclear configuration and chromatin morphology of the oocytes were evaluated under confocal laser scanning microscopy. The cells were assigned to 1 of the following meiotic stages: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Data were analysed by ANOVA with Fisher's PLSD test. In experiment 1, no significant difference were observed in the rates of cells maturing to the MI and MII stages, but that in the 10 ng mL–1 of TGF-a group (56.3%) were larger than in the other treatment groups (38.8–51.0%). The frequencies of MII stage cells in the 5, 10, and 50 µg mL–1 of IGF-1 treatment groups (9.8, 13.3, and 12.2%, respectively) were significantly higher than in the 0.5 µg mL–1 of IGF-1 group and the control group (5.3 and 2.2%, respectively). In experiment 2, the frequency of MI and MII cells in the control, 1 ng mL–1 of TGF-a plus 0.5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1, and 100 ng mL–1 of TGF-a plus 50 µg mL–1 of IGF-1 group were 44.1, 36.1, 63.5, 70.8, and 50.8%, respectively. The frequency of MII cells in the control group and the same treatment groups were 2.8, 7.2, 10.4, 15.3, and 10.8%, respectively. Both frequencies in the 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1 group were significantly higher than in the control group. The TGF-a may act in a paracrine fashion on the surrounding granulosa cells, and IGF-1 may play multiple roles in cellular metabolism, proliferation, growth, and differentiation in canine oocyte maturation, as has been reported for many other species. In conclusion, these results demonstrate that a synergistic effect between TGF-a and IGF-1 produces an increased rate of in vitro maturation to the MI and MII stages in canine oocytes.

scholarly journals Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽  
2020 ◽  
pp. 1-6
Author(s):  
Ji-Eun Park ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Choon-Keun Park
Keyword(s):  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

scholarly journals Pengaruh Suplementasi Ekstrak Daun Kelor (Moringa pterygosperma Gaertn) terhadap Ekspansi Kumulus Oosit dalam Medium Maturasi In Vitro

2019 ◽  
Vol 11 (1) ◽  
pp. 43
Author(s):  
Nur Anindya Syamsudi ◽  
Widjiati Widjiati ◽  
Hendy Hendarto
Keyword(s):  
In Vitro Maturation ◽  
Leaf Extract ◽  
Cumulus Cells ◽  
Maximum Response ◽  
Control Group ◽  
Control Group Design ◽  
Cumulus Expansion ◽  
Maturation Medium ◽  
Moringa Leaf Extract

Background: Cumulus cells are mediators providing energy transport, micronutrients, and carrier molecules for oocyte development, and mediate the influence of hormones on the oocyte cumulus complex. Cumulus cells play a role during oocyte growth and maturation which shows the quality of oocyte maturation. The use of basic maturation medium with the addition of various substances and compounds has been extensively studied to study oocyte maturation in vitro. Utilization of drugs from natural ingredients has unique advantages because in general cheap prices, have lower toxicity and side effects, and good therapeutic potential. One of the plants that can be used as a source of natural antioxidants, namely Moringa leaves. Objective: Determine the effect of supplementation Moringa pterygosperm Gaertn extract on the expansion of oocyte cumulus in in vitro maturation medium. Method: This research was true experimental with Post Test Only Control Group Design approach. Divided into 4 groups, namely K: control group, P1: treatment of 10 mg / mL Moringa leaf extract, P2: treatment of 15 mg / mL Moringa leaf extract, and P3: treatment of 20 mg / mL Moringa leaf extract. Furthermore oocytes are matured for 24 hours. Cumulus expansion was evaluated according to the level of expansion achieved where the minimum response (+), medium response (++), and maximum response (+++). Statistical analysis used the One Way Anova test and continued post HoC LSD. Results: There was an influence of supplementation Moringa pterygosperm Gaertn extract on the expansion of moderate response cumulus (++) (p = 0.007) and maximum response (+++) (p = 0.000), but there was no effect on the expansion of cumulus minimal response (+) (0.065 ). Doses of 20 mg / mL are effective for cumulus expansion (+++), but not effective for cumulus expansion (+), (++), and control. Conclusion: Supplementation Moringa pterygosperm Gaertn extract affected the expansion of moderate response (++) and maximum response (+++) oocytes in in vitro maturation medium. Keywords: moringa leaf extract, cumulus expansion, in vitro maturation. ABSTRAK Latar Belakang: Sel kumulus merupakan mediator penyedia transpor energi, mikronutrisi, dan atau molekul pembawa untuk perkembangan oosit, dan menjadi mediasi pengaruh hormon pada kompleks kumulus oosit. Sel kumulus berperan selama pertumbuhan dan maturasi oosit yang menunjukkan kualitas maturasi oosit. Penggunaan medium maturasi dasar dengan penambahan berbagai zat dan senyawa banyak diteliti untuk mempelajari maturasi oosit secara in vitro. Pemanfaatan obat dari bahan alam memiliki kelebihan unik karena pada umumnya harga murah, memiliki toksisitas dan efek samping lebih rendah, serta potensi terapeutik baik. Salah satu tumbuhan yang dapat digunakan sebagai sumber antioksidan alami, yaitu daun kelor. Tujuan: mengetahui pengaruh suplementasi ekstrak daun kelor (Moringa pterygosperma Gaertn) terhadap ekspansi kumulus oosit dalam medium maturasi in vitro. Metode: Jenis penelitian ini adalah eksperimental murni dengan dengan pendekatan Post Test Only Control Group Design. Dibagi menjadi 4 kelompok, yaitu K: kelompok kontrol, P1: perlakuan 10 mg/mL ekstrak daun kelor, P2 : perlakuan 15 mg/mL ekstrak daun kelor, dan P3 : perlakuan 20 mg/mL ekstrak daun kelor. Selanjutnya oosit di maturasi selama 24 jam. Ekspansi kumulus di evaluasi sesuai dengan tingkat ekspansi yang dicapai dimana respon minimal (+), respon sedang (++), dan respon maksimum (+++). Analisa statistik menggunakan uji one way Anova dan dilanjutkan post HoC LSD. Hasil: Terdapat pengaruh suplementasi ekstrak daun kelor terhadap ekspansi kumulus respon sedang (++) (p=0.007) dan respon maksimal (+++) (p=0.000), namun tidak terdapat pengaruh terhadap ekspansi kumulus respon minimal (+) (p=0.065). Kesimpulan : Suplementasi ekstrak daun kelor (Moringa pterygosperma Gaertn) berpengaruh terhadap ekspansi kumulus oosit respon sedang (++) dan respon maksimal (+++) dalam medium maturasi in vitro. Kata kunci: ekstrak daun kelor, ekspansi kumulus, maturasi in vitro.

176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID

2014 ◽  
Vol 26 (1) ◽  
pp. 202
Author(s):  
K. Reynaud ◽  
S. Canguilhem ◽  
S. Thoumire ◽  
S. Chastant-Maillard
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Limiting Factor ◽  
Cytoplasmic Maturation

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (<1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (>4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P < 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P > 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.

Sign in / Sign up

Export Citation Format

Share Document