• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.15-19
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• 2010

Oocyte enucleation is essential for somatic cell nuclear transfer (SCNT) in the production of cloned animals or embryonic stem cells from adult somatic cells. Most studies of oocyte enucleation have been performed using micromanipulator-based techniques, which are technically demanding, time-consuming, and expensive. Several recent studies have used chemical-induced oocyte enucleation; however, each has been plagued by low efficiency and toxicity. In this study, I found that the co-treatment of murine oocytes with demecolcine and BMI-1026, a potent cdk1 inhibitor, resulted in a high enucleation rate (97%). This method is entirely independent of a micromanipulator and is suitable for the large-scale production of enucleated oocytes. This new method of enucleation will be useful in SCNT and in the development of handmade cloning techniques.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.267-273
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• 2004

Maturation of oocytes is maintained by complex procedures along with follicular genesis and is a critical step for embryonic development. Purine known as an oocyte maturation regulator is present in follicular fluid. In this study, the roles of guanosine as a strong inhibitor of GVBD and a modulator of cyclic GMP concentration in ooyctes were revealed. Denuded immature oocytes were treated with guanosine, and the maturation rates and cGMP concentration of oocytes were measured. GVBD was blocked in a concentration dependent manner by guanosine, but this effect was reversible. However, GVBD was lagged yet not significant by adenosine. Both guanosine and adenosine modified cGMP concentration in oocytes. The characteristic of the guanosine-treated oocyte was significantly higher cGMP compared with the adenosine-treated oocyes at initial time of the maturation. Based these results, guanosine may be a strong and reversible GVBD inhibitor. Although the precise mechanism of guanosine presently is unclear, the results suggest that guanosine may lead the accumulation of cGMP in oocyte cytoplasm, which in turn suppresses GVBD.

• Journal of Genetic Medicine
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• v.18 no.1
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• pp.1-7
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• 2021

The oocyte quality is of great importance in infertility as it reflects the follicle developmental potential and further affects the embryo development, clinical pregnancy outcomes. The analysis of gene expression in somatic cells is an important study to better clinical in vitro fertilization (IVF) outcomes in embryo selection reflecting the appropriate communication between the oocyte and somatic cells. Specifically, somatic cell transcriptomic technology can help assess biomarkers of oocyte and embryo ability. The present article aims to overview the basic aspect of folliculogenesis and review studies involving changes in candidate gene expression of cumulus or granulosa cell related to clinical outcomes in human IVF.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.251-256
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• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

• Development and Reproduction
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• v.1 no.1
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• pp.45-56
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• 1997

Befor fertilization, mammalian oocytes undergo meiotic maturation, which consists of nuclear and cytoplasmic differentiation. In this study, changes of $Ca^{2+}$ stores in mouse oocytes were examined during meiotic maturation and the role of $Ca^{2+}$ in the regulation of the maturation was investigated by using monoclonal antibodies against smooth endoplasmic reticulum $Ca^{2+}$-ATPase(SERCA-ATPase) and calreticulin. Observations were made under epifluorescence microscope and/or confocal laser scanning microscope. In immature oocytes which did not resume meiotic maturation, SERCA-ATPases were mostly localized in the vicinity of the germinal vesicle and calreticulins were distributed evenly throughout the cytoplasm. In mature oocytes, SERCA-ATPases were observed throughout the cytoplasm, butwere absent from the nuclear region. In contrast, calreticulins were localized mostl in the cortex of the oocyte and were absent from the cytoplasm. However, bright fluoresence stainings were wbserved in the perimeiotic spindle region of mature oocyte when labeled with antibodies against calreticulin. These results indicate that mouse oocytes undergo distinct rearrangement of the localization of $Ca^{2+}$-ATPases and calreticulins during meiotic maturation. Thus it can be suggested that redistribution of the $Ca^{2+}$ stores, as revealed by differential fluorescence stainings, is deeply involved in the regulatory mechanism of mammalian oocyte maturation.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.2
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• pp.123-127
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• 1995

In vitro embryo production (IVP) is affected by various factors during in vitro maturation, fertilization, and development. In this experiment, the effect of ovary type, quality of follicular oocyte, medium used for fertilization, presence of hormone in medium, sperm concentration on in vitro maturation and fertilization were examined for effective IVP. In vitro maturation was carried out using TCM-199 supplemented with 15% FCS and hormones in 5% $CO_2$ incubator for 24h. In vitro fertilization was performed with frozen-thawed sperm in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin, and 5mM/ml caffeine for 24h. The fertilized embryos were co-cultured on monolayer of cumulus cells in TCM-199. When oocytes were collected from functionally active and inactive ovaries, maturation rate was 76.9 and 7.7%, respectively. When oocytes were classified morphologically to good and poor grades, maturation rate was 75 and 58.8%, respectively. FSH + LH + $E_2$ (86.4%) showed higher maturation rate than control (53.0%) and FSH (73%). The fertilization rate was 28.2, 100 and 91.7% in $1.6{\times}10^5$, $5.0{\times}10^5$ and $10.0{\times}10^5$ sperm concentration per ml. When oocytes were fertilized in mTALP and BO media, fertilization and cleavage rates of oocytes in mTALP were higher (84.3 and 56.9%) than those (67.4 and 23.3%) in BO medium. In this experiment, in vitro maturation, fertilization and development of oocytes were affected by type of ovary, grade of oocyte, hormones, sperm concentration and fertilization medium.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.31.1-31.13
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• 2022

Background: Compared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes. Objectives: This study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes. Methods: Pig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM. Results: Regardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference. Conclusions: IVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.

• Development and Reproduction
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• v.6 no.1
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• pp.17-23
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• 2002

In oocyte retrieval, a vein anesthetic drug is commonly used for induction and maintenance of general anesthesia. Propofol and Thiopental sodium are frequently used for ultrasound-guided transvaginal oocyte retrieval. The present study aimed to assess the effects of Propofol and Thiopental on in vitro fertilization(IVF). Immature porcine oocytes were exposed to various concentrations ot Propofo1 and Thiopental sodium. The rates of oocyte maturation, fertilization and development were observed. The parthenogenetic effects of the anesthetics were also evaluated. The rate of oocyte maturation after exposure to high concentrations of the anesthetics for long time was significantly higher than that of the control. But the rate of fertilization after long-time exposure to the high concentration of the anesthetic drugs was significantly lower than that of the control. The results support that Propofo1 serves like other anesthetics described, as a parthenogenetic activator. Oocytes exposed to Thiopental sodium showed decreased rates of maturation and fertilization. These results suggest that usage of optimum concentration of anesthetic drug is important in increasing the rates of oocyte maturation, fertilization and development in IVF.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.7
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• pp.956-965
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• 2019

Objective: The main goal of this study was to provide a morphological indicator that could be used to select high-quality oocytes of appropriate meiotic and developmental capabilities in pig. The higher quality of immature oocytes, the higher success rates of in vitro maturation (IVM) and in vitro fertilization (IVF). Thus, prior to the IVM culture, it is important to characterize oocytes morphologically and biochemically in order to assess their quality. Two of the largest indicators of oocyte quality are the presence of cumulus cells and status of chromatin. To investigate the effects of porcine oocyte chromatin configurations on the developmental capacity of blastocysts, we assessed oocyte chromatin status according to follicle size and measured the developmental potency of blastocysts. Methods: To sort by follicle size, we divided the oocytes into three groups (less than 1 mm, 1 to 3 mm, and more than 3 mm in diameter). To assess chromatin configuration, the oocytes were assessed for their stages (surrounded nucleolus [SN] germinal vesicle [GV], non-surrounded nucleolus [NSN] GV, GV breakdown, metaphase I [MI], pro-metaphase II [proMII], and metaphase II [MII]) at different maturation times (22, 44, and 66 h). To assess the development rate, oocytes of each follicle size were subjected to parthenogenetic activation for further development. Finally, GV oocytes were grouped by their chromatin configuration (SN, SN/NSN, and NSN) and their global transcriptional levels were measured. Results: SN GV oocytes were more suitable for IVF than NSN GV oocytes. Moreover, oocytes collected from the larger follicles had a greater distribution of SN GV oocytes and a higher developmental capacity during IVM, reaching MII more quickly and developing more often to blastocysts. Conclusion: Porcine oocytes with high-level meiotic and developmental capacity were identified by analyzing the relationship between follicle size and chromatin configuration. The porcine oocytes from large follicles had a significantly higher SN status in which the transcription level was low and could be better in the degree of meiotic progression and developmental capacity.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.10-19
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• 2019

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: $79.9{\pm}3.8%$ vs G2: $57.5{\pm}4.6%$) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO ($0.1{\mu}M$, MT) treatment (G2: $68.4{\pm}3.2%$ vs G2 + MT: $73.9{\pm}1.4%$). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.