• Asian-Australasian Journal of Animal Sciences
• /
• 제13권5호
• /
• pp.587-594
• /
• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• 한국수산과학회지
• /
• 제29권1호
• /
• pp.44-50
• /
• 1996

조피볼락 (S. schlegeli)의 성분화 과정을 구명하기 위해 시원생식세포의 출현과 원시생식소 형성, 그리고 암${\cdot}$수의 성분화 과정을 조직학적으로 조사하였다. 시원생식세포는 출산후 2일, 전장 6.3mm 개체에서 장관과 중신사이의 섬유성 간충직에 묻혀 식별되었다. 출산 65일 전장 $5.2\~5.9cm$ 개체에서, 생식소의 주변 부위에 강 (cavity)을 형성하고 다수의 생식원세포로 구성된 생식소는 난소로 발달한다. 이 시기에 체세포들이 곡정세관을 형성하는 생식소는 정소로 분화된다. 출산후 115일, 전장 $7.0\~7.2cm$ 치어들에서, 생식소는 흑색소를 함유하는 정소와 흑색소가 없는 난소로 구분된다. 따라서 조피볼락은 초기 성분화 단계에 자상을 거치지 않고 난소와 정소로 분화되는 분화형의 자웅이체에 속한다. 출산후 12개월된 개체들의 성비는 1 : 1이었다.

• 한국양식학회:학술대회논문집
• /
• 한국양식학회 2006년도 수산관련학회 공동학술대회 발표요지집
• /
• pp.667-669
• /
• 2006

• Anatomy and Cell Biology
• /
• 제56권4호
• /
• pp.508-517
• /
• 2023

In cancer patients, chemo/radio therapy may cause infertility by damaging the spermatogenesis affecting the self-renewal and differentiation of spermatogonial stem cells (SSCs). In vitro differentiation of stem cells especially mesenchymal stem cells (MSCs) into germ cells has recently been proposed as a new strategy for infertility treatment. The aim of this study was to evaluate the proliferation and differentiation of SSCs using their co-culture with Sertoli cells and conditioned medium (CM) from adipose tissue-derived MSCs (AD-MSCs). Testicular tissues were separated from 2-7 days old neonate Wistar Rats and after mechanical and enzymatic digestion, the SSCs and Sertoli cells were isolated and cultured in Dulbecco's modified eagle medium with 10% fetal bovine serum, 1X antibiotic, basic fibroblast growth factor, and glial cell line-derived neurotrophic factor. The cells were treated with the CM from AD-MSCs for 12 days and then the expression level of differentiation-related genes were measured. Also, the expression level of two major spermatogenic markers of DAZL and DDX4 was calculated. Scp3, Dazl, and Prm1 were significantly increased after treatment compared to the control group, whereas no significant difference was observed in Stra8 expression. The immunocytochemistry images showed that DAZL and DDX4 were positive in experimental group comparing with control. Also, western blotting revealed that both DAZL and DDX4 had higher expression in the treated group than the control group, however, no significant difference was observed. In this study, we concluded that the CM obtained from AD-MSCs can be considered as a suitable biological material to induce the differentiation in SSCs.

• 한국수정란이식학회지
• /
• 제29권4호
• /
• pp.351-359
• /
• 2014

Phasianus colchicus is not only a beautiful bird but also a great value in science and under the threat of endanger. Hence, the aim of this study was to isolate the pheasant male germ cells (mGCs) and then induce them into elongated sperm-like cells in vitro. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the c-kit positive cells and c-kit negative cells examined by flow cytometry analysis (FCA) was 92.87% and 2.57%, respectively. Subsequently, the mGCs were induced for 48h in DMEM/F12 medium supplemented factors such as retinol acid, testosterone and bovine FSH, followed by 5 weeks in culture. We found that some elongated sperm-like cells appeared initially in vitro under inducement of stimulated factors. The elongated sperm-like cells showed in the expression of changed morphology and post-transcriptional marker such as spermatid associated (SPERT), spermatid perinuclear RNA binding protein (STRBP), round spermatid basic protein 1 (RSBN1) and SPER1L. Moreover, in DNA content identified assay, induced cells showed that the 1C DNA population markedly increased in differentiated group but it was not change in undifferentiated group. Successful in vitro differentiation of pheasant testicular germline cells into spermatids appears to offer extremely attractive potential for the conservation of endangered birds and treatment of male infertility.

• Toxicological Research
• /
• 제29권4호
• /
• pp.221-227
• /
• 2013

Embryonic stem (ES) cells have potential for use in evaluation of developmental toxicity because they are generated in large numbers and differentiate into three germ layers following formation of embryoid bodies (EBs). In earlier study, embryonic stem cell test (EST) was established for assessment of the embryotoxic potential of compounds. Using EBs indicating the onset of differentiation of mouse ES cells, many toxicologists have refined the developmental toxicity of a variety of compounds. However, due to some limitation of the EST method resulting from species-specific differences between humans and mouse, it is an incomplete approach. In this regard, we examined the effects of several developmental toxic chemicals on formation of EBs using human ES cells. Although human ES cells are fastidious in culture and differentiation, we concluded that the relevancy of our experimental method is more accurate than that of EST using mouse ES cells. These types of studies could extend our understanding of how human ES cells could be used for monitoring developmental toxicity and its relevance in relation to its differentiation progress. In addition, this concept will be used as a model system for screening for developmental toxicity of various chemicals. This article might update new information about the usage of embryonic stem cells in the context of their possible ability in the toxicological fields.

• 한국발생생물학회지:발생과생식
• /
• 제12권2호
• /
• pp.195-202
• /
• 2008

1998년 3월부터 1999년 2월까지 전라남도 대흑산도 연안에서 채집한 비단가리비를 대상으로 생식소중량지수, 생식세포 분화 및 난소주기를 조직, 세포학적 관찰에 의해 조사하였다. 초기 난황형성 난모세포에서, 골지체, 미토콘드리아 및 조면소포체들은 지방적 형성에 관여하였다. 후기난황형성난모세포에서 생식상피상에 존재하는 외인성 물질들 즉, 글리코겐 입자들 및 지방 과립상 물질들이 난황막의 미세융모를 통해서 난모세포의 난질로 통과해 들어갔다. 후기난황형성난모세포에서, 난황과립들과 다포체들은 단백질성 난황과립형성에 관여하였다. 난황형성과정은 내인성 자율합성과 외인성 타가합성에 의해서 일어난다. 보조세포들은 초기단계인, 전난황형성 난모세포 및 초기 난황형성 난모세포들의 형성 및 발달에 영양세포로서 기능을 한다. 생식소 중량지수의 월별 변화는 난소 발달단계들과 밀접한 관련을 가지며 변하였다. 본 종의 생식주기는 초기활성기(1~3월), 후기활성기(3~4월), 완숙기(4~8월), 부분산란기(6~8월), 퇴화 및 비활성기(8~12월)의 5단계로 구분되었다. 산란은 6~8월 사이에 일어나며, 주산란기는 수온이 높은 7~8월이었다.

• 수산해양교육연구
• /
• 제27권4호
• /
• pp.1031-1040
• /
• 2015

섬진강 기수역에 서식하는 피뿔고둥, R. venosa(Muricidae)의 생식세포분화와 난모세포 내에서의 난형성과정 중 난황형성의 미세구조적 연구를 위해 투과전자현미경 관찰로 조사하였다. 초기난황형성난모세포들에서 골지체와 미토콘드리아는 글리코겐 입자들, 지방적들과 난황과립들의 형성에 관여하였다. 후기난황형성난모세포들에서 조면소포체와 다포체들은 세포질 내에서 단백질성 난황과립들의 형성에 관여하였다. 여러 크기의 다포체들은 후기난황형성 난모세포 내에서 변형된 미토콘드리아들에 의해 형성되었다. 특히, 다른 복족류들의 결과들과 이매패류에서 일어나는 현상을 비교하여 보면, 이매패류의 경우에는 난황형성의 타가합성과 관련된 것으로, 난황막 위에 미세융모(microvilli)와 피질층에 피질과립들이 출현하고 있는데, 복족류의 경우는 난황형성난모세포내에서 난황형성 중 난모세포 밖으로부터 외생적으로 일어나지 않아 출현하지 않는 점이 이매패류나 두족류와 다른 점이다. 성숙란 내의 성숙난황과립은 3가지 성분으로 이루어져 있다 : (1) 난황과립의 중앙부에 결정중심이 있고, (2) 주변부에는 전자밀도가 밝은 피질층이 있고, 그리고 이들을 (3) 한계막이 둘러싸고 있다. 최종적으로 복족류 종들의 난모세포들 내에서의 난황형성과정은 타가합성과정(외생적 endocytosis)이 발견되지 않고 단지 난모세포 자체의 내생적 자율합성과정을 거쳐 일어나는 특징을 보였다. 피뿔고둥은 난황형성을 위해 난황형성과정 중, 염분농도가 높고 낮음으로 인해 내생적 자율합성과 외생적 타가합성이 변경된 현상은 발견되지 않았다. 교미시기와 산란 활성은 수온 및 염분농도 상승과 관련이 있다.

• 한국발생생물학회지:발생과생식
• /
• 제17권1호
• /
• pp.9-16
• /
• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

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