• 한국발생생물학회지:발생과생식
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• 제2권1호
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• pp.45-52
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• 1998

강화인자 검출법을 이용 X염색체에 P[1ArB]이 형질전환되어 극세포 및 경게세포에서 표시유전자 lacZ를 발현하는 노랑초파리 (EDL 149)를 이용하여 난자형성과정 동안의 경계새포의 분화 및 이동을 조사하였다. 경계세포는 9기 난포의 선단에 위치한 난포 세포로부터 분화하여 9기와 10기에 이동하는 것을 확인하였다. 난소내 \beta -galactosidase의 활성은 우화 후 처음 4일간 급격히 증가하는 것을 확인하였으며 이 시기는 난포 내에서 경계세포가 분화하는 시기와 일치하였다. EDL149의 P[1ArB]삽입의 동형접합체의 난포 내에서 일부 경계세포의 불완전한 이동 또는 지연이 관찰되었다. 감수분열을 진행중인 정소내 세포 및 더듬이에서 확인된 lacZ 유전자의 발현양상은 P[1ArB]의 삽입부위가 난소특이 유전자부위가 아니지만 경계세포 이동의 조절에 역할을 하는 유전자임을 암시한다. 이 형질전환초파리 및 삽입위치 부근의 유전자는 발생중 진행되는 세포이동의 연구에 좋은 모델로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1347-1354
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• 2006

The aims of this study were to establish a simple and effective method for isolating male germline stem cells (GSCs), and to test the possibility of using these cells as a new approach for male infertility treatment. Testes obtained from neonatal and adult mice were manually decapsulated. GSCs were collected from seminiferous tubules by a two-step enzyme digestion method and plated on gelatin-coated dishes. Over 5-7 days of culture, GSCs obtained from neonates and adults gave rise to large multicellular colonies that were subsequently grown for 10 passages. During in vitro proliferation, oct-4 and two immunological markers (Integrin ${\beta}1,\;{\alpha}6$) for GSCs were highly expressed in the cell colonies. During another culture period of 6 weeks to differentiate to later stage germ cells, the expression of oct-4 mRNA decreased in GSCs and Sertoli cells encapsulated with calcium alginate, but the expression of c-kit and testis-specific histone protein 2B(TH2B) mRNA as well as the localization of c-kit protein was increased. Expression of transition protein (TP-l) and localization of peanut agglutinin were not seen until 3 weeks after culturing, and appeared by 6 weeks of culture. The putative spermatids derived from GSCs supported embryonic development up to the blastocyst stage with normal chromosomal ploidy after chemical activation. Thus, GSCs isolated from neonatal and adult mouse testes were able to be maintained and proliferated in our simple culture conditions. These GSCs have the potential to differentiate into haploid germ cells during another long-term culture.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.55-66
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• 2002

배아줄기세포(embryonic stem cell, ESC)는 미분화상태로 지속적인 계대가 가능하며, 정상 핵형과 전 분화능(pluripotency)을 가져 생체내-외에서 분화 유도시 삼배엽성의 모든 세포로 분화 가능하다. ESC를 feeder 세포 없이 부유배양하면 배아체(embryoid body, EB)를 형성하고, 초기 배아 발생과 유사한 분화 양상을 갖는다. ESC의 분화 유도가 초기배아 발생처럼 생식호르몬(GTH: FSH, LH; steroids)의 영향을 받는지는 불명하다. 본 연구는 ESC가 분화과정중 생식호르몬처리에 의해 그들 수용체가 발현되는가를 알아보고자 하였다. 순계혈통 생쥐인 C57BL/6J에서 과배란 유도후 포배를 수획하고, 유사분열적으로 불활성화된 feeder 세포와 공배양하여, 계대배양 하는 중 배아줄기세포주(JHYl)를 확립하였다. JHY1의 alkaline phosphatase 활성과 SSEA-1, 3, 4 발현을 통해 ESC임을 확인하였다. Feeder 세포 없이 ESC를 계대배양 후 호르몬처리(FSH LH E$_2$, P$_4$, T)하에서 5일 동안 부유배양하여 배아체를 형성시키고, 이후 7일 동안 부착배양하여 분화를 유도하였다. GTH와 스테로이드의 수용체 발현 실험에서 ESC에 E$_2$ 처리에 의한 LHR의 발현 증가를 제외한 나머지 호르몬 처리군에서 ESC보다 낮은 생식호르몬의 수용체 발현이 관찰되었다. 생식호르몬을 농도별 수용체 발현 정도는 증감되지 않았다. 미분화 ESC 표지유전자인 Oct-4는 호르몬 처리군에서도 발현되었다. 각 배엽의 표지유전자들(영양세포, handl; 외배엽성, keratin와fgf-5; 중배엽성, enolase와 $\alpha$ -globin; 내배엽성, gata-4와 $\alpha$ -fetoprotein) 등의 발현 양상을 조사한 결과 호르몬 처리후 내배엽성 표지유전자외에는 발현 증가가 관찰되지 않았다. 즉 생식호르몬에 의해 gata-4, $\alpha$-fetoprotein의 발현이 증가되는 것으로 보아 내배엽성 계열로의 분화 유도가 이루어진 것으로 사료된다.

• Journal of Animal Science and Technology
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• 제66권4호
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• pp.635-644
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• 2024

Spermatogonial stem cells originate from gonocytes and undergo self-renewal and differentiation to generate mature spermatozoa via spermatogenesis in the seminiferous tubules of the testis in male mammals. Owing to the unique capacity of these cells, the spermatogonial stem cell transplantation technique, which enables the restoration of male fertility by transfer of germlines between donor and recipient males, has been developed. Thus, spermatogonial stem cell transplantation can be used as an important next-generation reproductive and breeding tool in livestock production. However, in large animals, this approach is associated with many technical limitations and inefficiency. Furthermore, research regrading spermatogonial stem cell transplantation in stallions is limited. Therefore, this review article describes the history and current knowledge regarding spermatogonial stem cell transplantation in animals and challenges in establishing an experimental protocol for successful spermatogonial stem cell transplantation in stallions, which have been presented under the following heads: spermatogonial stem cell isolation, recipient preparation, and spermatogonial stem cell transplantation. Additionally, we suggest that further investigation based on previous unequivocal evidence regarding donor-derived spermatogenesis in large animals must be conducted. A detailed and better understanding of the physical and physiological aspects is required to discuss the current status of this technique field and develop future directions for the establishment of spermatogonial stem cell transplantation in stallions.

• Applied Microscopy
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• 제31권2호
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• pp.143-156
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• 2001

본 연구는 황소개구리의 정소내 생식세포분화 단계와 정자변태과정중의 정자의 형태적 특징을 알아보기 위하여 조사한 결과 다음과 같은 결론을 얻었다. 황소개구리의 정자형성과정 중 생식세포는 제1정원세포, 제2정원세포, 제1정모세포, 제2정모세포 및 정자세포로 구성되어져 있으며, 이들 생식세포의 분화단계는 세포의 형태적 특정을 기초로 하여 총 8단계로 구분되어졌다. 제1정원세포를 제외한 정모세포발생 단계에서부터 이탈 전까지의 정자세포는 정낭내에 존재하고 있었다. 정자변태과정은 3단계로 구분되어졌다. 성숙기의 정자의 첨체는 낭상이고, 두부의 모양은 양끝이 가는 원통형이었으며, 꼬리는 단지 축사로만 구성되어져 있었다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• ALGAE
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• 제19권3호
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• pp.191-199
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• 2004

Morphological and phonological characteristics of brown alga Spatoglossum crassum Tanaka new to Korea were described based on the field and the indoor cultured plants. The taxonomic characteristics of the plants were agreed to those from the type locality-submerged reproductive organs in cortex, anatomical features, and absence of phaeophycean hairs on the surface. But they have rudimentary midrib on lower portion of thallus. We can observe the young plants on November, adult ones in June, and senile ones in August. This species has an annual life-cycle in the field, starting with germ lings in early November. The differentiation of thallus is quite different from other species of genera in tribe Zonarieae, e.g. Zonaria and Homoeostrichus. Three different tissues, meristoderm, cortex and medulla are discerned. The outmost cortical one celled layer as a meristoderm produce cortex by unequal periclinal division. In the apical cell division, the primary inner cells are developed into 3-4 cell layered medulla of thallus. The distribution of this species extends from Korea to Shizuoka Peninsula (34°40'N) Japan, which is the type locality of this species.

• Reproductive and Developmental Biology
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• 제32권4호
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• pp.223-227
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• 2008

Human embryonic stem (ES) cells retain the capacity for self-renewal, are pluripotent and differentiate into the three embryonic germ layer cells. The regulatory transcription factors Oct4, Nanog and Sox2 play an important role in maintaining the pluripotency of human ES cells. The aim of this research was to identify unknown genes upregulated in human ES cells along with Oct4, Nanog, and Sox2. This study characterizes an unknown gene, named chromosome 1 open reading frame 31 (C1orf31) mapping to chromosome 1q42.2. The product of C1orf31 is the hypothetical protein LOC388753 having a cytochrome c oxidase subunit VIb (COX6b) motif. In order to compare expression levels of C1orf31 in human ES cells, human embryoid body cells, vascular angiogenic progenitor cells (VAPCs), cord-blood endothelial progenitor cells (CB-EPCs) and somatic cell lines, we performed RT-PCR analysis. Interestingly, C1orf31 was highly expressed in human ES cells, cancer cell lines and SV40-immortalized cells. It has a similar expression pattern to the Oct4 gene in human ES cells and cancer cells. Also, the expression level of C1orf31 was shown to be upregulated in the S phase and early G2 phase of synchronized HeLa cells, leading us to purpose that it may be involved in the S/G2 transition process. For these reasons, we assume that C1orf31 may play a role in on differentiation of human ES cells and carcinogenesis.

• Applied Microscopy
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• 제31권2호
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• pp.157-165
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• 2001

정상 성인 고환의 버팀세포(Sertoli cell)는 비분열세포이며, 정세관(seminiferous tubule)단면에서 비교적 불분명하게 관찰되고, 정세관 세포 성분의 $10\sim15%$를 차지하고 있다. 전자현미경적으로 버팅 세포는 특징적인 핵소체와 원형질막 및 세포질 소기관을 갖고 있다. 원형질막은 사춘기에 발달한 두 종류 즉 버팅세포와 버팀세포 및 버팅세포와 생식세포 사이의 세포연접을 가지고 있다. 그러나 태이에서 버팅세포의 정확한 미세구조에 대한 기술은 드물다. 이에 본 저자는 태아 고환의 발생 제 14주부터 제27주 사이의 17예를 수집하여 정상 미세구조를 확인하고, 태아기 버팀세포의 분화 양상을 알아보고자 하였다. 태아기에서 버팀세포와 생식세포 및 버팀세포와 버팀세포 사이의 세포연접은 부착반점과 비슷한 구조로 이루어져 있었고, 이들은 관찰 대상인 태령 제14주부터 관찰되었다. 태아기 버팅세포의 세포소기관의 발달은 전반적으로 미약하였다. 비교적 풍부하게 사립체가 태령 제14주부터 관찰되었고, 무과립세포질세망이 소수, 그리고 과립세포질세망이 비교적 풍부하게 관찰되었다. 지방소포의 수는 비교적 일정하게 관찰되었고, 포도당입자는 발생 단계에 따라 점차 증가하는 소견을 보였다. 미세섬유와 Charcot-Bottcher의 결정소체는 본 연구대상에서는 관찰되지 않았다. 결론적으로, 태아기의 버팀세포에서는 어른에서 관찰되는 특징적인 소견들이 관찰되지 않았으며, 어른과는 다소 다른 전자현미경 소견을 나타냈다. 하지만 버팅세포의 분화양상을 정확히 알기 위해서는 태령 제27주 이후부터 사춘기까지의 연구가 추가되어야 할 것으로 생각한다.

• 한국발생생물학회지:발생과생식
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• 제1권2호
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• pp.189-202
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• 1997

The hypothalamic peptide GnRH plays a central role in the regulation of the mammalian reproductive axis. Recent studies suggested that GnRH stimulates or inhibits the ovarian steroidogenesis and gametogenesis directly. Our previous report indicated that GnRH gene is expressed in adult rat ovary as well as in hypothalamus and that the expressed GnRH may induce the follicular atresia and apoptosis of ovarian granulosa cells in rat. Therfore, we studied whether GnRH gene is expressed in the mouse fetal ovary, when the germ cells are degenerating by apoptosis during gonad diffeerentiation. Mouse fetal gonads were obtained on the 12, 15,18 and 20th day of gestation from the mother mice superovulated (10 IU PMSG and 10 IU hCG) and mated. The morphological changes of fetal ovaries were examined histochemically by hematoxylin-eosin staining. The fetal sex was confirmed by PCR methods for sexing. RT-PCR methods were used to examine the expression of GnRH gene and the sex steroid hormones were determined by conventional radioimmunoassays. The levels of estradiol (E) and progesterone (P) were increaseduntil 18th day of gestation and then E was decreased just before parturition. The morphological changes of fetal gonadal tissue sections showed the ovarian development and coincided with the result of PCR analysis for sexing using ovary- or testis- specific oligonucleotide primers. Immunoreactive GnRH in placenta was decreased gradually until the end of gestation but fetal brain and ovarian GnRH were increased. The level of GnRH gene expression was increased during fetal ovarian development from 12 till 18th day and decreased suddenly on 20th day just before birth. From these results, it is suggested that ovarian GnRh may play a regulatory role on the germ cell differentiation of fetal ovary.