• 한국임상수의학회지
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• 제27권1호
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• pp.46-49
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• 2010

In this study, we investigated the number of follicles, oocyte recovery rate and oocyte competence after in vitro maturation according to the size of follicle. And equine oocyte competence after in vitro maturation was investigated in terms of the diameter of follicle with criteria of maturation: nuclear stage after Hoechst staining. The average number of follicles per ovary with middle size (11-20 mm, 2.68) was higher than those of small (5-10 mm, 0.74) and large size follicle (> 21 mm, 1.63), therefore medium follicle (53.1%) had higher proportion than other size of follicles. The average numbers of follicle per ovary was 5.05. The rate of oocyte recovery in small (54.5%) and middle follicle (50%) was higher than that in large follicle (40.9%). After culture for 48 h in Medium 199, 50%, 45.5%, and 44.4% of oocytes from the follicles with diameters of 5-10, 11-20, > 21 mm, respectively reached the metaphase II stage. This is the first report showing number of follicle, oocyte recovery rate according to follicular size, and in vitro oocyte maturation in Jeju mare in Korea. To fulfill in vitro equine embryo production, further studies such as the seasonal effect, in vitro fertilization etc is need.

• 한국동물학회지
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• 제10권1호
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• pp.1-9
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• 1967

본 실험은 4 일간의 성주기가 뚜렷한 성체인 golden hamster로부터 미성숙인 난자를 적출하여 화학적배양액내에서 그의 성숙을 유도시키고 성주기에 따른 미성숙 난자의 성렬율을 관찰하는 것을 주요 목적으로 하여 행하여졌다. 한 개의 난소로부터 5-6개의 미성숙 난자를 적출하여 5% bovine serum albumin이 섞인 TC Medium 199에 넣어서$CO_2$-incubator를 이용하여 6 시간내지 24시간 동안 37$^{\circ}C$를 유지해가며, 배양액이 직접 공기와접촉하는 것을 막기 위해 유동성인 paraffin oil로 배양액을 덮고서 배양을 완수했다. 실험의 결과를 다음과 같이 완수했다. 1. Hamster의 난자의 성숙분렬을 유발시키는데 가장 적합한 배양액은 BMOC, Eagle's Medium , Waymouth's Medium 그리고 TC Medium 199의 네가지 가운데 TC Medium199이었다. 즉 이 배양액을 이용했을 때 난자가 높은 성렬도를 보여주었다. 2. 5% bovine serum alumn을 TC Medium 199에 섞어주었을 때 미성숙 난자가 성순분렬을 유기하는 율이 가장 높았다. 3. 발정기에 있는 난소로부터 얻은 난자가 가장 현저하게 높은 율로 성숙분렬을 보여주었다. 반대로 발정후기의 난자는 배양 시작 후 단시간 내에 대부분이 퇴화하였으며 발정기에 가까워 갈수록 퇴화율이 줄어들었다. 이것은 노포가 성숙분렬을 억제하는 물질을 생성하리라고 여겨지고 있긴 \ulcorner만, 동시에 이 노포는 또 한편 난자의 배란뒤에까지도 생명력을 유지할수 있는 능력을 발정기에 이르기까지의 기간동안 난자에게 부여하며, 난자는 이 능력을 배란전까지 축적하게 되며 이 때문에 노포로부터 유리되어 나온 난자가 장기간 그 생명력을 유지해나가는 것이라고 추정된다. 4. Paraffin oil 로 공기를 차단하여서 배양한 난자나 혹은 watch glass를 이용하여 공기와 접촉시킨 난자에서나 모든 비슷한 율로 성숙분렬을 보여주었다. 이로 보아 조작이 까다로운 paraffin oil을 이용하는 방법보다는 손쉽게 배양할 수 있는 watch glass 의 방법이 오히려 유용하다고 할 수 있다.

• Molecules and Cells
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• 제42권3호
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• pp.228-236
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• 2019

CD4 T cells differentiate into $ROR{\gamma}t/IL$-17A-expressing cells in the small intestine following colonization by segmented filamentous bacteria (SFB). However, it remains unclear whether SFB-specific CD4 T cells can differentiate directly from naïve precursors, and whether their effector differentiation is solely directed towards the Th17 lineage. In this study, we used adoptive T cell transfer experiments and showed that naïve CD4 T cells can migrate to the small intestinal lamina propria (sLP) and differentiate into effector T cells that synthesize IL-17A in response to SFB colonization. Using single cell RT-PCR analysis, we showed that the progenies of SFB responding T cells are not uniform but composed of transcriptionally divergent populations including Th1, Th17 and follicular helper T cells. We further confirmed this finding using in vitro culture of SFB specific intestinal CD4 T cells in the presence of cognate antigens, which also generated heterogeneous population with similar features. Collectively, these findings indicate that a single species of intestinal bacteria can generate a divergent population of antigen-specific effector CD4 T cells, rather than it provides a cytokine milieu for the development of a particular effector T cell subset.

• 농업과학연구
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• 제43권1호
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• pp.52-60
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• 2016

In animal reproduction, the quality of oocytes and embryos has been evaluated by the expression of specific molecules. Follistatin (FST), which was isolated from follicular fluid, binds and bio-neutralizes the TGF-${\beta}$ superfamily members. Previous studies using the bovine model showed FST could be an important molecular determinant of embryo developmental competence. However, the effect of FST treatment on porcine embryo developmental competence has not been established. In this study, the effect of exogenous FST on porcine embryo developmental competence was investigated during in vitro culture. FST (10 ng/ml) treatment induced a significant decrease in the rate of cell arrest at the 4-cell stage. The expression levels of DNA-methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) were decreased in 4-cell stage embryos. FST treatment also resulted in significant improvements in developmental competence of embryos in terms of blastocyst formation rate and OCT-4 mRNA levels, the latter being related to pluripotency. In conclusion, during in vitro culture, FST treatment significantly ameliorated 4-cell block during embryonic development and improved embryo developmental competence. Therefore, FST treatment may potentially have a functional role in porcine embryogenesis that is broadly applicable to enhance in vitro embryo development.

• 한국가축번식학회지
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• 제20권4호
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• pp.413-421
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• 1997

체외에서 포유동물의 난자와 정자의 배양에 관한 연구는 난자의 성숙과정과 수정현상에 대한 많은 새로운 정보를 제공하였다. 동시에 체외수정의 연구로부터 얻은 결과는 또다른 의문을 제기하였다. 특히 동결융해정액을 이용한 돼지체외수정의 경우 낮은 정자의 침입율과 전핵형성율 및 높은 다정자침입(polyspermy)율은 아직도 해결해야할 문제점으로 남아있다. 돼지난자의 성숙에 관한 연구의 성과는 수정후 낮은 전핵형성율을 개선시켰으나 타동물종에 비하면 아직도 매우 낮게 나타나고 있다. 한편 동결정액의 처리를 위하여 caffeine이나 Ca2+와 같은 물질을 수정용 배지내에 첨가하는 등 수정능력획득의 유기를 위하여 여러 가지 방법이 연구되고 있지만 정자의 침입율은 아직도 낮고, polyspermy의 발생율은 높게 나타내고 있다. 따라서 정자의 침입율을 향상시키고 polyspermy를 억제하기 위하여 난관세포와의 공동배양, 난포액을 첨가한 배양액 내에서 정자의 전배양 및 정자농도의 조절은 매우 효과적인 방법으로 이용되어왔다. 그러나 수정란의 체외생산성 향상과 이와 관련된 연구를 보다 효과적으로 수행하기 위해서는 위에서 지적한 문제에 영향을 미치는 요인에 대한 보다 근본적인 이해가 요구된다.

• 한국수정란이식학회지
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• 제30권4호
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• pp.327-333
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• 2015

The objectives of the present study were to select an effective basic medium including its hormone and protein supplementation for IVM of oocytes of indigenous zebu cows. The ovaries of cows were collected from slaughter house and the follicular fluid was aspirated from 2 to 8 mm diameter follicles. The COCs with more than 3 cumulus cell layers and homogenous cytoplasm were selected for maturation. The oocytes were matured in media for 24 hrs at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The maturation of oocytes was evaluated by examining the presence of first polar body under microscope. An efficient basic medium was determined after culturing COCs in either TCM 199 or SOF medium in Experiment 1. An efficient hormone supplementation was determined after culturing COCs in either FSH or gonadotrophin supplemented TCM 199 in Experiment 2. An efficient protein supplementation was determined after culturing COCs in either FBS or Oestrous cow serum (OCS) supplemented TCM 199 in Experiment 3. The oocyte recovery rate per ovary was 3.35. The overall rate of IVM was 74.6%. The maturation rate was $75.5{\pm}3.9$ and $62.2{\pm}20.2%$ in TCM and SOF medium, respectively (P>0.05). The maturation rate of oocytes was significantly higher ($76.6{\pm}13.2%$) in FSH supplemented medium than gonadotrphin supplemented counterpart ($69.7{\pm}10.8%$) (P<0.05). The maturation rates of oocytes were $81.7{\pm}12.9$ and $85.7{\pm}12.7%$ in medium supplemented with FBS and OCS, respectively (P>0.05). In conclusions, both TCM 199 and SOF supplemented with either FBS or OCS, and FSH may be used as medium for IVM of indigenous zebu oocytes in Bangladesh.

• Reproductive and Developmental Biology
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• 제34권1호
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• pp.55-62
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• 2010

Matrix metalloproteinases (MMP) play important roles in extracellular matrix (ECM) remodeling during ovarian follicular development, oocytes development and ovulation. In an attempt to investigate the effect of MMP activation in development cumulus-oocytes complexes, we examined the localization and expression of MMP, and monitored MMP expression profile. Cumulus-oocytes complexes were collected and matured in vitro for 24 hr, 36 hr and 48 hr. A mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-3 was detected in all culture medium regardless of CC, DC and CDCs. Activity of MMP-2 in the DC progressively was increased from 24 hr to 48 hr. But MMP-9 was not detected in all culture medium. The localization of MMP-2 was also measured by immunohistochemistry analysis. The MMP-2 and TIMP-2 was detected in cumulus cell and oocyte zone pellucida. Expression of MMP-2 protein in the COCs was progressively increased from 24 hr to 48 hr. However, MMP-9 protein was progressively decreased from 24 hr to 48 hr. And TIMP-2 protein was most highly expressed in the CDCs 36 hr. Expression of TIMP-3 protein in the CDCs was progressively increased from 24 hr to 48 hr. In conclusion, these results suggest that MMP-2 plays a role in maintaining normal maturation and development by controlling the ECM inhibitor concentration on cumulus cell and oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제21권2호
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• pp.177-182
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• 1994

ICR female mice aged 3 to 4 weeks, were stimulated with 7.5 IU PMS injection. At 48-52h post-PMS injection, ovaries were dissected out and oocytes-cumulus complexes(OCCs) were divided into three groups, cumulus-free oocytes(O), cumulus-free oocyte cocultured with cumulus cells(O+C) and OCC. The oocyte were cultured in TCM199 containing various protein sources, FCS, BSA or PVP with gonadotropins(Gns) for 24h. Spermatozoa were collected from cauda epididymis and capacitated in T6 + BSA for 2h. After oocyte maturation in vitro(IVM) in different experimental groups, matured oocytes were inseminated with the capacitated spermatozoa in T6 + BSA for 6h. In the groups of IVM in TCM + BSA or PVP, fertilization(IVF) did not occur efficiently. However, increased fertilization was found in TCM+ FCS group. The oocytes groups, with cumulus cells showed decreased polyspermy in FCS group (O; 31.8 %, O + C; 12.2 %, OCC; 16%), the addition of Gns did not prevent polyspermy in all three groups. The rates of fertilization increased in zona-free oocytes in PVP group. This results showed that culture system for IVM and IVF could be improved. Furthermore, PVP can be used for the substitution of protein source during maturation, and its low rate of fertilization has been found due to zona hardening which occurred in FCS-free medium.

• Animal cells and systems
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• 제3권4호
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• pp.385-391
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• 1999

Changes in the levels of prostaglandian F$_{2a}$ (PGF$_{2a}$) and E$_2$ (PGE$_2$) in culture medium during in vitro ovulation of Rana dybowskii follicles were examined. The ovulation was induced by frog pituitary homogenate (FPH) or TPA (12-O-tetradecanoylphorbol-13-acetate, a protein kinase activator) and the levels of PGs were measured by radioimmunoassay. When the ovarian follicles were cultured, only a few oocytes were ovulated by 12 h, but half of them were ovulated by 24 h in response to FPH, whereas around 30% of oocytes were ovulated by 12 h and maximum ovulation (around 50%) occurred by 24 h in response to TPA. Without any stimulation (control), no ovulation occurred. TPA elevated the level of PGF$_{2a}$ to high levels when compared to control (basal levels), but the increase by FPH was less evident. Likewise, the levels of PGE$_2$ increased markedly in response to TPA, but rather decreased by FPH treatment. Interestingly, PGF$_{2a}$ induced ovulation but PGE$_2$ suppressed FPH- or PGF$_{2a}$-induced oocyte ovulation. Basal levels of PGs Increased steadily during culture. When theca/epithelium (THEP) layer and granulosa cell-enclosed oocytes (GCEOs) were separated by microdissection and cultured independently, higher levels of both PGs were secreted by THEP than by GCEOS. Synthesis of PGs by follicle or follicular components was strongly suppressed by exogenous cAMP or indomethacin. These results suggest that: 1) PGF$_{2a}$ plays an important role in Rana ovulation, 2) protein kinase C is involved in PGs production, and 3) thecal epithelium layer is responsible for the PGs production in Rana.

• 한국가축번식학회지
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• 제17권1호
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• pp.57-68
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• 1993

This stduy was carried out to find a reliable method for the production of in vitro fertilized embryos having more excellent development capacity and freezability in the rabbit and cattle. The greatest number of rabbit oocytes was recovered 6hrs after HCG injection(P<0.05). The maturation rate in vitro was slightly higher in the oocytes(6-h-oocytes) from 6h than those (8-h-oocytes)from 8 hrs after HCG injection and the beneficial effect of FSH during oocyte maturation was significantly great in the oocytes from large follicles. The cleavage rate into 2-to-6-cell stage was not differ between the 6-h-oocytes and 8h-oocytes, but the cleavage of these oocytes was greatly promoted by FSH addition to maturation medium and the cleavge of 8-h-oocytes matured without FSH was significantly low. The embryo development into 16-cell to morula was not promoted by the co-culture with rabbit oviduct epithelial cells. The freezability by embryo stages was ovidusly high at 4-cell and morula stage in 6-h-oocytes and the viability of 16-cell embryos from 8h-oocytes was similar to that of morula stage. The implantation sites after surgical tranfer of fresh rabbit embryos were not implanted. In bovine experiment, the in vitro development into 16-cell and morula after in vitro maturation and fertilization in the follicular oocytes was slightly improved by the co-culture with granulosa cells compared to that with oviduct epithelial cells and the frozen-thawed viability rate of these embryos ranged from 14 to 40%. The excellent fresh embryos were transferred nonsurgically to 6 recipients, but were not pregnant.