In oocytes from different species, MPF, a complex of Cdk1 and cyclin B, is the master regulator of cell cycle. The activity of MPF is regulated by the phosphorylations mediated by Wee1B kinase and Cdc25B phosphatase. Although a regulation of MPF activity by these inhibitory phosphorylations are well established, a dogma in the cell cycle is that MPF activity is regulated by the dynamics of cyclin B during the metaphase II (MII) arrest (also known as CSF arrest). However, growing evidences suggest that Wee1B-mediated Cdk1 phosphorylation is also critical to trigger the progression of cell cycle during the onset of anaphase. Therefore, in the present study, we investigated the role of Cdc25B phosphatase during MII arrest. Cdc25B is present in MII arrested oocytes as a hyperphosphorylated form and disruption of its function either by antibody or siRNA injection induces the progression of cell cycle to interphase. Moreover, the hyperphosphorylated form, which has been known as an active form of Cdc25B, is dephosphorylated during the anaphase onset. Interestingly, this dephosphrylation occurred ahead of cyclin B degradation. Conversely, overexpression of Cdc25B prevents metaphase to anaphase transition induced by calcium stimulation. Therefore, our findings provide novel paradigm in cell cycle that MPF activity during metaphase arrest is regulated by the balance between Cdk1 inhibitory kinases, Wee1, and the counteracting phosphatases, Cdc25. When cells exit from metaphase, Cdc25 is inactivated and Wee1 is reactivated and thereby Cdk1 kinase activity is rapidly and transiently decreased. This initial decrease of Cdk1 activity is further promoted by the proteolytic degradation of cyclin B, which ensures irreversible progression of cell cycle to interphase. Thus, the concerted effort of phosphorylation/dephosphorylation of Cdk1 and synthesis/degradation of cyclin B play roles in fine-tuning the activity of Cdk1 during metaphase to anaphase transition.

현재, 사춘기 전후 시기의 수컷 흰쥐의 성 성숙에 대한 연구가 활발히 이루어지고 있으나, 전반적인 사춘기 시기에 이루어지는 수컷 흰쥐 생식기관의 발달에 대한 연구는 부족한 실정이다. 이에 본 연구에서는 수컷 흰쥐의 성 성숙 발달 지표인 Anogenital distance(AGD)에 근거하여 사춘기 개시시기인 생후 25일부터 완전한 성 성숙이 이루어진 생후 50일 경까지 수컷 흰쥐에서 이루어지는 생식기관의 조직학적 발달을 조사하였으며, 이를 완전한 성체인 생후 70일의 수컷 흰쥐의 생식기관과 비교하였다. 생후 25일의 수컷 흰쥐를 5일 간격으로 생후 50일까지 조사하였으며, 성체인 생후 70일의 수컷 흰쥐도 함께 조사하였다. 오전 10시에 체중을 측정한 후 일괄 희생하여 생식기관 무게 및 AGD를 측정하였다. 정소를 포함한 부속생식기관들은 Hematoxylin & Eosin 염색법을 사용하여 조직학적 조사를 시행하였다. 체중은 생후 25일부터 생후 70일까지 유의하게 증가(P<0.01)하였으며, 정소와 부정소의 무게 또한 생후 25일부터 생후 70일까지 유의하게 증가(P<0.01)하였다. 저정낭은 생후 25일부터 생후 45일까지 점차적으로 증가(P<0.05)하다가 생후 50일부터 급격하게 증가하였다(P<0.01). 전립선의 경우 생후 25일부터 생후 35일까지 점진적으로 증가(P<0.05)하다가 생후 40일부터 급격하게 증가(P<0.01)하였다. AGD는 생후 25일부터 생후 70일까지 유의하게 증가(P<0.01)하였다. 조직학적 조사 결과, 정소는 생후 25일부터 점진적인 성숙으로 인해 세정관의 크기가 커짐이 관찰되었고, 생후 45일까지는 세정관 내 정자를 관찰할 수 없었으나, 생후 50일부터 세정관 내 정자를 확인할 수 있었다. 부정소 역시 점차적인 성숙으로 인해 생후 25일부터 도관의 내강이 커져 면적이 크게 증가하는 것을 확인하였으며, 생후 50일부터 도관 내 정자를 관찰할 수 있었다. 전립선과 저정낭의 경우 생후 25일부터 생후 70일까지 내강의 면적이 증가하여 점진적인 성숙이 이루어지는 것을 확인하였다. 본 연구에서는 AGD 측정을 통해 생후 25일부터 꾸준한 정소하강이 발생하여 생후 50일부터는 완전한 성 성숙이 이루어짐을 확인하였다. 또한 조직학적 연구를 통해 생식기관에서 이루어지는 성 성숙과 실제로 정자가 형성되는 시기를 확인하였다. 이후 지속적인 연구를 통해 수컷 포유류의 사춘기 개시에 이르기까지의 생식기관의 성숙 과정을 보다 정밀하게 조사할 필요가 있으며, 이를 통해 수컷 포유류의 사춘기 개시에 관여하는 다양한 조절 인자의 기능을 규명할 수 있을 것으로 기대한다.

시상하부-뇌하수체-부신(HPA) 축은 생리적, 심리적 스트레스와 관련된 호르몬의 분비를 조절하는 신경 내분비 축이다. 이전의 보고에 따르면 신체적 및 정신적 스트레스에 의한 HPA 축반응이 사춘기 발달과 관련이 있다고 알려져 있다. 본 연구는 암컷 흰쥐의 사춘기 전후 시기에시상하부-뇌하수체-부신 축에서 스테로이드 호르몬합성 유전자들의 활성 변화를 날짜별로 조사한 것이다. 생후 29일의 암컷 흰쥐를 사춘기 개시 이후 시기인 43일까지 2일 간격으로 희생하였다. HPA 축에서 스테로이드합성 관련 유전자들의 발현 변화를 조사하기 위해 semi-quantitative RT-PCR을 실행하였으며, 방사면역측정법(RIA)를 통해 혈청 내 코르티코스테론(corticosterone) 수준을 측정하였다. 시상하부에서 CRH를 발현을 조사한 결과 생후 35일까지는 다소 증가하다가 37일부터 감소하는 경향을 보인 후 생후 43일(p<0.05)에 다시 유의하게 증가하였다. GR 발현은 유의한 양상은 나타나지 않았다. 뇌하수체에서 ACTH mRNA 수준은 생후 33일까지 다소 증가하다가 35일부터 43일까지 점차적으로 감소하는 경향은 보이지만, 유의성은 없었다. 부신에서 StAR mRNA 수준은 점차적으로 감소하다가 생후 35일(p<0.05), 37일(p<0.05)에서 유의하게 감소하였고, 생후 39일에 다소 증가하였다가 다시 감소하여 43일(p<0.01)에서 유의하게 감소하였다. CYP11A1의 발현 역시 StAR와 유사한 양상을 보이며, 생후 35일(p<0.05), 37일(p<0.05)에서 유의하게 감소하는 양상을 보였다. 또한 3β-HSD의 발현은 전체적으로 감소하여 생후 35일(p<0.05)에서 39일(p<0.01)까지 유의하게 감소하였고, 생후 41일에 다소 증가한 후 43일(p<0.01)에서 유의하게 감소하였다. CYP11B1과 CYP21A의 발현을 조사한 결과, CYP11B1은 생후 31일(p<0.01)부터 35일(p<0.01)까지 크게 감소하고, 37일에서 증가한 후 다시 감소하여 생후 39일(p<0.05), 43일(p<0.05)에서 유의하게 감소하였다. CYP21A 발현은 생후 37일(p<0.01)까지 크게 감소하고 39일 부터 증가하는 양상을 보였다. 혈중 코르티코스테론의 경우 생후 31일(p<0.01)부터 35일(p<0.01)까지 유의하게 증가하고 37일부터 감소하는 경향을 보였다. 본 연구를 통해 사춘기 개시 전후 시기에 HPA 축에서 스테로이드합성 관련 유전자들의 발현 패턴이 동적으로 변화하며, 부신에서 효소들이 생후 35일경에 대체로 감소하는 양상을 확인하였다. 이는 사춘기 개시에 관여하는 HPG 축과 부신 축인 HPA 축간의 상호 조절 시스템이 존재하며 사춘기 개시 시점에 부신에서의 스테로이드합성이 어떠한 영향을 받을 수 있다는 가능성을 시사한다.

스테로이드 호르몬은 다양한 생리작용의 조절자로서 잘 알려져 있으며, 생리적 활성에 따라 5그룹 (mineralocorticoids, glucocorticoids, estrogens, progestagens, androgens)으로 나뉜다. 그 중 estrogen류는 특히 암컷 포유류의 이차성징을 유도함이 잘 알려져 있다. 본 연구는 사춘기 전후 암컷 흰쥐난소에서의 steroidogenesis 조절 효소의 유전자 발현에 대해 정밀 조사한 것이다. 미성숙한 암컷 흰쥐를 생후 29일에서 43일까지 2일 간격으로 희생한 뒤 total RNA를 추출하여 semi-quantitative RT-PCR을 시행하여 steroidogenesis 관련유전자인 StAR(steroidogenic acute regulatory protein), CYP11A1(side-chain cleavage enzyme), CYP17(17α-hydroxylase), 3β-HSD(3β-hydroxysteroid dehydrogenase), 17β-HSD(17β-hydroxysteroid dehydrogenase), CYP19(Aromatase)의 발현 양상을 조사하였다. 연구에 사용된 암컷 흰쥐는 대체로 37일에 질구개방이 일어났다. 유전자 발현 조사에서 StAR는 생후 41일, 43일에 유의하게 증가(p<0.05, p<0.01)하였다. CYP11A1은 43일에 유의하게 증가(p<0.01)하였다. CYP17의 경우, 35일과 39일에서 유의한 감소(각 p<0.05)를 보였다. 3β-HSD, 17β-HSD, CYP19의 경우는 유의한 변화를 보이지 않았다. 전반적으로 스테로이드 합성 관련 유전자 모두 생후 35일에 감소하였다가 암컷의 성 성숙 지표인 질구개방이 대체로 일어난 37일을 기준으로 유전자 발현이 급증됨을 확인하였다. 이는 사춘기 개시를 위한 다량의 sex steroid 합성을 위한 것으로 추정된다. 이러한 결과는 사춘기 전후시기 난소의 성 성숙에 관한 기초자료로 사용될 수 있을 것으로 사료된다.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfo-conjugation of estrogens at the 3-hydroxyl position. Sulfated estrogens lose their ability to interact with the estrogen receptor (ER). Previous studies have reported that testicular expression of EST is under the regulation of LH and androgen. In an effort to understand the biological significance of estrogens in the testis, we analyzed the EST gene expression in the developing mouse testis and Leydig cells and its regulation by estrogen receptor alpha (ERα). Male mice at postnatal day (PND) 1, 7, 14, 28, and 56 and ERα flox/flox Cyp17iCre male mice which show deletion of ERα specifically in Leydig cells were used for this study. Testes and Leydig cells isolated from these mice were subjected to quantitative RT-PCR analysis and immunohistochemistry. In addition, 17β-estradiol (E2), ERα-selective agonist PPT, ERβ -selective agonist DPN, and ER antagonist ICI 182,780 were treated in primary adult Leydig cell culture. These cultured cells were subjected to quantitative RT-PCR analysis. In testis, EST mRNA level was excessively low by PND 14 and markedly increased from puberty (PND 28) onward. In the interstitium, EST mRNA was not detected by PND 14 but considerably expressed from PND 28 onward. EST immunoreactivity was moderate in the interstitium by PND 14. Strong EST immunoreactivity was found in the interstitium from PND 28 onward. In ERαflox/flox Cyp17iCre mouse testis and Leydig cells, EST mRNA level was significantly lower than wild type (ERαflox/flox). In primary adult Leydig cell culture, the expression of EST mRNA was increased by E2 and PPT, but was not changed by DPN. The expression of EST in the testis is developmentally regulated. In adult Leydig cells, EST could play an important role in the steroidogenesis by modulating the activity of estrogens. Estrogen as well as LH and androgen may play a role in the regulation of EST expression in Leydig cells via ERα signaling.

Mature mammalian oocytes are ovulated at the metaphase II stage of meiosis and complete the cell cycle by fertilization with sperm. During fertilization sperm release an egg activation protein, pho-spholipase C zeta (PLCZ) into oocyte cytoplasm, PLCZ hydrolyze PIP2 into IP3 and DAG. The elevation of IP3 concentration induces Ca2+ release from endoplasmic reticulum (ER) by binding to the IP3 receptor (IP3R) on the membrane of ER. Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca2+]i oscillations or oocyte activation. Purified recombinant human PLCZ1 (hPLCZ1) protein evaluated its [Ca2+]i oscillation activity in mouse and human oocytes. Here we investigated that produced mouse PLCZ-specific antibodyrecognized the PLCZ protein in mouse testes. PLCZ antibody was raised in rabbits against 19-mer sequence at the C-terminus (MENKWFLSMVRDDFKGGKI) of mouse PLCZ protein. Sperm were fixed in 3.7% paraformaldehyde followed by permeabilization. Sperm were incubated in 5% normal goat serum (NGS) and then incubated overnight with anti-mouse PLCZ. Peanut agglutinin (PNA)-lectin was used for detection of the acrosome. Mouse testes from 6~8 weeks old ICR mouse were fixed in 10% formalinand serial sectioned at 5~8um. Testes tissues were immunostained with anti PLCZ antibody and peanut agglutinin(PNA) for acrosome staining. Produced anti mouse PLCZ antibody recognized 74 kDa protein in western and PLCZ is localized to the post-acrosomal region of mouse sperm and to the equatorial region of bull sperm. Mouse PLCZ protein wasdetected on spermatocytes, spermatid, but not on spermatogonia in seminiferous tubules. Some residual bodies on sperm neck and tail showed strong signal of PLCZ, but this staining was still present with antigenic peptide pretreatment to reduce non specific antibody reaction. Also this antibody reacted with the apical region (arrowheads) of principal cells, where secretory vesicles accumulate on the epididymal tissue. But antigenic peptide pretreatment did not remove this apical region staining. This study presents PLCZ protein is localized on the post-acrosomal region or equatorial region of mouse and bull sperm head. Also PLCZ protein in mouse testes expressed from spermatocytes to mature sperm on later stage of spermatogenesis.

The Egr family of zinc finger transcription factors is rapidly induced by various mitogens and regulates cell growth, differentiation, and apoptosis. While it is clear that loss of Egr1 leads to anovulatory infertility due to LHβ deficiency in female mice, molecular function of Egr1 in male reproduction has not been clearly investigated. Here, we demonstrate that Egr1 acts as an intrinsic transcription factor in Leydig cells to regulate their proliferation and steroidogenesis in the testis as well as an extrinsic factor for male reproduction via LHβ transcription in the pituitary. Egr1 is predominantly expressed in spermatogonia and Leydig cells in immature testes and later detected in some of these cell types in mature testes. The fertility potential of Egr1(-/-) male mice is relatively deteriorated even at 2 month-old age and aggravated with aging. The incidence of abnormalities of seminiferous tubules such as Sertoli cell only was dramatically increased with aging. The number and mean size of Leydig cells were significantly reduced in Egr1(-/-) testes. The impairment of Leydig cells is consistent with significant reduction in levels of testosterone and expression of factors critical for steroidogenesis such as StAR in Egr1(-/-) testes. Exogenous administration of hCG rapidly and transiently induced Egr1 expression in Leydig cells culture in vitro. hCG could reinstate reduced mean size of Leydig cells but not reduced number of Leydig cells and aberrantly low StAR expression, suggesting that Egr1 has critical functions for Leydig cell proliferation and their steroidgenesis. In addition, daily sperm production and in vitro fertilization (IVF) competence were significantly reduced, and apoptosis was facilitated in these mice. Furthermore, hCG administration to compensate for relatively low LH levels in Egr1(-/-) males could not restore the compromised reproductive phenotypes such as IVF competence and apoptosis in these mice. Interestingly, expression of Egr2, a member of Egr family, is significantly elevated in Egr1(-/-) Leydig cells suggesting that genetic compensation of Egr2 may alleviate phenotypic aberration of Egr1(-/-) male testes. Collectively, these results suggest that Egr1 act as an intrinsic transcription factor required for proliferation and steroidogenesis of Leydig cells to govern spermatogenesis in the testis.

After vasectomy (VAX), the flux and composition of the epididymal fluid are modified, leading to sequelae to the epididymis. In an effort to understand molecular pathophysiology of the epididymis following VAX, we investigated the changes of gene expression and sperm functions in the epididymis of vasectomized mice. After VAX, the epithelial cell height was significantly decreased in cauda epididymis, resembling the those of vas deference. This suggests that VAX evokes alteration of segmental characteristics of epididymal epithelium. Of note, these was an increase in luminal diameter in corpus and cauda epididymis, indicating the alteration of fluid homeostasis in epididymal lumen and protein synthesis and secretion. Also, the formation of sperm granuloma and infiltration of inflammatory cells were noted in lumen of epididymis at 8 weeks postvasectomy, indicating the activation of immune response in epididymis. The serum TNF-α levels and epididymal TNF-α and IL-1β mRNA levels were significantly increased in VAX mice. Microarray analysis demonstrated that claudin (CLDN) 10 and cystic fibrosis transmembrane conductance regulator (CFTR) were downregulated after vasectomy. In contrast, angiotensin II receptor type 2 (Agtr2) was up-regulated after vasectomy. Taking into account the functional importance of angiotensin system in epididymal epithelia and muscle tissue, VAX may alter the secretory function of epithelia and sperm transport via alteration in angiotensin system. Importantly, the IgG type of anti-sperm antibody (ASA) were markedly increased in the blood of vasectomized mice. Together this indicates that VAX provokes local inflammation in epididymis as well as systemic inflammation, which in turn change the blood epididymal barrier, an important element for epididymal immunological privilege. In addition, the number of sperm in cauda epididymis was increased but the sperm motility was decreased after vasectomy. The spontaneous acrosome reaction was increased by vasectomy after capacitation. This suggests that VAX affects sperm functions as well as sperm maturation. In conclusion, bilateral vasectomy lead to local immune response of epididymis, causing immunologic infertility in men.

Early growth response-1(Egr-1)은 zinc finger transcription factor로 세포분화, 생장 및 사멸을 조절한다. Egr-1 KO 수컷 생쥐의 정소에서는 maturation arrest, Sertoli cell only 등의 비정상 세정관이 유의적으로 증가한다. Glial cell Derived Neurotrophic Factor(GDNF)는 고농도에서는 줄기세포 자가 재생, 저농도에서는 정원세포를 분화효과를 갖는다. 본 연구에서는 발생중인 생쥐 정소에서 Egr-1 발현을 확인하고, GDNF에 의한 spermatogonial stem cell의 stemness 조절 신호전달 기작연구의 일환으로 Egr-1 유도현상을 규명하고자 하였다. ICR 생쥐의 생후 1, 2, 4, 8주령 정소를 획득하여 RT-PCR과 면역조직화학법을 통해 Egr-1 발현을 확인하였다. 한편 생쥐의 생후 5일된 정소를 획득하여 THY1 항체를 이용한 MACS 방법으로 spermatogonial stem cell을 분리하였다. GDNF(1, 100 ng/ml)를 0, 0.5, 1, 2, 6시간 처리하고 Egr-1 mRNA 발현을 분석하였다. 생후 3~12개월령 Egr-1 KO 수컷 생쥐와 Hetero 수컷 생쥐의 정소를 획득하여 H&E 염색 후 정소 내 seminiferous tubule을 비교하였다. Egr-1 mRNA는 생후 1, 2주령까지 높게 발현하다가 성숙함에 따라 발현이 감소하였다. 면역조직화학적 분석 결과, Egr-1은 gonocytes, (stem)spermatogonia, 분열 중인 peritubular cells과 Leydig cells에서 발현하였다. MACS로 분리한 spermatogonial stem cell에서 GDNF 처리 시 Egr-1 mRNA 발현은 0.5시간과 1시간에 유의적으로 증가하였고, 6시간 후 다시 감소하였다. Egr-1은 정소에서 gonocytes, spermatogonial stem cells, peritubular cells, Leydig cells에서 주로 발현된다. MACS로 분리한 spermatogonial stem cell에서 GDNF에 의해 짧은 시간에 Egr-1 mRNA가 유도되는 것으로 보아, Egr-1은 GDNF에 의한 spermatogonial stem cell의 niche 유지에 중요한 기능을 담당하는 상위 전사인자로 사료된다.

Tooth bud is initiated by dental epithelium thickening and invaginated epithelium forms cap-like structure with condensed underlying mesenchyme. From this cap stage, specific epithelial cells form a cluster of non-dividing cells, primary enamel knot (PEK), and this PEK expresses abundant signaling molecules and functions as a signaling center for tooth development. Recently, hundreds of genes involved in tooth development have been reported by advanced genome-wide screening technology, but still remained unidentified genes obstruct elucidation of the precise molecular mechanisms underlying tooth development. In this work, we examined the expression patterns and developmental functions of a novel tooth germ-expressing gene, Dachshund1 (Dach1). Dach1, known as the cell fate regulator via controlling the transcription in various cell types, expressed in PEK region from cap stage to bell stage. In order to evaluate the developmental functions of Dach1 in tooth development, we cultivated the lower first molar at E12.5 for 2 and 4 days with or without treatments of antisense-oligodeoxynucleotides (AS-ODNs) against Dach1. After the knocking down of Dach1, morphological changes and cell physiology such as proliferation, cell death and migration patterns were examined. In addition, the expression levels of PEK-expressing genes such as Bmp2, Bmp7, Fgf4, Shh and Wnt5a were examined by using RT-qPCR and in situ hybridization methods. Based on these results, we suggest that Dach1 would involve in mice tooth morphogenesis through regulating the expressions of PEK related genes and cellular events to form the proper tooth structural formation.

Tdrd family members contain Tudor domain repeat which is found in polar granules in Drophila. Tdrd12 is one of Tdrd family members. Tdrd12 contains a DEAD-box and a Tudor domain. However, the molecular mechanism and physiological function of Tdrd12 has not been described. To examine the expression pattern of Tdrd12, RT-PCR and Northern blot analysis were performed using total RNAs extracted from tissues; liver, intestine, heart, brain, kidney, lung, brain, uterus, ovary, and testis. The full-length of Tdrd12 was amplified from total RNA from mouse testis and cloning into the cloning vector. Cloned PCR products were purified,sequenced and analyzed using the ABI Prism Sequencer 3130XL. To look into Tdrd12 protein location, rabbit antibodies against mouse Tdrd12 were made using two epitopes: 1st epitope: (318~334)- SQRPNEKPLRLTEKKDC and 2nd epitope: (737~750)- LEAKEDKKARRPLC, and its specificity was tested using tissue extracts including the gonad. Here, we identified that Tdrd12 mRNA is detected in the ovary and testis, but not in other tissues. The size of its transcript is about 4.5kb on the northern blot. Antibody against Tdrd12 detects about 150 kDa protein on the western blot analysis. Immunostaining assay shows that Tdrd12 is localized at the spermatid in the seminiferous tubules. The current study is the first to investigate Tdrd12 expression is limited in the gonad. Thissuggest that Tdrd12 plays a role in the gonad like other known Tdrd family members, Tdrd1, Tdrd6, Tdrd7, and Tdrd9.

The spermatogenesis and oogenesis-specific helix-loop-helix transcription factor 2 (Sohlh2) is exclusively expressed in germ cells of male and female gonad. Sohlh2 acts as a transcriptional factor via its specific DNA binding site, E-box to regulate target genes such as Lhx8, Zp genes, Ngn3. Sohlh2 localize in the female oocyte and in the male spermatogonia. However, the regulatory mechanism of Sohlh2 was poorly understood. In this study, we examine the patterns interacting with Sohlh2. First, we performed immunoprecipitation with the antibody against Sohlh2 protein extracts from the testis. Two-dimensional SDS-gel showed sexual distinguishable protein including Fkbp12, 13, 3, 59. Among them, Fkbp3 is a member of the immunophilin protein family, which play a role in immunoregulation and basic cellular processes involving protein folding and trafficking. Protein is a cis-trans prolyl isomerase that binds the immunosuppressants FK506 and rapamycin. It has a higher affinity for rapamycin than for FK506 and thus may be an important target molecule for immunosuppression by rapamycin. In the expression analysis of Fkbp3 is detected in the multiple tissues; intestine, stomach, kidney, spleen, liver, heart, brain, lung, uterus, ovary, testis. Here, we identified that Fkbp3 mRNA is detected in the ovary and testis, kidney, liver, heart, brain, lung. Immunostaning assay shows that Fkbp3 is localized at the spermatogonia in testis. In further studies, in order to confirm the interaction between Fkbp3 and Sohlh2, we will perform immunoprecipitation.

Hepatocytes derived from human embryonic stem cells (hESCs) may be a useful source for the treatment of diseased or injured liver. However, a low survival rate of grafted hepatocytes and immune rejection are still major obstacles to be overcome. We previously showed that secreted proteins (secretome) from hESC-derived hepatocytes had a potential therapeutic power in the tissue repair of injured liver without cell transplantation. The purpose of the present study was to discover key protein(s) in the secretome of hESC-derived hepatocytes using proteomic analysis and to study the tissue repair mechanism which may be operated by the secretomes. Purified indocyanine green+ hepatocytes derived from hESCs displayed multiple hepatic features, including expression of hepatic genes, production of albumin, and glycogen accumulation. The nano-LC/ESI-QTOF-MS analysis identified 365 proteins in the secretome of hESC-derived hepatocytes and the protein functional network analysis was conducted using the MetaCore TM from GeneGO. In addition, 20 tissue regeneration-related transcription factors (TFs) were extrapolated through further proteomic analysis. After intraperitoneal injection, the secretome significantly promoted the liver regeneration in a mouse model of acute liver injury. Protein functional network analysis on the secretome-induced regenerating liver confirmed 20 transcription factors (TFs) which were identified in the ICGhigh cells. The upreguation of these tissue repair-related TFs were validated by qPCR and western blotting on the regenerating liver tissues. These results demonstrate that application of the secretome analysis in combination with the protein functional network mapping would provide a reliable tool to discover new tissue-regenerating proteins as well as to expand our knowledge of the mechanisms of tissue regeneration.

MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.

Use of nature-derived matrices of a part of body tissues has been used to repair damaged tissues in practical terms. Recently, the same idea has also been applied to regenerate whole organs including the heart, liver, lung, and pancreas etc. Thus, so-called bio-artificial organ technology becomes a promising way of overcoming the lack of donor organs and immune rejections in organ transplantation if we can obtain recipient stem cells. Although the regenerated heart in vitro so far may demonstrate some typical organ's responses in vitro and vivo, it is still far from a fully functional organ for transplantation. We initiated a study to look at changes occurring during the generation of bio-artificial organ using the mouse model. Adult hearts were dissected out and perfused for acellularization with SDS-containing buffer and washed several times. Enzymatic treatment also evaluated the acellular purity by isolating genomic DNA and total RNA before and after DNase and RNase treatments. For recellularization, differentiating H9C2 cell or cells derived from P19 EC cells along with mesenchymal stem cells were seeded on the finally obtained heart matrix several times before submerging culture for generating the heart. Histological analyses revealed that complete removal of cellular components. The intensive staining of alcian blue (pH 1.5 and 2.5) suggests that acid mucopolysaccharides, glycocomponents and sulfate-containing saccharides are widely spread within the heart matrix. There was little DNA and RNA in the heart matrix after the enzymatic treatments as judging by the DAPI or PI staining. Cell seeding and subsequent submerging culture showed substantial heart tissue development as evidenced by immunocytochemistry and RT-PCR in the recellularized and grown heart. From these results, we suggest that each procedure for bio-artificial organ has to be carefully examined to improve the entire process.

Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48 h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.

For the study of population genetic structure with mtDNA, it is essential to measure genetic diversity at each mtDNA regions. Also, to evaluate the variation according to the each region should follow as well as to see if there are differences. In this study, we delved into the variations and dendrogram among samples of seven mtDNA regions (NDⅡ, NDⅤ, NDⅣ, NDⅣL, NDⅥ, NDⅠ, 12SrRNA) from wild Pacific abalone, Haliotis discus hannai collected in Yeosu, Korea. The region with the highest genetic variation was NDⅣ region (Haplotype diversity = 1.0000, Nucleotide diversity = 0.010823) with two to five times higher variation than the others. Furthermore, the study to see if there is a difference between the regions of samples showed that similar aspects of dendrogram in NDⅡ and NDⅠ(divergence of 90% and 87%), which forms a group with hd4, 7, 8 and 10 at bootstrap support, based on 1000 replications. Also, pair-wise FST between clusters within the regions showed high values; 0.4061 (P=0.0000), 0.4805 (P=0.0000) respectively. Therefore we can infer that it is the most efficient and accurate way to analyze the region of NDⅣ with the highest variation in addition to the regions of NDⅡ and NDⅠ, which formed clusters with high bootstrap value, for study of population genetic structure in this species.

Olive flounder (Paralichthys olivaceus) is a most important aquaculture species in Korea. Like most marine fishes, olive flounders are stomachless at first feeding and aquired gastric function during the metamorphosis, so food was mainly digested by pancreatic enzyme from first feeding to premetamorphosis. However, comprehensive analysis of pancreatic and gastric digestive enzyme of olive flounder at early developmental period is still unclear. In the expression study of pancreatic and gastric digestive enzyme by real-time PCR at early developmental stage, pancreatic enzyme such as chymotrypsinogen 2, preproelastase 2 and 4, pancreatic protein somatomedin-B domain (PPSB) mRNA expression were initiated at first feeding and strongly expressed in the pancreas developmental stage, while gastric digestive enzyme signal was not at all detected during same period. Although, trypsinogens were secreted from pancreas and have similar amino acid sequence, trypsinogen 3 expression induction was detected both pancreas and stomach developmental stage, while trypsinogen 2 expression was significantly increased only post-metamorphosis period. Pepsinogen mRNA expression was only detected at metamorphosis according to stomach differentiation. Lipid digestive enzyme, lipase and intestine fatty acid binding protein 1 (I-FABP 1), were already reached a certain level at beginning of hatching and more increased during early developmental stage and then gradually decreased before metamorphosis. These results suggested that feed ingestion of olive flounder was exclusive charged by pancreatic digestive enyme, lipid digestive enzyme and trypsinogen 3 from first feeding and then fully swiched by gastric digestive enzyme and trypsinogen 2 from metamorphosis period.

참굴 Crassostrea gigas는 우리나라의 매우 중요한 양식패류로 연간 양식에 필요한 종묘는 약 1,800만연 이상으로 대부분 자연채묘에 의한 천연종묘로 공급되고 있으며, 이중 10~15%를 인공종묘로 사용하고 있다. 그러나 인공종묘의 여러 가지 장점에도 불구하고, 천연종묘보다 생산단가가 높은 단점이 있다. 따라서 경제성이 있는 우량한 인공종묘의 생산, 보급을 위한 연구가 시급하다. 인공종묘의 생산단가를 줄이기 위해서는 우선 모패의 사육관리에 소요되는 경비를 줄이거나, 우량한 배우자를 사용하여 건강한 종묘를 생산, 폐사율을 줄임으로써 생산량을 증가시키는 방법이 있다. 모패의 사육관리에 소요되는 비용 절감과 우량 어미간 선택교배에 의한 우량종묘 생산을 동시에 해결할 수 있는 방법이 정자의 냉동보존 방법이다. 본 연구는 참굴의 정자 냉동보존시 적정 결빙억제제(cryoprotective agent, CPA) 및 농도를 탐색하고자, CPA 종류 및 농도별 냉동보존 효과를 파악하고 해동 후 정자의 세포 손상 등을 조사하였다. 실험에 사용한 참굴의 정액은 참굴 인공종묘배양장에서 모패로 사용 중인 2년생(각장 90.9±2.5 mm, 전중 62.3±3.0 g, 30마리) 어미굴로부터 채취하였다. CPA 종류 및 농도별 냉동효과를 조사하기 위한 희석액으로는 여과해수(33 psu)를, CPA는 dimethyl sulfoxide(DMSO), ethylene glycol (EG), glycerol 및 methanol을 사용하였다. 사용 농도는 전체 희석액에서 CPA의 최종농도가 각각 5, 10, 15, 20%가 되도록 하였다. CPA 및 농도별 냉동/해동 참굴 정자의 세포 손상을 파악하기 위하여 냉동보존 중인 참굴 정자를 해동하여 주사전자현미경(scanning electron microscope, SEM)으로 관찰하였다. CPA 종류 및 농도별 냉동보존 참굴 정자의 해동 후 생존율을 측정한 결과, DMSO로 냉동보존 한 참굴 정자의 생존율은 모든 농도에서 50% 이상이었으나, EG로 냉동보존 한 참굴 정자의 생존율은 농도 15%를 제외하고는 50% 이하 이었다. Glycerol과 methanol을 CPA로 사용하여 냉동보존한 참굴 정자 의 생존율은 모든 농도에서 20% 이하로 낮게 나타났다. CPA 종류 및 농도별 냉동보존 참굴정자의 해동 후 운동성을 측정한 결과, DMSO로 냉동보존한 참굴 정자의 SAI는 모든 농도에서 1.5 이상이었으나, 다른 CPA를 사용하여 냉동보존 한 참굴 정자의 SAI는 모두 1.0 이하로 운동성이 낮았다. CPA 종류 및 농도별 냉동/해동 후 참굴 정자의 형태를 SEM으로 관찰한 결과, 모든 종류의 CPA에서 농도가 낮아짐에 따라 정자 두부의 변형과 꼬리의 절단이 발생한 정자의 관찰빈도가 높았다. 냉동/해동 정자의 세포 손상은 DMSO, EG, methanol, glycerol 순으로 적었으며, 농도는 15, 20, 10, 5% 순으로 적었다. 이상의 결과를 종합한 결과, 참굴 정자의 냉동보존 시적정 CPA 및 농도는 15% DMSO이었다.

Three species of Nortamea concinua (NC) and Haliotis discus hannai (HDH) from Tongyeong and Sulculus diversicolor supertexta (SDS) are widely distributed on the coast of the Yellow Sea, southern sea and Jeju Island in the Korean Peninsula under the innate ecosystem. There is a need to understand the genetic traits and composition of three mollusk species in order to evaluate exactly the patent genetic effect. PCR analysis was performed on DNA samples extracted from a total of 21 individuals using seven decamer oligonucleotides primers. Amplification products were separated by electrophoresis in 1.4% agarose gels (Bioneer Corp., Daejeon, Korea) with TBE (0.09 M Tris, pH 8.5; 0.09 M borate; 2.5 mM EDTA), using 100 bp DNA ladder (Bioneer Corp., Daejeon, Korea) as DNA molecular weight marker and detected by staining with ethidium bromide. Seven primers, BION-55 (5’-GTCACGGACG-3’), BION-50 (5’-CAAGCGA GGA-3’), BION-75 (5’-GAGGTCCACA-3’), BION-35 (5’-AGCGGCTAGG-3’), BION-61 (5’-GAC CGCTTGT-3’), BION-69 (5’-GCATCCACCA-3’) and BION-66 (5’-TGGTGGACCA-3’) were shown to generate the unique shared loci to each species and shared loci by the three species which could be clearly scored. A hierarchical clustering tree was constructed using similarity matrices to generate a dendrogram, which was facilitated by the Systat version 10 (SPSS Inc., Chicago, IL, USA). 236 specific loci, with an average of 56.3 per primer, were identified in the NC species. 142 specific loci, with an average of 44.7 per primer, were identified in the HDH species. Especially, 126 numbers of shared loci by the three species, with an average of 18 per primer, were observed among the three species. Especially, the decamer primer BION-75 generated 7 unique loci to each species, which were identifying each species, in 700 bp NC species. Interestingly, the primer BION-50 detected 42 shared loci by the three species, major and/or minor fragments of sizes 100 bp and 150 bp, respectively, which were identical in all samples. As regards average bandsharing value (BS) results, individuals from HDH species (0.772) exhibited higher bandsharing values than did individuals from NC species (0.655). In this study, the dendrogram obtained by the seven decamer primers indicates three genetic clusters: cluster 1 (CONCINNA 01~ CONCINNA 07), cluster 2 (HANNAI 08~HANNAI 14), cluster 3 (SUPERTEXTA 15~SUPERTEXTA 21). Comparatively, individuals of HDH species were fairly closely related to that of SDS species, as shown in the hierarchical dendrogram of genetic distances.