scholarly journals The effect of vitrification after warming on the expressions of p38, CDK1, and cyclin B in immature goat oocytes followed by in vitro maturation

2020 ◽  
Vol 13 (10) ◽  
pp. 2126-2132
Author(s):  
A. A. Muhammad Nur Kasman ◽  
Budi Santoso ◽  
Widjiati Widjiati
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclin B ◽  
Mitogen Activated Protein Kinase ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Mitogen Activated Protein ◽  
Oocyte Meiotic Maturation

Background and Aim: The combination of vitrification techniques and in vitro maturation can reduce oocyte competence. Mitogen-activated protein kinase and maturation-promoting factor are significant in oocyte meiotic maturation regulation. This study aimed to analyze vitrification's effect, after warming followed by in vitro maturation, on the expressions of protein 38 (p38), cyclin-dependent kinase 1 (CDK1), and cyclin B and oocyte maturation level. Materials and Methods: Immature goat oocytes were soaked in vitrification and warming solutions. The procedure was followed by in vitro maturation and in vitro maturation without post-warming vitrification as a control. These oocytes, along with their cumulus, were vitrified using hemistraw in liquid nitrogen. Oocyte maturation was carried out in a maturation medium that was added with 10 μg/mL of FSH, 10 μg/mL of LH, and 1 μg/mL E2 for 22 h. The expressions of p38, CDK1, and cyclin B were observed using immunocytochemical methods, which were assessed semiquantitatively according to the modified Remmele method. The oocyte maturation level was observed using the aceto-orcein staining method based on the achievement of chromosomes up to the metaphase II stage and/or the formation of the polar body I. Results: p38 expression in vitrified oocytes after warming, followed by in vitro maturation, increased insignificantly (p≥0.05), with the acquisition of 3.91±2.69 and 2.69±0.50 in the control oocytes. CDK1 expression in vitrified oocytes decreased significantly (p≤0.05) after warming, followed by in vitro maturation, with the acquisition of 2.73±1.24 and 7.27±4.39 in the control oocytes. Cyclin B expression in vitrified oocytes decreased insignificantly (p≥0.05) after warming, followed by in vitro maturation, with the acquisition of 3.09±1.4 and 4.18±2.61 in the control oocytes. The proportion of vitrified oocyte maturation levels after warming, followed by in vitro maturation, decreased significantly (p≤0.05), with the acquisition of 45.45% and 77.27% in the control oocytes. Conclusion: This study concluded that vitrification after warming resulted in an insignificant increase in p38 expression, a significant decrease in CDK1 expression, an insignificant decrease in cyclin B expression, and a significant reduction in oocyte maturation levels.

Treatment of allicin improves maturation of immature oocytes and subsequent developmental ability of preimplantation embryos

Zygote ◽  
2017 ◽  
Vol 25 (4) ◽  
pp. 480-488 ◽  
Author(s):  
Sang-Gi Jeong ◽  
Seung-Eun Lee ◽  
Yun-Gwi Park ◽  
Yeo-Jin Son ◽  
Min-Young Shin ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Treated Group ◽  
Developmental Competence ◽  
Mitogen Activated Protein Kinase ◽  
Preimplantation Embryos ◽  
Maturation Medium ◽  
Mitogen Activated Protein ◽  
Porcine Oocyte

SummaryAllicin (AL) regulates the cellular redox, proliferation, viability, and cell cycle of different cells against extracellular-derived stress. This study investigated the effects of allicin treatment on porcine oocyte maturation and developmental competence. Porcine oocytes were cultured in medium supplemented with 0 (control), 0.01, 0.1, 1, 10 or 100 μM AL, respectively, during in vitro maturation (IVM). The rate of polar body emission was higher in the 0.1 AL-treated group (74.5% ± 2.3%) than in the control (68.0% ± 2.6%) (P < 0.1). After parthenogenetic activation, the rates of cleavage and blastocyst formation were significantly higher in the 0.1 AL-treated group than in the control (P < 0.05). The reactive oxygen species level at metaphase II did not significantly differ among all groups. In matured oocytes, the expression of both BAK and CASP3, and BIRC5 was significantly lower and higher, respectively, in the 0.1 AL-treated group than in the control. Similarly, the expression of BMP15 and CCNB1, and the activity of phospho-p44/42 mitogen-activated protein kinase (MAPK), significantly increased. These results indicate that supplementation of oocyte maturation medium with allicin during IVM improves the maturation of oocytes and the subsequent developmental competence of porcine oocytes.

scholarly journals Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes

Animals ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 163
Author(s):  
Payungsuk Intawicha ◽  
Li-Kuang Tsai ◽  
Shih-Ying Yen ◽  
Neng-Wen Lo ◽  
Jyh-Cherng Ju
Keyword(s):  
Oocyte Maturation ◽  
Mammalian Cells ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
Mitogen Activated Protein ◽  
Porcine Oocyte

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

O-231 In vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation with recombinant AMH in the maturation medium

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
J Bedenk ◽  
N Jančar ◽  
E Vrtačnik-Bokal ◽  
I Virant-Klun
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Time Lapse ◽  
Human Oocyte ◽  
Hormonal Stimulation ◽  
Maturation In Vitro ◽  
Maturation Medium ◽  
Maturation Rate

Abstract Study question Does the addition of recombinant AMH to the in vitro maturation (IVM) medium improve the maturation of GV oocytes after controlled ovarian hormonal stimulation? Summary answer Our results show that the addition of recombinant AMH to the in vitro maturation medium improves the maturation rate of GV oocytes. What is known already Anti-Müllerian hormone (AMH) is an important hormone involved in the process of sex differentiation during embryonic development. At the transition to the 21. century, more and more researchers have studied the role of AMH in ovarian function, especially its impact on folliculogenesis. AMH is becoming one of the main biomarkers of ovarian reserve and ovarian-specific disease, however, little is known about its effect on human oocyte maturation. Therefore, we matured immature GV (germinal vesicle) oocytes in IVM medium with recombinant AMH to assess its effect compared to the conventional IVM procedure with FSH and hCG. Study design, size, duration In this two-year prospective study, we compared the maturation rate of four groups of immature (GV) oocytes matured in maturation medium with added i) AMH (n = 15), ii) AMH+FSH+hCG (n = 44), iii) FSH+hCG (conventional; n = 22), and iv) hormone-free maturation medium (control; n = 15). Each oocyte was matured in vitro for a maximum of 28 hours and monitored by time-lapse microscopy to assess the time of GV breakdown (MI) and extrusion of the polar body (MII). Participants/materials, setting, methods Ninety-six GV oocytes of 46 patients (aged &lt; 38 years, involved in the ICSI programme) after short antagonist protocol of controlled ovarian hormonal stimulation were included after written informed consent. IVM of oocytes was performed in the MediCult IVM System (LAG and IVM medium, Cooper Surgical, Denmark) with added hormones, and in a CO2 incubator equipped with the PrimoVision time-lapse microscope (Vitrolife, Sweden). Main results and the role of chance IVM medium with added recombinant AMH gave the best result with all (100 %) oocytes matured in vitro. In conventional IVM medium with FSH and hCG, the oocyte maturation rate was poorer, with 68 % of oocytes matured in vitro. An even lower oocyte maturation rate (34 %) was observed in IVM medium with AMH, FSH and HCG, which might be explained by the antagonistic action of these hormones. In a group of control oocytes, 25 % of oocytes matured in vitro. The mean time to GV breakdown (MI stage) was 3.7 hours and to polar body release (MII stage) 20,5 hours. The time to MI stage was quite comparable in all groups of oocytes (3.5, 3.8 and 3.7 hours). There was a tendency for the polar body to be released later if AMH was added to the maturation medium (21.5 and 20.2 vs. 19.9 hours) but differences were not statistically significant, as revealed by Student’s t-test. In the control group of oocytes, these times were prolonged (4.2 and 22.2 hours) due to slow spontaneous maturation. These preliminary results demonstrate that AMH could directly affect the oocyte maturation in vitro. Limitations, reasons for caution The limitation is the relatively small number of oocytes included; GV oocytes accounted for less than 10 % of all oocytes in the in vitro fertilisation (ICSI) programme. Moreover, the proportion of GV oocytes spontaneously matured to MI stage before the start of the experiment and were therefore not included. Wider implications of the findings Based on our data, we believe that AMH directly affects human oocyte maturation in vitro. Despite the common knowledge that AMH regulates the recruitment of growing ovarian follicles, it appears that the addition of AMH to the maturation medium can improve the human oocyte maturation in vitro. Trial registration number 0120-546/2018/6

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.

293 EFFECT OF DIFFERENT CONCENTRATIONS OF LH, FSH, AND E2 ON THE MATURATIONAL RATE OF INDIGENOUS SOUTH AFRICAN CATTLE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
K. P. M. Lekola ◽  
J. W. Ng'ambi ◽  
N. Nkadimeng ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale
Keyword(s):  
South African ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
African Cattle ◽  
Maturation Medium ◽  
Maturation Rate

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture

2012 ◽  
Vol 24 (5) ◽  
pp. 656 ◽  
Author(s):  
Islam M. Saadeldin ◽  
Ok Jae Koo ◽  
Jung Taek Kang ◽  
Dae Kee Kwon ◽  
Sol Ji Park ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Early Period ◽  
Threshold Effect ◽  
Developmental Competence ◽  
Control Group ◽  
Maturation Medium ◽  
Paradoxical Effects

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus–oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10–6 M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4 × 10–6 M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine–paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

scholarly journals Pengaruh urea dalam media maturasi in vitro terhadap tingkat maturasi oosit sapi

2021 ◽  
Vol 10 (2) ◽  
pp. 46
Author(s):  
Sepvian Dewi Kurniawati ◽  
Suryanie Sarudji ◽  
Widjiati Widjiati
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Metaphase Plate ◽  
Petri Dish ◽  
Control Group ◽  
Maturation Medium ◽  
First Polar Body ◽  
Maturation Rate ◽  
Follicle Aspiration

This study was aimed to determine the effect of urea in maturation medium on in vitro oocyte maturation rate. The medium used was TCM-199 added with Hepes, NaHCO3, Kanamycin 0.15 IU/mL, PMSG, 0.15 IU/mL hCG, and 10% FBS. Cumulus oocyte complexes (COCs) of cows derived from follicle aspiration were divided into three groups. In control group (P0), the COCs were matured in vitro in a maturation medium without urea addition, meanwhile in the P1 and P2 groups, the medium was added with urea 20 and 40 mg/dL, respectively. Each petri dish contained three drops of maturation medium (300 µl/drops) according to the groups. Microdrops were coated with mineral oil and then incubated in a 5% CO2 incubator, at 39 ˚C with maximum humidity. Aceto-orcein staining was conducted to evaluate the maturation of oocytes based on the achievement of metaphase II phase that is indicated by the presence of metaphase plate and/or first polar body. The result showed that the oocyte maturation rates of P0, P1, and P2 were 51.25, 52.43 (p >0.05), and 46.88 % (p <0.05) respectively. It could be concluded that the presence of urea at 40 mg/dL in maturation medium reduced the percentage of bovine oocyte maturation in vitro.

scholarly journals 271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 285
Author(s):  
J.I. Park ◽  
Y. Jang
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Chi Square ◽  
Cell Stage ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Humidified Air ◽  
Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

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