• 한국발생생물학회지:발생과생식
• /
• 제5권2호
• /
• pp.131-136
• /
• 2001

초파리 발생과정에서 세포사멸에 관여하는 유전자인 reaper(rpr), grim, dcp-1, diap1, diap1, diap2의 발현양상을 경제적 RT-PCR 방법으로 분석하였다. 세포사멸 유도 유전자인 rpr, grim의 발현양상은 발생단계에 따른 ecdysone titer 변화 양상과 매우 유사하였다. Effector caspase인 dcp-1 전사체는 초기 배와 암컷 성체에서 높은 발현을 보였다. 반면에 세포사멸 억제인자인 diap1과 diap2 전사체는 세포사멸 유도 인자인 rpr과 girm 전사체와 서로 상반적인 양상으로 발현되었다. 또한, 유주 3령 유충의 발생단계 별로 침샘조직과 성체원기조직에서 rpr, diap2, dcp-1의 전사체의 양적 변동을 분석하였다. rpr, diap2의 전사체양은 두 조직에서 서로 상반적으로 변화하였다. 이 결과는 정상 발생과정에서 세포죽음 관련유전자들의 발현이 ecdysone 신호에 의해 조절됨을 암시해 주었다.

• Fisheries and Aquatic Sciences
• /
• 제18권2호
• /
• pp.211-220
• /
• 2015

We investigated the differences in the expression of the neurohormones kisspeptin (Kiss) and gonadotropin-inhibitory hormone (GnIH) and cytochrome P450 aromatase (P450arom), gonadotropin hormones (GTHs), and sex steroids in the goldfish Carassius auratus exposed to light-emitting diodes (LEDs). The expression levels of Kiss1, Kiss2, G-protein-coupled receptor 54 (GPR54), GTHs, GnIH, and P450arom were compared between the control (white light) and LED-treated goldfish. Furthermore, we measured the plasma levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The levels of Kiss1 mRNA and protein; Kiss2, GPR54, and $GTH{\alpha}$ protein; GTH mRNA; and plasma FSH and LH in the hypothalamus and cultured hypothalamus cells were significantly higher in the green and purple LED treatment groups than in the other groups. These results suggested that red LEDs inhibit the sex maturation hormones, Kiss, GPR54, GTHs, and P450arom, and that GnIH plays a role in the negative regulation of reproductive function in goldfish.

• 한국발생생물학회지:발생과생식
• /
• 제24권2호
• /
• pp.135-147
• /
• 2020

Polyvinylidene fluoride (PVDF) is a stable and biocompatible material that has been broadly used in biomedical applications. Due to its piezoelectric property, the electrospun nanofiber of PVDF has been used to culture electroactive cells, such as osteocytes and cardiomyocytes. Here, taking advantage of the piezoelectric property of PVDF, we have fabricated a PVDF nanofiber scaffolds using an electrospinning technique for differentiating human embryonic stem cells (hESCs) into neural precursors (NPs). Surface coating with a peptide derived from vitronectin enables hESCs to firmly adhere onto the nanofiber scaffolds and differentiate into NPs under dual-SMAD inhibition. Our nanofiber scaffolds supported the differentiation of hESCs into SOX1-positive NPs more significantly than Matrigel. The NPs generated on the nanofiber scaffolds could give rise to neurons, astrocytes, and oligodendrocyte precursors. Furthermore, comparative transcriptome analysis revealed the variable expressions of 27 genes in the nanofiber scaffold groups, several of which are highly related to the biological processes required for neural differentiation. These results suggest that a PVDF nanofiber scaffold coated with a vitronectin peptide can serve as a highly efficient and defined culture platform for the neural differentiation of hESCs.

• Reproductive and Developmental Biology
• /
• 제33권3호
• /
• pp.157-162
• /
• 2009

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of $\alpha$-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-$\alpha$/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.

• 한국발생생물학회지:발생과생식
• /
• 제22권4호
• /
• pp.309-318
• /
• 2018

The present study was conducted to investigate the regulatory mechanism of plasminogen activators (PAs) activation by $17{\beta}$-estradiol ($E_2$) and progesterone ($P_4$) in porcine uterine epithelial cells (pUECs). pUECs were collected from porcine uterine horn and cultured at 80% confluence. Then, 0.1% (v/v) DMSO, 20 ng/mL $E_2$, and $P_4$ with or without fetal bovine serum (FBS) treated to cultured cells for 24 hours. The supernatants were used for measurement of PAs activity and expression of urokinase-type PA (uPA), tissue-type PA (tPA), uPA specific receptor (uPAR), and type-1 PA inhibitor (PAI-1) mRNA were analyzed by real-time PCR. The expression of PAs-related genes was not affect by steroid hormones in both of serum treatment groups. However, PAs activity was increased by treatment of $E_2$ compared to 0.1% DMSO treatment in serum-free group (p<0.05). Then, $E_2$ and $P_4$ were diluted with 0.002% (v/v) DMSO for reduction of its effect and treated to cultured cells without FBS. Only tPA mRNA was significantly increased by $E_2$ treatment (p<0.05). PAs activity was enhanced in $E_2$ treated group compared to control groups (p<0.05). These results indicate that serum-free condition is more proper to evaluate effect of steroid hormones and activation of PAs in pUECs was mainly regulated by estrogen. These regulation of PAs activation may be associated with uterine remodeling during pre-ovulatory phase in pigs, however, further studies are needed to investigate precise regulatory mechanism.

• 한국발생생물학회지:발생과생식
• /
• 제25권4호
• /
• pp.235-244
• /
• 2021

Interferon Regulatory Factor 3 (IRF3) is a member of interferon-regulated transcription factor family and is known to play an important role in the innate immune response against viral infections. In this study, the expression of IRF3 in different tissues, developmental stages, and stocking densities of olive flounder was investigated. The expression of IRF3 was observed to gradually increase in early-stage juvenile fish. The highest expression was observed in later-stage juvenile fish when immune tissues were formed. High IRF3 expression was observed in the muscles and the brain tissues. The expression of IRF3 was observed in fish at different stocking densities after viral hemorrhagic septicemia virus (VHSV) infection. It yielded an interesting expression pattern in the muscles and the brain tissues of fish stocked at low density. These observations can be used as basic data for the study of the expression of immune response-related genes against viruses based on stocking density and immune systems in other fish species.

• 한국발생생물학회지:발생과생식
• /
• 제15권3호
• /
• pp.265-272
• /
• 2011

It is well known that adipose tissue or body fat has been proved as a crucial component of brain-peripheral axis which can modulate the activities of reproductive hormonal axis in female mammals including rodents and human. Concerning the male reproduction, however, the role of adipose tissue has not been thoroughly studied. The present study was carried out to elucidate the effect of a high-fat (HF) diet on the reproductive system of postpubertal male rats. The HF diet (45% energy from fat, HF group) was applied to male rats from week 8 after birth for 4 weeks. The blood glucose levels, body and tissue weights were measured. Histological studies were performed to assess the structural alterations in the reproductive tissues. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus and pituitary, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Body weights (p<0.01) and blood glucose levels (p<0.01) of HF group were significantly higher than those of control animals. Similarly, the weights of epididymis (p<0.05), prostate (p<0.01), seminal vesicle (p<0.01) in HF group were higher than control levels. The weights of testis were not changed. The weights of kidney (p<0.001) and spleen (p<0.01) were significantly higher than control levels while the adrenal and pancreas weights were not changed. There were only slight alterations in the microstructures of accessory sex organs; the shape of luminal epithelial cells in epididymis from HF group were relatively thicker and bigger than those from control animals. In the semi-quantitative RT-PCR studies, the mRNA levels of hypothalamic GnRH (p<0.05) in HF group were significantly higher than those from the control animals. The mRNA levels of kisspeptin in HF group tend to be higher than control levels, the difference was not significant. Unlike the hypothalamic GnRH expression, the mRNA levels of pituitary $LH{\beta}$ and $FSH{\beta}$ were significantly decreased in HF group (p<0.05). The present study indicated that the 4-weeks feeding HF diet during the postpubertal period can alter the hypothalamus-pituitary (H-P) neuroendocrine reproductive system These results suggest that the increased body fat and the altered leptin input might disturb the H-P reproductive hormonal activities in male rats, and the changed activities seem to be responsible for the changes of tissue weights in accessory sex organs.

• 한국발생생물학회지:발생과생식
• /
• 제21권4호
• /
• pp.407-415
• /
• 2017

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)-related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: $32.5{\pm}10.1%$ vs control: $77.8{\pm}5.3%$). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, $200{\mu}M$) and melatonin ($0.1{\mu}M$), in BIP/GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, $p-eIF2{\alpha}$, $eIF2{\alpha}$, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

• 한국발생생물학회지:발생과생식
• /
• 제15권4호
• /
• pp.373-383
• /
• 2011

성 호르몬과 유사한 기능을 하는 것으로 알려진 내분비교란물질은 흰쥐 흉선세포에 세포자연사를 일으켜 흉선의 기능을 감소시키는 것으로 보고되고 있으나, 그 작용 기전에 대해서는 잘 알려져 있지 않다. 따라서 본 연구에서는 내분비 교란물질 중 하나인 Tributyltin(TBT)를 흰쥐에 투여한 후 흉선의 상피세포가 지방세포로 분화되는지를 조사하고, 이로 인한 T 세포의 세포자연사와 연관성을 조사함으로써 TBT가 흉선기능에 미치는 영향을 알아보고자 하였다. 3주령 된 암컷 흰쥐에 각각 TBT 1, 10, 25 mg/kg/day를 1주일 동안 경구 투여한 후, 흉선을 획득하여 무게와 세포 수를 측정하였고, 파라핀 절편으로 H&E 염색, TUNEL 분석을 수행하였다. 또한 real-time PCR 방법으로 상피세포 표지 유전자들, 지방세포유도 유전자들과 세포자연사 관련 유전자들을 비교 분석하였으며, 유세포 분석기를 통해 세포자연사 정도를 조사하였다. 흉선의 무게와 세포 수는 대조군과 비교하여 TBT의 농도가 높을수록 유의하게 감소하였다. H&E 염색 결과, TBT의 농도가 증가할수록 흉선의 크기가 감소하고 피질과 수질의 경계가 흐려짐을 관찰하였다. TUNEL 염색 결과에서는 대조군에 비해 TBT를 투여한 군에서 세포자연사가 증가하였으며, 유세포 분석결과에서도 TBT 농도 의존적으로 세포자연사가 증가하였다. Real-time PCR 결과, 상피세포 표지 유전자인 EVA, IL-7, AIRE, KGF는 TBT의 농도가 증가할수록 발현양이 유의하게 감소한 반면, 지방세포 유도와 관련된 유전자인 $PPAR{\gamma}$, aP2, PEPCK, CD36은 TBT의 농도가 증가할수록 발현양이 유의하게 증가하였다. 세포자연사와 관련된 유전자인 $TNF{\alpha}$, TNFR1도 TBT의 농도가 증가할수록 발현양이 유의하게 증가하였다. 위의 결과를 종합하여 볼 때, 내분비교란물질인 TBT는 흉선 상피세포에 직접적으로 작용하여 지방세포로 분화 유도시킴으로써 흉선의 기능을 상실케 만들면서 T 세포의 발달에 영향을 미치는 것으로 보인다. 이러한 결과는 지속적으로 노출되는 TBT가 흉선의 기능을 떨어뜨림으로써 면역력 저하를 유발시킬 수 있음을 제시하고 있다.

• Reproductive and Developmental Biology
• /
• 제31권3호
• /
• pp.181-186
• /
• 2007

DNA 결합 단백질 억제(Inhibitor of DNA binding protein) 또는 분화억제(Inhibitor of differentiation) 인자인 Id는 네 종류(Id1-4)가 있으며, 세포의 증식과 분화, 혈관 형성 및 세포자가사멸 등에 정 또는 부의 조절인자로서 널리 알려져 있다. 하지만 아직까지 번식생리 분야에서 이들 유전자들의 발현이나 기능에 관해 알려진 것은 거의 없다. 따라서 본 연구에서는 쥐 난소에서 난포의 발달에 따른 Id3 mRNA의 발현 양상을 살펴보고자 실시하였다. 쥐에게 PMSG주사 후, 3, 6, 12, 24, 36 및 48시간째에 난소를 회수하여 고정, 탈수 및 파라핀 포매를 실시하였다. In situ hybridization 실험을 위하여 anti-sense와 sense Id3 cRNA 탐침자를 제작한 다음 난소 절편에 반응시켰다. 반응이 끝난 난소 절편은 NTB-2 유광제에 노출시킨 후 현상액에 반응시켰다. 모든 처리가 끝난 슬라이드는 H&E 염색을 실시한 다음 현미경하에서 hybridization 감도를 1+에서 4+로 구분하여 평가하였다. 난자에서는 Id3 mRNA 감도가 원시와 제1차 난포에서 ${\geq}2+$으로 확인되었으나, 제2차, 우성 및 배란전 난포에서는 1+ 이하로 관찰되었다. 한편, 과립막세포에서는 우성과 배란 전 난포에서 Id3 mRNA가 3+ 또는 4+로 강하게 발현되는 것을 확인하였다. 이상의 결과를 종합하여 보면, Id3 mRNA는 난포의 발단 단계 또는 난포세포에 따라 특이적으로 발현하는 것으로 보아 난포의 발달과 밀접한 연관이 있을 것으로 사료된다.