• Journal of Embryo Transfer
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• v.25 no.4
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• pp.251-258
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• 2010

In the present study, effects of concentration and time of culture in presence of roscovitine on nuclear maturation and meiotic spindle configuration, chromosomal alignment were examined in porcine oocytes. In experiment 1, porcine cumulus oocyte complexes (COCs) were cultured at $39^{\circ}C$ in a 5% $CO_2$ atmosphere in North Carolina State University 23 (NCSU-23) supplemented with 25, 50, 75 or $100\;{\mu}M$ roscovitine for 22 h and then were cultured for additional 22 h after removal of roscovitine. Nuclear maturation and morphology of the meiotic spindle and chromosomal alignment were examined to determine the optimal concentration of roscovitine in oocyte maturation. In experiment 2, COCs were cultured in NCSU-23 supplemented with $50\;{\mu}M$ roscovitine for 17, 20, 27 or 42 h and then an additional 22 h without roscovitine was followed to determine the optimal time of culture. The optimal concentration of roscovitine to arrest and resume meiosis of porcine oocyte was $50\;{\mu}M$ by examining nuclear status (p<0.05) and normal spindle and chromosome configuration. The optimal time of culture in presence of roscovitine to arrest meiosis of porcine oocyte was 17 h (p<0.05), although MII rates and normal morphology of the meiotic spindle and chromosomal alignment were not significantly different among various times of culture. In conclusion, the optimal concentration and time of culture in presence of roscovitine to arrest porcine oocytes are $50\;{\mu}M$ and 17 h, respectively.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.169-173
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• 2010

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2~6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.283-289
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• 2004

This study was conducted to determine the effects of incubation time after cooling on mouse meiotic spindle and chromosome alignment and the optimal incubation time for their restoration. Oocytes at the metaphase II were obtained from superovulated mice. Control oocytes were held at 37$^{\circ}C$ during the experiment. Oocytes were rapidly cooled to $0^{\circ}C$, held for 30 minutes, warmed and incubated at 37$^{\circ}C$ for 5, 15, 30, 60 and 120 minutes, respectively. The morphological features of spindle and chromosomes in oocytes were evaluated by immunofluorescent staining. Meiotic spindle of control oocytes exhibited a normal-looking bipolar configuration(barrel-shaped) and highly fluorescent microtubles. The chromosomes were clustered in a discrete bundles at metaphase plate. Disassembly of meiotic spindle and chromosome dispersion were occurred immediately after chilling of oocyte. Fluorescence intensity index(FIS), normal chromosomes aligned and normal spindle configuration were compared according to incubation time at 37$^{\circ}C$. Restoration of a barrel-shaped spindle and normal chromosome alignment was occurring after 5 minutes incubation at 37$^{\circ}C$, improved as a incubation time increased, and decreased gradually after 120 minutes incubation(P<0.05). The optimal incubation time for restoration of meiotic spindle and chromosomes in cooled oocytes was 60 minutes.

• 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.

• Korean Journal of Plant Taxonomy
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• v.35 no.1
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• pp.47-55
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• 2005

During the course of the field trip to Mt. Baekdu and Manchurian region especially along the border of North Korea, young flower buds were collected and fixed to analyze the meiotic configuration of chromosomes and to discuss the cytotaxonomic characteristics of these areas. A total of 29 taxa with 31 populations of vascular plants were analyzed chromosomally. Among them, 26 taxa with 28 populations were collected from Mt. Baekdu which include Papaver radicata var. pseudoradicata, Bupleurum euphorbioides, Oplopanax elatus, Silene jenisseensis, and Gentiana jamesii and three taxa collected from Manchurian region which include Scrophularia buergeriana, Ostericum grosseserratum and Callistephus chinensis. The first report of chromosome numbers were provided here for those species of Artemisia lagocephala as 2n = 9II, Cacalia komaroviana as 2n = 30II, Chrysanthemum zawadskii var. alpinum as 2n = 27II and Rodiola rosea as 2n = 11II,

• Development and Reproduction
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• v.18 no.2
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• pp.117-125
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• 2014

Vitrification method is widely used in oocyte cryopreservation for IVF but the birth rates are lower than that of the fresh oocyte. One of the known main reasons is structural instability of meiotic spindle and chromosome systems of mature oocyte. To get the best way for keeping competence of matured oocytes, we studied the best conditions for vitrification focused on equilibration times. The mature oocytes were underwent vitrification with current popular method and analyzed the survival rates, microtubule stability and DNA integrity. The survival rates of recovered oocyte are almost same between groups and are more than 93%. The structural configuration of meiotic spindle was well kept in 10 min equilibration group and the stability rate was almost same with that of control. The chromosomal breakdown was observed in all experimental groups, but the chromosomal stability was higher in 10 min equilibration group than the other groups. The 10 min equilibration group showed best condition compared with the other groups. Based on these results, the equilibration time is one of the key factors in successful keeping for competence of mature oocyte. Although, more fine analysis about the effects of physical stress on oocyte during vitrification is needed to define the optimal condition, it is suggested that the optimal equilibration time to get competent oocyte in mouse is 10 min. Information acquired this study may provide insight into intracellular structural events occurring in human oocytes after vitrification and application for cryopreservation of human oocyte.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.11 no.6
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• pp.2124-2128
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• 2010

In this study, we determined effects of the mitogen-activated protein kinase (MAPK) inhibitor, U0126 on meiotic maturation, microtubule organization and actin filament assembly in the porcine oocyte. The phosphorylated MAPK was first detected at 12 h after the initiation of maturation cultures, fully activated at 24h, and remained until metaphase II. Treatment of germinal vesicle (GV) stage oocytes with $20{\mu}M$ U0126 completely blocked MAPK phosphorylation, but germinal vesicle breakdown (GVBD) was normally proceeded. However, the oocytes didn‘t progress to the metaphase I. The inhibition of MAPK resulted in abnormal spindles. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, MAPK activity plays an important regulatory role in GV chromatin configuration and meiotic progress in porcine oocyte maturation.

• Journal of Embryo Transfer
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• v.33 no.1
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• pp.1-11
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• 2018

Cellular cyclic adenosine-3' 5'-monophosphate (cAMP) modulator is known as meiotic inhibitor and can delays spontaneous maturation in IVM experiment. Among many cAMP modulators, the role of Pituitary adenylate cyclase activating polypeptide (PACAP) on IVM isn't known. The purpose of this study is to improve the maturation of oocytes derived from follicles ${\leq}3mm$ in diameter through PACAP as meiotic inhibitor during pre-in vitro maturation (pre-IVM). First, we checked PACAP and its receptors in cumulus cells and, to establish the optimal phase and concentration of PACAP for pre-IVM, we conducted chromatin configuration assessments. As a result, the rate of GV (Germinal Vesicle) according to duration of pre-IVM was significantly decreased 12 h and 18 h after IVM (87.1 and 84.1%, respectively) compared to 0 h (99.4%). When COC was cultured for 18 h, the GV rate in the $1{\mu}M$ of PACAP treatment group (82.1%) was significantly higher than any other PACAP treatment groups (60.5, 64.1, 74.4 and 69.9 %, respectively). So, we divided into four groups as follows; MF (the conventional IVM group, obtained from follicle from 3 to 6 mm in diameter), SF (the conventional IVM group, obtained from follicle ${\leq}3mm$ in diameter), Pre-SF(-)PACAP (IVM group including 18 h pre-IVM without $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter) and Pre-SF(+)PACAP (IVM group including 18 h pre-IVM with $1{\mu}M$ of PACAP, obtained from follicle ${\leq}3mm$ in diameter). To examine the effect of PACAP during pre-IVM, we investigated analysis of nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. In cumulus cells, PACAP receptors, ADCYAP1R1 and VIPR1 were detected but were not detected in oocytes. After IVM, the Pre-SF(+)PACAP had the highest Metaphase II rate (91.7%) among all groups (P<0.05). The GSH levels in the MF and Pre-SF(+)PACAP were significantly higher than in the other groups (P<0.05) and ROS levels was no significant difference among all groups. In conclusion, these results indicated that even though the oocytes were derived from SF, pre-IVM application of PACAP improved meiotic and cytoplasmic maturation by regulating intracellular oxidative stress.

• Korean Journal of Animal Reproduction
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• v.24 no.2
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• pp.143-154
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• 2000

Most eggs initiated the fertilization processes but arrested at specific stages. The stages included failure of the oocyte to exit from the meiotic metaphase-II with or without sperm penetration, failure of appropriate sperm aster formation, inability to form proper male and female pronuclei, failure of suitable pronuclear apposition, and failure to form proper number of either male or female pronuclei. Various images of defective microtubule organization and chromatin configuration during IVF and ICSI procedures were observed. We discussed the data with previous research results during normal fertilization in humans and other mammals. In conclusion, various aberrant patterns in microtubule assembly and chromatin configuration, which were assessed in the present study, could be used as criteria to improve assisted reproductive technology in clinics. However, further cellular and molecular characterization is needed to clarify these aberrant patterns of cytoskeletal assembly.

• Korean Journal of Veterinary Research
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• v.34 no.4
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• pp.735-744
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• 1994

Catheters were placed into the external jugular veins of immature female rats. On the following day (day 28 of age), the animals were injected subcutaneously with pregnant mare serm gonadotropin(PMSG): 4IU(control) or 20IU(superovulation). Each animal was sequentially bled at Ohr and 12hr and subsequently at 6hr intervals until sacrifice at 72hr after PMSG. The superovulatory dose of PMSG significantly(P<0.05) increased the ovulatory response by 4.0 fold above controls. On the other hand, superovulated oocytes displayed considerably different stages of meiotic maturation: prophase I (14.7%), anaphase I (36.2%), telophase I (10.3%), metaphase I/II (32.4%), while in control rats a majority of the oocytes examined(94.0%) consistently showed a metaphase II configuration. Serum luteinizing hormone(LH) levels were determined by RIA. Both groups exhibited a similar time relationship with two distinct peaks: an initial slight rise at 0-18hr and a second sharp rise at 54-60hr. However, there was a marked change in the magnitude of LH levels between the two groups. In superovulated animals, prior to the second peak, overall LH levels were significantly(P<0.001) higher than controls. In contrast, at the peak occurring at 60hr, LH concentrations were significantly(P<0.001) reduced by 54% below that of control. Additionally, a maximum increase of mean ${\Delta}LH$ between two peaks was much less in superovulated as compared to control rats. The initial prolonged elevation of serum LH before 54hr in superovulated rats was found to result from actual cross-reaction of the injected PMSG with LH antibody in the assay, while a precipitous second elevation between 54hr and 60hr resulted primarily from an endogenous LH surge. This study clearly defines time-course features of serum LH in PMSG-treated rats. The overall results indicate that, following superovulatory treatment with PMSG, the increased ovulatory response is primarily associated with PMSG-derived intrinsic gonadotropin, and that the recovery of immature or asynchronously mature oocytes at ovulation may reult from the circulatory alteration of LH activity characterized by an initial prolonged elevation of serum LH and its subsequent attenuation.