• Reproductive and Developmental Biology
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• 제28권2호
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• pp.101-105
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• 2004

This study was carried out to evaluate the effects of glucose and sodium phosphate on in vitro development of porcine oocytes matured and fertilized in vitro. When the culture medium was supplemented with various concentrations of glucose, the higher proportions (23 and 26%) of oocytes developed to morular or blastocyst stages were at the concentrations of 2.78 and 5.56 mM than 0 (9%; P<0.05) and 11.12 mM (18%). In experiment to evaluate effect of sodium phosphate during in vitro development of porcine oocytes, a significantly (P<0.05) higher proportions of embryos developed to morular or blastocyst stages was obtained with sodium phosphateof 0.28 (25%) and 0.53 (27％) mM than 0 (15%), 1.05 (19%) and 2.10 (10%) mM. On the other hand, when oocytes were cultured in medium with (0.53 mM) sodium phosphate, the proportions of developed embryos were significantly (P<0.05) higher in medium without (29%) that than with (14%) 5.56 mM glucose. However, a higher proportion of embryos developed to morular or blastocyst stages were obtained in medium with (23%) that than without (8%) glucose (P<0.05). The minimum essential medium (MEM) added to the culture medium were higher regardless of presence of sodium phosphate and glucose on the development of embryos. Although sodium phosphate and glucose could support morular and blastocyst development to a limited extend (10∼24%), significantly higher proportion (36%) at morular or blastocyst stages was obtained by MEM adding in the medium with sodium phosphate and glucose. These results suggest that the early development of in vitro fertilized porcine oocytes can be maintained efficiently by glucose and sodium phosphate when they were cultured in medium with MEM.

• 대한수의학회지
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• 제63권4호
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• pp.40.1-40.7
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• 2023

To optimize the most efficient method for porcine in vitro maturation (IVM), we compared the effects of supplementing extracellular vesicles (EVs) derived from porcine follicular fluid (pFF). The cumulus oocyte complexes were grouped into 4 groups with different supplementations as following: pFF (G1), pFF-depleted EVs (G2), EVs (G3) and control (G4) groups. After IVM with different supplementations, maturation rates and the developmental competences of porcine oocytes and blastocyst development were investigated. Additionally, glutathione (GSH) and reactive oxygen species (ROS) levels were measured in mature oocytes. The EVs were isolated and characterized with cryo-TEM and nanoparticle tracking analysis. The pFF significantly affected the maturation rate, whereas the presence of EVs did not show notable difference in the maturation rates. Although there were numerical increases in the measured parameters in EV and pFF-depleted EVs groups, no significant differences were observed between them. The EV group showed similar oocyte maturation rate for both positive and negative control groups. The GSH was not different among the groups, but ROS levels were significantly lower in pFF-supplemented group when compared with other groups with the highest level in the control group. G2 group wasn't significantly different G1 and G3 group. G3 group wasn't significantly different from G2 and G4 group. This suggests that EVs in IVM medium which probably effected partially to protect against oxidative stress and potentially enhance the quality of oocytes. This study indicates that the EVs in pFF play a significant role in improving the efficiency of oocyte maturation in porcine.

• 한국가축번식학회지
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• 제21권4호
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• pp.381-387
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• 1997

This study was conducted to investigate the effects of fetal bovine serum(FBS), calf serum(CS) and human serum(HS) on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows : 1. The maturation rates of oocytes cultured in medium containing FBS 5, 10, 20 and 30% were 47.0, 63.5, 48.4 and 43.2%, respectively. There were significantly higher than those of non-treated group(25.3%). And the highest maturation rate was the 10% treatment. 2. The maturation rates of oocytes cultured in medium containing CS 5, 10, 20 and 30% were 55.2, 56.6, 59.4 and 46.5%, respectively. There were significantly higher than those of non-treated group(25.3%). And the highest maturation rate was the 20% treatment. 3. The maturation rates of oocytes cultured in medium containing HS 5, 10, 20 and 30% were 74.5, 78.2, 73.1 and 68.6%, respectively. There were significantly higher than those of non-treated group(29.6%). And the highest maturation rate was the 10% treatment.

• 한국가축번식학회지
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• 제22권3호
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• pp.265-276
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• 1998

In an attempt to evaluate the function of MAP kinase of porcine oocytes and to develop a method of assessment for kinase activity, we used MBP as a substrate to detect the MAP kinase activity of porcine oocytes matured in in vitro. The MAP kinase which had lower activity during the first 20 hours of culture started to show an increased amount of activity at 25 hours at which a collapse in nuclear membrane was induced. Significant (P<0.05) a, pp.ared at 30 hours of being cultured. The gel phosphorylation method, MBP which has been known to be a substrate for kinase such as cdc2 kinase, was phosphorylated at two positions corresponding to ERK 1 (44kDa) and ERK2 (42 kDa) which are known as mammalian MAP kinase. The existence of MARKK and MAP kinase were identified with western blotting at 0 hour culture of immature GV oocytes. The amount of those proteins did not increase during 40 hours of culture, which suggest that the increase of MAP kinase activity was caused by phosphorylaton rather than due to change in protein amount. MAPKK and MAP kinase were shown to be dephosporylated with deactivated at M 1 stage by inhibition of protein synthesis with cycloheximide added at the strat following the cultrue. We have reulsts that indicate the existedence of MAP kinase cascade which was activated simultaneously with start of porcine oocyte maturation (GVBD).

• Reproductive and Developmental Biology
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• 제36권4호
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• pp.237-241
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• 2012

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although it has been suggested that EGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier; however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribes mRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylated in the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminal domain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptional elongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, and phosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes, two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for the IIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained strongly in the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatin of prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II began to accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to the blastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, and that transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNA polymerase II.

• 한국가축번식학회지
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• 제22권4호
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• pp.411-417
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• 1998

본 연구는 난관액과 oviductal conditioned medium 이 다정자 침입과 체외배발달에 미치는 효과를 규명하기 위하여 수행하였다. 배양액내 난관액과 oviductal conditioned medium 의 첨가는 다정자침입율과 난자내 침입한 평균정자수를 감소시켰다 (P＜0.05). 정자와 난관액 그리고 oviductal conditioned medium 과 l.5, 3, 4.5 시간 공배양후 첨체반응의 성적은 대조구에 비하여 증가하였다. 체외수정후 체외발달 배양액에 난관액이나 oviductal conditioned medium 을 첨가하여 192 시간동안 배양했을 때 상실배와 배반포배발달율이 난관액과 oviductal conditioned medium 첨가구에서 유의적으로 높았다 (P＜0.05). 이러한 연구결과는 난관액과 OVCM 등 난관 유래물질은 다정자침입율과 난자내 침입한 평균정자수를 감소시키며 체외발달율을 높인다고 사료된다.

• 한국발생생물학회지:발생과생식
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• 제21권2호
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• pp.131-138
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• 2017

This study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta ($Sec61{\beta}$), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVM I) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVM II). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, $Sec61{\beta}$, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVM I or IVM II stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, $Sec61{\beta}$ and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but $Sec61{\beta}$ and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, $Sec61{\beta}$, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of $Sec61{\beta}$ and COPG2 could be changed by EGF in the porcine oocytes during maturation.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Reproductive and Developmental Biology
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• 제33권1호
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• pp.29-33
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• 2009

The brilliant cresyl blue (BCB) has been used to select the developmental competent oocytes in pigs, goats and cows. Growing oocytes have a higher level of active glucose-6-phosphate dehydrogenase(G6PDH) compare to mature oocytes and are rarely stained compared to mature oocytes, because G6PDH converts BCB to colorless. First polar body extrusion regard as a guideline of meoisis completion. Selection of polar body extrude oocyte is more developmental competent to blastocyst than unselected. This study was conducted to compare the BCB test to the polar body extrusion on selection of developmental competent porcine oocytes for the production of blastocyst. Cumulus-Oocytes complex were exposed to 26uM BCB stain diluted in NCSU-23 for 90 min. There was no significant difference embryo development to blastocysts between BCB treated and not treated($19.58{\pm}1.99$ vs $18.75{\pm}2.27%$), which means there was no detrimental effect of BCB exposure to oocytes. Normal fertilization is not differed among treatment groups from 70.0 to 78.4% development to blastocyst, beside polyspermy did not. To compare two different selection methods, BCB test and polar body extrusion, evaluate the developmental competent of IVP embryos. BCB+PB+(blue stained and polar body extruded, $20.71{\pm}0.45%$) and BCB-PB+(colorless and polar body extruded, $20.04{\pm}l.29%$) groups are significantly (p<0.05) higher developed than those of BCB+PB-(blue stained and no polar body, $13.24{\pm}0.73%$) and BCB-PB-(colorless and no poladbody, $7.25{\pm}0.77%$). These results showed that selection of polar body extruded oocytes method is more efficient than that of BCB test.

• 한국수정란이식학회지
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• 제9권1호
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• pp.117-125
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• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.