• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.343-351
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• 1996

The objectives of this study are to determine cortical granule distribution during in vitro maturation, parthenogenetic activation and in vitro fertilization of oocytes, and to investigate effects of microfilament inhibitor on the cortical granule distribution during in vitro maturation and fertilization of oocytes in the pig, The corti-cal granule distribution were imaged with fluor-escent labeled lectin under laser scanning confocal microscope or detected by transmission electron microscope. At germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively thick area on the oolema. Microfilaments were also observed in a thick uniform area around the cell cortex. Following germinal vesicle break down,microfilaments concentrated to the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerization and prevented movement of cortical granules to the cortex. Cortical granule exudate following sperm penetration was evenly distributed in the entire perivitelline space. Therefore, these results suggested that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilization of porcine oocytes. (Key words cortical granule, porcine, maturation, fertilization).

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.29-33
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• 2002

This study was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 24 hrs post onset of maturation, the oocytes were cultured $3{\sim}13{\mu}M\;Ca^{2+}$ for 5 min., $5-8{\mu}g/ml$ cytoclacin for 6 hrs, 0.5~2.0 mM 6-dimethylaminopurine(DMAP) for 3 hrs alone or combination. The activated oocytes were cultured in TCM-199 media at 5% $CO_2$, 95% air, $38^{\circ}C$. The cleavage rate after 48 hrs culture of oocytes treated with $3-13{\mu}M\;Ca^{2+}$, $5-8{\mu}g/ml$ cytoclacin and 0.5~2.0 mM DMAP for 5 min., 6 hrs and 3 hrs were 9.6%~20.0%, 0.0%~7.3% and 9.4%~21.8%, 0.0%~7.3% and 9.1%~21.8% and 0.0%~7.3%, respectively. When oocyte were treated with $10{\mu}M\;Ca^{2+}$, $10{\mu}g/ml$ cytoclacin and 2.0 mM DMAP the blastocyst formation rate was significantly higher than other group. The cleavage rate after 48 hrs culture of oocytes treated with $Ca^{2+}$ + cytoclacin, $Ca^{2+}$ + DMAP, cytoclacin + DMAP were 75.9%~93.5% and 9.7%~19.0%, respectively. When oocytes were treated with $Ca^{2+}$ followed by DMAP, the blastocyst formation rate was significantly higher than other group(p<0.05). When necleus transferred embryos co-cultured with bovine serum albumin(BSA), epithemal growth factor(EGF) and calf serum(CS), the developmental rate to blastocyst were higher than control group.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.111-122
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• 2017

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in $75{\mu}M$ of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

• Korean Journal of Veterinary Research
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• v.59 no.2
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• pp.81-88
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• 2019

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF ($113.0-114.8{\mu}m$) were significantly larger than that of no IVG-SAF ($111.8{\mu}m$). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.121-129
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• 2022

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an N6-methyladenosine (m⁶A) RNA modification regulator and a key determinant of prem-RNA processing, mRNA metabolism and transportation in cells. Currently, m⁶A reader proteins such as hnRNPA2/B1 and YTHDF2 has functional roles in mice embryo. However, the role of hnRNPA2/B1 in porcine embryogenic development are unclear. Here, we investigated the developmental competence and mRNA expression levels in porcine parthenogenetic embryos after hnRNPA2/B1 knock-down. HhnRNPA2/B1 was localized in the nucleus during subsequent embryonic development since zygote stage. After hnRNPA2/B1 knock-down using double stranded RNA injection, blastocyst formation rate decreased than that in the control group. Moreover, hnRNPA2/B1 knock-down embryos show developmental delay after compaction. In blastocyste stage, total cell number was decreased. Interestingly, gene expression patterns revealed that transcription of Pou5f1, Sox2, TRFP2C, Cdx2 and PARD6B decreased without changing the junction protein, ZO1, OCLN, and CDH1. Thus, hnRNPA2/B1 is necessary for porcine early embryo development by regulating gene expression through epigenetic RNA modification.

• Animal Bioscience
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• v.37 no.6
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• pp.1007-1020
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• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.39 no.1
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• pp.48-57
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• 2024

Background: Cadmium (Cd) is toxic heavy metal that accumulates in organisms after passing through their respiratory and digestive tracts. Although several studies have reported the toxic effects of Cd exposure on human health, its role in embryonic development during preimplantation stage remains unclear. We investigated the effects of Cd on porcine embryonic development and elucidated the mechanism. Methods: We cultured parthenogenetic embryos in media treated with 0, 20, 40, or 60 µM Cd for 6 days and evaluated the rates of cleavage and blastocyst formation. To investigate the mechanism of Cd toxicity, we examined intracellular reactive oxygen species (ROS) and glutathione (GSH) levels. Moreover, we examined mitochondrial content, membrane potential, and ROS. Results: Cleavage and blastocyst formation rates began to decrease significantly in the 40 µM Cd group compared with the control. During post-blastulation, development was significantly delayed in the Cd group. Cd exposure significantly decreased cell number and increased apoptosis rate compared with the control. Embryos exposed to Cd had significantly higher ROS and lower GSH levels, as well as lower expression of antioxidant enzymes, compared with the control. Moreover, embryos exposed to Cd exhibited a significant decrease in mitochondrial content, mitochondrial membrane potential, and expression of mitochondrial genes and an increase in mitochondrial ROS compared to the control. Conclusions: We demonstrated that Cd exposure impairs porcine embryonic development by inducing oxidative stress and mitochondrial dysfunction. Our findings provide insights into the toxicity of Cd exposure on mammalian embryonic development and highlight the importance of preventing Cd pollution.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.65-70
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• 2006

This study investigated apoptosis and in vitro development of parthenogenetic preimplantation porcine embryos. In vitro matured oocytes for $42{\sim}44h$ were used. Apoptotic cell death was analyzed by using a terminal deoxynucleatidyl transferase mediated deoxyuridine 5-triphosphate nick-end tabling (TUNEL) assay. In experiment 1, oocytes were activated with two electric pulses (CH) of 1.2 kV/cm for $30{\mu}sec$ (E), E + 6-dimethylaminopurine (6-DMAP) or E + cycloheximide (CH) and cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$. In experiment 2, oocytes were activated by E and cultured in PZM-3 or NCSU-23 under a gas atmosphere of 20% $O_2$ ($5%\;CO_2$, in air) or 5% $O_2$ $(5%\;CO_2,\;5%\;O_2\;90%\;N_2)\;at\;38.5^{\circ}C$. Oocytes activated with E+6-DMAP or E+CH showed higher blastocyst rates (36.3% and 32.5%) compared to E alone (27.7%). The frequency of apoptosis according to treatments were 5.3%, 7.7% and 7.1% respectively. Oocytes activated with E alone showed lower (P<0.05) frequency of apoptosis compared to other groups. In experiment 2, parthenotes cultured in PZM-3 showed slightly higher blastocyte rates (28.2% and 29.7%) compared to NCSU-23 (22.6% and 24.4%) regardless of atmosphere. Blastocysts generated in PZM-3 showed lower (P<0.05) apoptosis rate under 20% $O_2$ (9.2% vs 16.9%), whereas those in NCSU-23 had slightly lower apoptosis rate under 5% $O_2$ (14.0% vs 18.4%). This result represents that activation method and culture condition could affect the frequency of apoptosis as well as in vitro developmental rate.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.213-220
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• 2009

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ${\mu}g/ml$) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or $20{\sim}80{\mu}g/ml$ FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes ($85{\sim}89%$) was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage ($86{\sim}94%$) and mean number of cells in blastocyst ($33{\sim}37$ cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 ${\mu}g/ml$ FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 ${\mu}g/ml$ FSH,i numectivelo. In SCNT, fusion ($78{\sim}83%$) of cell-cytoplast couplets and siosequent embryo cleavage ($82{\sim}88%$) were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 ${\mu}g/ml$ FSHr(25% vs. $11{\sim}18%$). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 ${\mu}g/ml$ FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.127-134
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• 2010

This study was to investigate the effect of flavonoid treatment on in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos, and their pregnancy and delivery rate after embryo transfer into recipient. In experiment 1, to optimize the flavonoid concentration, parthenogenetic day 2 ($\geq$ 2-cell) embryos were cultured in 0 (control), 1, 10 and $20\;{\mu}M$ flavonoid for 6 days. In the results, in vitro development rate was the highest in $10\;{\mu}M$ flavonoid group (57.1%) among treatment groups (control, 49.5%; $1\;{\mu}M$, 54.2%; $20\;{\mu}M$, 37.5%), and numbers of total and ICM cells were significantly (p<0.05) higher in $10\;{\mu}M$ flavonoid group than other groups. We found that $10\;{\mu}M$ flavonoid treatment can significantly (p<0.05) decrease the apoptotic index and derive high expression of anti-oxidant, anti-apoptotic, cell growth and development marker genes such as Mn-SOD, Survivin, Bax inhibitor, Glut-5, In-tau, compared to control group. In experiment 2, to produce the cloned Jeju Black Cattle, beef quality index grade 1 bull somatic cells were transferred into enucleated bovine MII oocytes and reconstructed embryos were cultured in $10\;{\mu}M$ flavonoid added medium. When the in vitro produced day 7 or 8 SCNT blastocysts were transferred into a number of recipients, $10\;{\mu}M$ flavonoid treatment group presented higher pregnancy rate (10.2%, 6/59) than control group (5.9%, 2/34). Total three cloned Jeju Black calves were born. Also, two cloned calves in $10\;{\mu}M$ flavonoid group were born and both were all healthy at present, while the one cloned calf born in control group was dead one month after birth. In addition, when the result of short tandem repeat marker analysis of each cloned calf was investigated, microsatellite loci of 11 numbers matched genotype between donor cell and cloned calf tissue. These results demonstrated that the flavonoid addition in culture medium may have beneficial effects on in vitro and in vivo developmental capacity of SCNT embryos and pregnancy rate.