• Journal of Embryo Transfer
• /
• v.11 no.2
• /
• pp.179-191
• /
• 1996

In-vitro culture has provided new inforrnation on mechanisms of oocytes rnaturation and results obtained in vitro have led to new questions. In porcine, follicular and oocyte size have the crucial importance for the oocytes maturation. The addition of hormones to the culture medium was found to accelerate and facilitate meiotic maturation. The presence of some factors in serum trigger the resumption of meiosis and support the maturation of oocytes in vitro. The maturation rate of porcine oocytes was also increased by supplementation of porcine follicular fluid to the culture medium. The growth factors can stimulate nuclear maturation and enhances cytoplasnic maturation of oocytes by interaction with gonadotropins. The maturation-promoting factor brings about GVBD and the subsequent maturational events in oocytes. However, cAMP can block the spontaneous meiotic maturation of oocytes in culture. The understanding of these influences is a prerequisite to enhancing in vitro maturation of porcine oocytes.

• Reproductive and Developmental Biology
• /
• v.28 no.3
• /
• pp.187-190
• /
• 2004

Protein kinase C exists as a family of serine/threonine kinases which are broadly classified into three groups as cPKC nPKC and aPKC depending on their cofactor requirements. Previous studies have shown that the role of PKC in the process of mouse oocyte maturation. For example, phorbol 12-myristate 13-acetate which is known as an activator of cPKC and nPKC inhibits germinal vesicle break down and 1st polar body extrusion in maturing oocytes. In this study, the effect of thymeleatoxin, a specific activator of cPKC not nPKC, was tested comparing with PMA to address the roles of cPKC and nPKC during mouse oocyte maturation. Cumulus-oocyte complex were cultured in M16 medium for 6 or 12 hr with each of these PKC activators to investigate the effect of germinal vesicle breakdown (GVBD) or the extrusion of 1st polar body. IC/sup 50/ of GVBD were at concentrations of 50nM in PMA and 400nM in thymeleatoxin and of 1st polar body extrusion were 20nM in PMA and 200nM in thy- meleatoxin. The results suggest that activation of nPKC is more closely related to the inhibition of GVBD and 1st polar body extrusion than activation of cPKC. Additionally, we found that the oocytes inhibited 1st polar body extrusion with PMA or thymeleatoxin were arrested in metaphase I of first meiosis.

• Proceedings of the Korean Society of Developmental Biology Conference
• /
• 2009.02a
• /
• pp.49-57
• /
• 2009

The cloning of canids was succeeded in 2005, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay of successful somatic cell nuclear transfer (SCNT)was due to the unique reproductive characteristics of the female dogin comparison to other domestic mammals, such as ovulation of immature canine oocyte and a requirement of 25 days for the completion of meiosis within the oviduct (Holst & Phemister, 1971). When the technology for the recovery of in vivo matured oocyte was established, the application of cloning also became possible and cloned dog offspring were obtained. This report summarizes the progress of technical procedures that are required for cloning canids and the application of this technique. The first cloned dog, Snuppy, was achieved using an in vivo-matured oocyte which was enucleated and transferred with an adult skin cell of male Afghan hound. After establishment of a criterion of well-matured oocyte for the improvement of SCNT efficiency, we obtained three cloned female Afghan hound and a toy poodle cloned from 14 year-old aged Poodle using SCNT through this factor. To date, cloned dogs appeared to be normal and those that have reached puberty have been confirmed to be fertile. Through application of canine SCNT technique, first, we demonstrated that SNCT is useful for conserving the breed of endangered animal from extinction through cloning of endangered gray wolves using inter-species SCNT and keeping the pure pedigree through the cloning of Sapsaree, a Korean natural monument. Secondly, we showed possibility of human disease model cloned dog and transgenic cloned dog production through cloning of red fluorescent protein expressing dog. Finally, SCNT can be used for the propagation of valuable genotypes for making elite seed stock and pet dog. In summary, dog cloning is a reproducible technique that offers the opportunity to preserve valuable genetics and a potential step towards the production of gene targeted transgenic cloned dogs for the study of human diseases.

• Reproductive and Developmental Biology
• /
• v.34 no.3
• /
• pp.217-221
• /
• 2010

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro condition is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. This study analyzed change of proteins in follicular fluids during the porcine follicular development. Follicular fluids were collected from follicles of diameter 1~2 mm, 2~6 mm and 6~10 mm in ovary of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. We confirmed totally 27 same spots, 1 spot from follicle fluid of 2~6 mm follicle and 5 spots from follicle fluid of 6~10 mm in diameter were analyzed by MALDI mass spectrometry and searched on NCBInr. In results, spot No. 28 from 2~6 mm follicle was Ig lambda chain C region, and spot No.32 and 33 from 6~10 mm was Apolipoprotein A-(APOA4). Spot No.29 and 31 were failed to analyze. These results indicate that the porcine oocyte during in vitro maturation depend on specific different expressed proteins may play an important roles in the sequence of molecular events in porcine oocyte maturation and follicular development.

• Clinical and Experimental Reproductive Medicine
• /
• v.44 no.1
• /
• pp.15-21
• /
• 2017

Objective: The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of $Ca^{2+}$ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). Methods: MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a $Ca^{2+}$ chelator to investigate the effect of $Ca^{2+}$ oscillations on their maturation. Results: As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the $Ca^{2+}$ chelator-treated group. Conclusion: The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by $Ca^{2+}$ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular $Ca^{2+}$ oscillations driven by fertilization.

• Development and Reproduction
• /
• v.16 no.1
• /
• pp.59-67
• /
• 2012

Previously, we found that $Zap70$ (Zeta-chain-associated protein kinase) expressed in the mouse oocytes and played significant role in completion of meiosis specifically at MI-MII (metaphase I-II) transition. Microinjection of $Zap70$ dsRNA into the cytoplasm of germinal vesicle oocyte resulted in MI arrest, and exhibited abnormalities in their spindles and chromosome configurations. The purpose of this study was to determine the mechanisms of action of $Zap70$ in oocyte maturation by evaluating downstream signal networking after $Zap70$ RNAi (RNA interference). The probe hybridization and data analysis were used by Affymetrix Gene Chip Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. Total 1,152 genes were up (n=366) and down (n=786) regulated after $Zap70$ RNAi. Among those genes changed, we confirmed the expressional changes of the genes involved in the regulation of actin cytoskeleton and MAPK (mitogen-activated protein kinase) signaling pathway, since the phenotypes of $Zap70$ RNAi in oocytes were found in the changes in the chromosome separation and spindle structures. We confirmed the changes in gene expression in the actin skeletal system as well as in the MAPK signaling pathway, and concluded that these changes are main cause of the aberrant chromosome arrangement and abnormal spindles after $Zap70$ RNAi.

• Clinical and Experimental Reproductive Medicine
• /
• v.48 no.4
• /
• pp.352-361
• /
• 2021

Objective: The study assessed the developmental potential of germinal vesicle (GV) oocytes subjected to in vitro maturation (IVM) after prematuration culture with cilostamide (a phosphodiesterase-3 inhibitor) and the impact of cilostamide exposure on the morphology of meiosis II (MII) oocytes and subsequent embryo quality. Methods: In total, 994 oocytes were collected from 63 patients. Among 307 GV oocytes, 140 oocytes were selected for the experimental group and 130 oocytes for the control group. The denuded GV-stage oocytes were cultured for 6 hours with cilostamide in the experimental group and without cilostamide in the control group. After 6 hours, the oocytes in the experimental group were washed and transferred to fresh IVM medium. The maturational status of the oocytes in both groups was examined at 26, 36, and 48 hours. Fertilization was assessed at 18 hours post-intracytoplasmic sperm injection. Embryo quality was assessed on days 3 and 5. Results: In total, 92.1% of the oocytes remained in the GV stage, while 6.4% converted to the MI stage (p<0.01) after cilostamide exposure. In both groups, more MII oocytes were observed at 36 hours (25.8% vs. 21.5%) than at 26 hours (10.8% vs. 14.6%) and 48 hours (13% vs. 7.9%) (p>0.05). With the advent of cilostamide, blastocyst quality was better in the experimental group than in the control group (p<0.05). Conclusion: Cilostamide effectively blocked nuclear maturation and promoted cytoplasmic growth. Prematuration culture with cilostamide enabled synchronization between cytoplasmic and nuclear maturity, resulting in better blastocyst outcomes.

• Clinical and Experimental Reproductive Medicine
• /
• v.40 no.1
• /
• pp.1-6
• /
• 2013

Objective: Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system. Methods: Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were also fused to the GFP expression vector. The recombinant vectors were transfected into HEK-293T cells plated onto coated glass coverslips. The transfected cells were stained with 4',6-diamidino-2-phenylindol and photographed using a fluorescence microscope. Results: Mutants containing the HD region as well as full-length Obox4 were clearly localized to the nucleus. In contrast, the other mutants of either the N-terminal or C-terminal region without HD had impaired nuclear localization. We also found that the N-terminal and C-terminal of the Obox HD contributed to nuclear localization and the entire HD was necessary for nuclear localization of Obox4. Conclusion: Based on the results of the present study, we demonstrated that the intact HD region of Obox4 is responsible for the nuclear localization of Obox4 protein in cells.

• Clinical and Experimental Reproductive Medicine
• /
• v.1 no.1
• /
• pp.49-54
• /
• 1974

In the present studies, effect of progesterone on the germinal vesicle break-down of the mouse oocytes cultured in the micro tube was investigated. The results obtained are as follows: As dose of progesterone in the medium rose, accordingly the break-down of the germinal vesicle was suppressed. It was found that $ED_{50}$ was 15.7 ${\mu}g$/ml, and $ED_{90}$ 60.7 ${\mu}g$/ml of progesterone. The dose suppressing the oocyte maturation was apparently higher than that on the rabbit or on the mouse embryonal development. The inhibiting effect of progesterone on the GVBD was reversible. The germinal vesicle of the oocytes were broken down immediately in the medium upon removal of the hormone. Progesterone stops meiosis at any stage upon administration, while dbe AMP or theophylline supresses only the break-down of the nuclear membrane. Recovering of the meiotic division of the oocytes once exposed to progesterone was delayed a little. The inhibiting action of progesterone was not altered by adding more pyruvate or in the presence of higher concentration of the mineral ions in the culture medium.

• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.6
• /
• pp.781-787
• /
• 2017

Objective: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.