• 한국가축번식학회지
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• 제11권1호
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• pp.73-84
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• 1987

This experiment was carried out to investigate the effects of hormone addition(FSH, HCG, estrogen and progesterone) and composition (BSA and FCS) of mKRB on the in vitro maturation and fertilizability of follicular oocytes of the Korean native cattle. The ovaries were removed at a slaughterhouse, returned to laboratory in a thermostat (30-35$^{\circ}C$) within 4 hr, and collected by aspirating normal follicles which had diameters of 1 to 6 mm. The oocytes with cumulus cells were cultured for 8, 16, 24 and 30 hr in a modified KRB solution containing BSA or FCS and hormones. The in vitro matured oocytes in mKRB containing FCS, FSH and steroids were transferred in the rabbit uterus for examination of their in vivo fertilizability with bovine sperm preincubated 4 to 6 hr in the rabbit uterus. 1. The mean number of oocytes collected per cattle was 6.5 from 1-3mm follicles, 1.3 from 4-6mm follicles, and total was 7.7. 2. The meiotic division at 16hr-cuture in the oocytes from 1-3mm follicles was slightly stimulated by the addition of FSH in mKRB + BSA solution compared with the control. At 30hr-culture, their maturation rates(%Met II) were also increased by FSH of 1 $\mu\textrm{g}$/ml(38.4%) and 5$\mu\textrm{g}$/ml(35.7%) as compared with the control (21.4%). The maturation rate at 30hr-culture in the oocytes from 4-6mm follicles was 53.8% and 57.1% by the FSH addition of 1$\mu\textrm{g}$/ml and 5$\mu\textrm{g}$/ml, respectively. These rates were similar with the control(57.1%), but higher than those of oocytes from 1-3mm follicles. 3. The meiotic division at 16hr-culture in the oocytes from 1-3mm follicles was stimulated by the HCG addition of 1IU/ml and 5IU/ml. However, the maturation rate at 30hr-culture was greatly decreased by the HCG addtion (26.6% and 13.3%) compared with the control(53.3%) and these rates (30.8%) in the oocytes from 4-6mm follicles were also lower than that fo the control(58.3%). 4. Low maturation rate (37.5%) of the oocytes cultured in mKRB containing BSA and 5IU/ml HCG was increased (55.0%) when 15% FCS with HCG was added to mKRB instead of BSA. 5. When 16hr-cultured oocytes in mKRB containing BSA and gonadotropins (5$\mu\textrm{g}$/ml FSH and 5IU/ml HCG) were transferred in the medium without gonadotropins and recultured for 16hr, the maturation rate of HCG-treated oocytes was greatly improved. 6. The maturation rates of oocytes were greatly affected by steroids. The combined addition of FCS+FSH+estrogen or +progesterone to mKRB increased the maturation rate compared with the combination of BSA+FSH or FCS+FSH in mKRB. 7. The fertilization rate, presence of pronuclei, was increased by the combination of FCS+FSH+p in mKRB as compared with that (5.6%) of BSA+FSH and the rates of FCS+FSH+steroids ranged from 12.5 to 17.6%.

• Asian-Australasian Journal of Animal Sciences
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• 제30권6호
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• pp.781-787
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• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제17권3호
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• pp.317-324
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• 2004

The present study was conducted to investigate the involvement of nitric oxide (NO) in cumulus expansion, oocyte mortality and meiotic maturation of porcine cumulus enclosed oocytes (CEOs) cultured in two different models when gonadotropins, including follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) were presented or not. And the interaction between NO and $\beta$-mercaptoethanol ($\beta$-ME), a free radical scavenger was also investigated. Two models refer to spontaneous maturation model and hypoxanthine (HX) medium model. All the 3,433 eligible CEOs were incubated at $39^{\circ}C$ and the cumulus expansion, oocyte morphology and nuclear phase were evaluated 44 h after incubation. (1) In spontaneous maturation model, NO stimulates the cumulus expansion and $\beta$-ME delayed it. NO doesn't affect the oocyte meiotic resumption but inhibits the oocytes to develop to metaphase II. (2) In HX medium model, NO or $\beta$-ME doesn't affect the expansion in the absence of gonadotropins, but in the presence of gonadotropins, NO or $\beta$-ME inhibits the expansion. In the presence of gonadotropins, NO inhibits the oocyte meiotic resumption and it especially inhibits the oocyte to develop to metaphase II, and $\beta$-ME reverses such inhibitory effects. The cooperation of gonadotropins and $\beta$-ME stimulates the meiotic resumption and especially, promotes the CEOs to develop to metaphase II in both models. Moreover, HX might contribute to the fragility of oocyte zona pellucida and gonadotropins, nitric oxide and $\beta$-ME could alleviate it separately, and cooperatively. It is concluded that NO exerts different functions in two models and $\beta$-ME affected the functions of NO in different models.

• 한국가축번식학회지
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• 제20권4호
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• pp.395-412
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• 1997

포유동물의 난자는 제 2 감수분열 중기에서 배란되어 수정된다. 일반적으로 포유동물에서의 수정은 inositol triphosphate (IP3) 또는 cyclic adenosine diphosphoribose (cADPr)에 의해 조절되어지는 세포질내 칼슘변화에 의해 일어난다. 난활성이 일어나면 세포질내 고농도의 MPF (maturation promotion factor)는 감소하고, 전핵이 형성되고, cytoskeleton이 재구성되고, 단백질합성이 조절되어진다. 이러한 모든 과정이 정상적으로 일어난 후 수정란은 종특이한 발달경로로 배발달을 시작한다. 본 논문에서는 돼지난자의 인위적인 난활성 유도방법에 대해서 설명하고자 한다. 신호전달(signal transduction) 경로에 의한 하나의 기전이 돼지난자에서 확인되었는데, 이 경로의 자극은 난활성으로 이루어진다. 또한 전기자극에 의한 난활성방법에 의해 12일까지 정상배 발달이 이루어진다고 보고되고 있다. 향후 연구에서는 정자가 어떠한 기전에 의해서 배발달을 시작하게 하는지를 밝혀내야 한다고 본다.

• 한국발생생물학회지:발생과생식
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• 제17권2호
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• pp.141-147
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• 2013

Keeping the intact germinal vesicle (GV) is essential for maintaining the capacity of mammals including human. It is maintained by very complex procedures along with folliculogenesis and is a critical step for getting competent oocyte. So far, a few mechanisms involved in folliculogenesis are known but GV arrest mechanisms are largely unrevealed. Cyclic AMP, a adenosine derived substance, have been used as inhibitor of germinal vesicle breakdown as a putative oocyte maturation inhibitor. In this study, we examined the potency of adenosine as GV maintainer and a possible signaling mediator for that. A1, A2b, and A3 were detected in cumulus cells of cumulus enclosed-oocyte (CEO). Intact of germinal vesicle was not kept like in follicle but the spontaneous maturation was inhibited by exogenous adenosine. It is inhibited with concentration dependent manners. Intracellular calcium level of cumulus was extensively increased after adenosine treatment. Based on these results it is suggested that one of the pathway for GV arrest by adenosine and its receptors is calcium mediated signaling pathway in CEO.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.240-240
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• 2004

The modification of in vitro maturation (IVM) conditions should be required to efficient production of matured porcine oocyte in vitro. Estradiol-17β (E₂) as steroid hormone exists in ovarian follicular fluid and plays a major role in ovulation. It has been reported that estradiol as well as other steroids are involved in keeping the oocytes in meiotic arrest. (omitted)

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.123-123
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• 2003

This study was conducted to determine the ability of nuclear development of canine oocytes depend on the kind of maturation media and addition of serum sources. Ovaries were collected from a bitches at various stages of estrus cycle by an ovariohysterectomy. Oocytes were collected of cumulus oocytes complexes after slicing of ovaries with blade. The maturation medium was containing 0.6 mM/ml cysteine, 0.2 mM pyruvic acid, 20 ng/ml $E_2$ and 1 $\mu g/ml$ rbST Exp. 1, the oocytes were matured in four different maturation medium as follows: 1) TCM-199, 2) DMEM, 3) NCSU37 and 4) modified-NCSU37 with 10% FBS. Exp. 2: the oocytes were matured in mNCSU37 supplemented with different protein sources (10% FBS, 10% EDS, 0.3% BSA and 0.1% PVA) to select the optimal one. Oocytes were matured in a humidified atmosphere containing 5% $CO_2$ at $39{\circ}C$ for 72 hrs. The maturation rate were analyzed by Duncan's multiple range test using General Linear Models procedure in SAS. The rates of meiotic resumption to MI-MII depend on different culture media were achieved with TCM-199 (5.2%), DMEM (5.0%), NCSU37 (7.2%) and m-NCSU37 (5.9%), respectively. The rates of meiotic resumption to MI-MII according to addition of protein source were 10% FBS (13.3%), 10% EDS (25.0%), 0.3% BSA (25.0%) and 0.1% PVA (15.4%), respectively. In conclusion, the results obtained showed that in vitro maturation media and protein supplement to m-NCSU37 culture medium tested did not promote the final steps of IVM in canine oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제49권2호
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• pp.149-158
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• 2022

Objective: Optimizing culture media for the incubation of immature oocytes is a vital strategy to increase the oocyte maturation rate during in vitro maturation (IVM) programs. This study evaluated the IVM and fertilization rates of human germinal vesicle (GV) and metaphase I (MI) oocytes using two different maturation media (commercial and homemade) with or without growth differentiation factor 9-β (GDF9-β). supplementation. Methods: Immature oocytes from intracytoplasmic sperm injection (ICSI) cycles were collected and assigned to one of two IVM culture media (commercial or homemade; cleavage-stage base). After maturation, MII oocytes were examined under an inverted microscope for the presence of the polar body, zona pellucida (ZP) birefringence, and meiotic spindle (MS) visualization after maturation in four conditions (commercial or homemade medium, with or without GDF9-β. ICSI was done for matured oocytes, and fertilization was confirmed by the visualization of two distinct pronuclei and two polar bodies. Results: No significant differences were found between the two culture media in terms of the time and rate of oocyte maturation or the rate of fertilization (p>0.05). Growth factor supplementation increased the 24-hour maturation rate for both GV and MI oocytes only in homemade medium. The maturation rate after 24 hours was higher for MI oocytes (p<0.05). Similar results were observed for MS visualization and ZP structure in both types of media (p>0.05). Conclusion: Higher rates of oocyte maturation and fertilization were observed after application of homemade medium supplemented with GDF9-β. Therefore, this combination may be recommended as an alternative for clinical IVM programs.

• 한국수정란이식학회지
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• 제7권2호
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• pp.125-131
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• 1992

The nudear changes of bovine oocytes during 24 hrs. of culture for mejotic maturation were examined. Bovine oocytes were collected from small(<2 mm), medium(2~6 mm) and large(>6mm) follicles and classified into three grades by their morphological characteristics. A total of 242 oocytes collected were obtained:from 184 small, 157 medium and 1 large follicles, respectively and were classified into 95 grade I, 155 grade H and 92 grade III oocytes. All the bovine oocytes collected and graded were washed with a basal medium and incubated in groups of 10 for 24 hrs in 5% $CO_2$ and 39$^{\circ}C$. The basal medium used was composed of TCM-199 supplemented with sodium bicarbonate, sodium pyruvate, streptomycin, penicillin G and 10% FCS. The oocytes were cultured in drops of 50,$\mu$l basal medium supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH and 1$\mu$g /ml estradiol-17$\beta$. The oocytes were fixed and examined on their chromosomal status by 1% acetorcein staining in the interval of 3 hrs. Most of the grade I oocytes developed to germinal vesicule stage at 0 to 3 hrs., germinal vesicle breakdown at 6 hrs., metaphase I at 9 to 15 hrs., anaphase I and telophase I at 18 hrs., and metaphase II and the first polar body at 24 hrs. after culture for meiotic maturation. However, it was found that compared to grade I oocytes, grade H and W oocytes reached earlier to germinal vesicle breakdown and most of them developed earlier to M II stage at 21 hrs. after culture.

• 농업과학연구
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• 제48권3호
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• pp.607-615
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• 2021

Cytokines are protein mediators that possess the ability to assist cell-to-cell communication in immune system-related activities. In general, pathogen endotoxins activate the release of inflammatory mediators, and with time, there is an increase in the cytokine levels in the body. Interleukin (IL)-6 mediates the acute-phase inflammatory response, and elevated IL-6 levels have been reported in peritoneal fluids of women with pelvic inflammation and endometriosis, thereby associating it with oocyte quality and infertility. To overcome subfertility or infertility in humans and animals, the present study was done to examine the effect of recombinant IL-6 on porcine oocytes matured in vitro and subsequently to determine the fertilization rate and embryo development. Porcine oocytes were incubated with varying concentrations of IL-6 (0 - 2 ㎍·mL^-1) for 44 h followed by in vitro fertilization and culturing of the oocytes. The oocytes or embryos were fixed with 3.7% paraformaldehyde (PFA) and stained with fluorescence dyes, and the meiotic spindle, chromosome organization, fertilization status and embryo development were subsequently assessed under a fluorescence microscope. We observed induction of an abnormal meiotic spindle alignment in the oocytes incubated with IL-6 compared to the control oocytes incubated without IL-6. Moreover, significantly decreased fertilization rates and embryo development were observed for oocytes incubated with IL-6 (p < 0.05). Thus, an increased IL-6 level during oocyte maturation could be associated with fertilization failure due to an aberrant chromosomal alignment and a disruption of the cortical granules. Taken together, our results indicate that successful assisted reproduction can be achieved by controlling the levels of inflammatory cytokines.