• Asian-Australasian Journal of Animal Sciences
• /
• 제32권8호
• /
• pp.1112-1121
• /
• 2019

Objective: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Methods: First, we developed an in vitro model to study the response of bovine cumulusoocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in $10{\mu}g/mL$ of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-${\kappa}B$); and the concentrations of interleukin (IL)-$1{\beta}$, tumor necrosis factor (TNF)-${\alpha}$, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Results: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-${\kappa}B$. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-$1{\beta}$, TNF-${\alpha}$, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. Conclusion: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

• 한국수정란이식학회지
• /
• 제15권1호
• /
• pp.15-21
• /
• 2000

These studies were undertaken to evaluate morphological normality and development competence in vitro of hyman oocytes following vitrificatioin using ethylene glycol and electron microscopic grid. Human immature oocytes retrieved from natural and stimulated cycles was vitrified at 0 or 48 h and 0, 8 to 15 or 24 to 28 h after maturation culture, respectively. In oocytes retrieved from unstimulated cycle, no signifciant differences were found in morphological normality (56 to 63%) and fertilization (31 to 37%) rates between the times of vitrification. In stimulated patients, however, more oocytes were morphologically normal when vitrified at 24 to 28 h than when vitrified at 0 or 8 to 15 h after maturation culture. Regardlesss of the hormonal stimulation, high cleavage rates(83 to 100%) were obtained in all treatment groups but did not differ significantly. Twenty to 43% of cleaved oocytes developed to the blastocyst stage at 6 days after IVF. These results suggest that vitrified oocytes from unstimulated and stimulated cycles could develop to the blastocyst stage, regardless of the stages of vitrification.

• 농업과학연구
• /
• 제45권3호
• /
• pp.493-498
• /
• 2018

There has been widespread warming and a general increase in summer temperatures over the Korean peninsula ($0.5^{\circ}C$/10 years from 2001 to 2010). South Korea is transforming into a subtropical region, and the productivity of livestock is affected by the climatic changes. In this study, we investigated whether the summer heat waves affect the developmental competency of Korean native cattle (Hanwoo), a taurine type of cattle with a small portion of indicine varieties. We collected oocytes during the summer (heat stress, HS) and autumn (non-HS condition) and examined the developmental competencies including in vitro maturation and preimplantation embryo development. No significant differences were observed between the HS and non-HS oocytes in nuclear maturation (extrusion of the polar body); however, the cleavage and blastocyst rates were significantly lower in the HS group than those in the non-HS group. The lower developmental competence of the HS oocytes compared to the non-HS is, in part, due to insufficient cytoplasmic maturation because of a higher production of Reactive oxygen species (ROS) levels as well as peri/cortical distributed mitochondria in the HS oocytes after in vitro maturation. Next, we examined the ROS and mitochondria distribution and found a significant increase in the levels of ROS in the HS oocytes and a polarized distribution (pericortical cytoplasm) of mitochondria in the HS oocytes. In summary, impaired cytoplasmic maturation of oocytes from exposure to HS affects the preimplantation embryo development by dysfunction of mitochondria. To improve reproductive performance, embryo transfer using cryopreserved embryos/oocytes is recommended in the hot summer season of South Korea.

• 한국수정란이식학회지
• /
• 제31권1호
• /
• pp.39-46
• /
• 2016

Cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig embryos, because of their greater susceptibility to cryoinjuries, have shown a reduced developmental competence. The aim of this study was to evaluate the survival status of vitrified-warmed porcine embryos. Forced blastocoele collapse (FBC) and non-FBC blastocysts are vitrified and concomitantly cultured in culture media which were supplemented with/without fetal bovine serum (FBS). Porcine vitrified-warmed embryos were examined in four different methods: group A, non-FBC without FBS; group B, non-FBC with FBS; group C, FBC without FBS; group D, FBC with FBS. After culture, differences in survival rates of blastocysts derived from vitrified-warmed porcine embryos were found in group A~D (39.5 (A) vs 52.5 (B) and 54.8 (C) vs 66.7% (D), respectively, p<0.05). Reactive oxygen species (ROS) level of survived blastocysts was lower in group D than that of another groups (p<0.05). Moreover, total cell number of survived blastocysts was higher in group D than that of other groups (p<0.05). Otherwise, group D showed significantly lower number of apoptotic cells than other groups ($2.0{\pm}1.5$ vs $3.2{\pm}2.1$, $2.8{\pm}1.9$, and $2.7{\pm}1.6$, respectively, p<0.05). Taken together, these results showed that FBS/FBC improves the developmental competence of vitrified porcine embryos by modulating intracellular levels of ROS and the apoptotic index during the vitrification/warming procedure. Therefore, we suggest that FBS and FBC are effective treatment techniques during the vitrification/warming procedures of porcine blastocysts.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2006년도 The 6th Intermational Symposium on Developmental Biotechnology
• /
• pp.95-96
• /
• 2006

• 한국수정란이식학회지
• /
• 제23권4호
• /
• pp.291-298
• /
• 2008

We separately cultured follicular oocytes collected from individual ovaries of slaughtered Korean native cows and examined both the embryonic development rate and pregnancy rate after embryo transplantation according to the meat yield and quality grades of the source beef carcass. Oocytes from meat yield grade B cows exhibited a higher fertilization rate and embryonic developmental rate to the eight-cell stage than oocytes from grade A or C animals (p<0.05), but there was no significant difference in rate of development to the blastocyst stage among meat yield grades A, Band C. The oocyte cleavage rate and development rate to the eight-cell stage from meat quality grade 3 cattle was higher than grades 1++, 1+, 1 and 2 (p<0.05). Embryos derived from grade animals displayed a development rate to the blastocyst stage of 19.4%, which was also higher than all other meat quality grades (p<0.05). Transplantation of in vitro-cultured oocytes from meat yield grade A ovaries led to a higher pregnancy rate (64.2%) than in vitro-cultured oocytes from meat yield grade B ovaries (56.5%), but there was no significant difference between the two groups in pregnancy or abortion rates. In conclusion, embryonic development rate and pregnancy rate has a close relation to meat quality grades of the source beef carcass, this results is to give information for the Korean native cows improvement of breed.

• 한국발생생물학회지:발생과생식
• /
• 제22권4호
• /
• pp.297-307
• /
• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• 한국동물생명공학회지
• /
• 제37권2호
• /
• pp.121-129
• /
• 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.

• Asian-Australasian Journal of Animal Sciences
• /
• 제30권4호
• /
• pp.585-592
• /
• 2017

Objective: The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods: Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results: The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture ($143.8{\pm}10.5$ to $159.2{\pm}14.8$) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups ($31.4{\pm}8.3$ to $33.4{\pm}11.1$). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05) between sexes. Conclusion: The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

• 한국수정란이식학회지
• /
• 제23권4호
• /
• pp.275-281
• /
• 2008

The ability to preselect the sex of piglets is advantageous in the pig industry. The objective of this study was to examine the feasibility of using intracytoplasmic sperm injection (ICSI) with sorted spermatozoa to produce piglets with a preselected sex. Pig embryos were produced by ICSI of frozen X- and Y-sperm that had been separated by flow cytometry. The developmental competence of the embryos was investigated in vitro and in vivo. The populations of X- and Y-spermatozoa were 52.7% and 47.3%, respectively in our samples. The in vitro development of ICSI embryos was enhanced by longer of in vitro maturation of oocytes ($44{\sim}48\;h$ vs. $40{\sim}43\;h$). Their cleavage ($65{\sim}70%$) and blastocyst formation ($9{\sim}12%$) rates were not significantly different between male and female ICSI embryos, or between sorted and unsorted sperm-derived embryos. One pregnancy was established in a recipient that was transferred with 110 female ICSI embryos, but the pregnancy was terminated on Day 89 of gestation. Our results suggest that the separation X- and Y-spermatozoa by flow cytometric sorting can be a useful tool in combination with ICSI for the production of pig embryos and piglets of preselected sex.