• 한국발생생물학회지:발생과생식
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• 제25권1호
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• pp.15-24
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• 2021

Benzo[a]pyrene (B[a]P) is a potent carcinogen and is classified as an endocrine-disrupting chemical. In mammalian testes, Sertoli cells support spermatogenesis. Therefore, if these cells are negatively affected by exposure to xenotoxic chemicals, spermatogenesis can be seriously disrupted. In this context, we evaluated whether mouse testicular TM4 Sertoli cells are susceptible to the induction of cytotoxicity-mediated cell death after exposure to B[a] P in vitro. In the present study, while B[a]P and B[a]P-7,8-diol were not able to induce cell death, exposure to BPDE resulted in cell death. BPDE-induced cell death is accompanied by the activation of caspase-3 and caspase-7. Depolarization of the mitochondrial membrane and cytochrome c release from mitochondria were observed in benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated cells. These results indicate that TM4 cells are susceptible to apoptosis in a caspase-dependent manner. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that aryl hydrocarbon receptor (AhR) expression was almost undetectable in TM4 cells and that its expression was not altered after B[a]P treatment. This indicates that TM4 cells are nearly AhR-deficient. In TM4 cells, the CYP1A1 protein and its activity were not present. From these results, it is clear that AhR may be a prerequisite for CYP1A1 expression in TM4 cells. Therefore, TM4 cells can be referred to as CYP1A1-deficient cells. Thus, TM4 Sertoli cells are believed to have a rigid and protective cellular machinery against genotoxic agents. In conclusion, it is suggested that tolerance to B[a]P cytotoxicity is associated with insufficient AhR and CYP1A1 expression in testicular Sertoli cells.

• 한국발생생물학회지:발생과생식
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• 제4권1호
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• pp.37-43
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• 2000

생쥐 정소에서 밀착결합단백질의 일종인 zonular occludens-1 (ZO-1)의 발현을 조사하였다. RT-PCR결과 ZO-1의 2가지 isoform인 ZO-1$\alpha$＋, ZO-1$\alpha$-의 발현을 확인하였다. 생쥐 신생 및 성체의 정소에서 분자량 225 및 2001 KDa의 2종의 ZO-1의 단백질항원의 발현을 확인되어 RT-PCR의 결과와 일치하였다. ZO-l$\alpha$＋에 대한 ZO-l$\alpha$-의 상대적 발현량은 성숙에 따라 증가하였다. 2종의 ZO-1항원을 동시에 인식하는 항체를 사용한 면역염색을 통해 세정관 외곽의 Sertoli세포 사이의 접촉부위 및 Sertoli 세포와 생식세포 접촉부위에서 ZO-1의 존재를 확인하였다. ZO-1은 세정관내 세포들 사이의 결합부위 및 세포질에서 공통적으로 발현되지만 성숙에 따라 Sertoli 세포의 결합부위에서 강한 신호가 검출되었다. 2종의 ZO-1 항원의 상대적 발현량의 변화 및 세정관 외곽의 분포의 강화는 기능적 혈액정소장벽의 출현 및 정자형성의 진행과 관련된 것으로 사료된다.

• 환경생물
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• 제17권3호
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• pp.285-292
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• 1999

플라스틱 제품의 가소제로 널리 사용되며, 최근 내분비 교란물질로 알려져 있는 di-(2-ethylhexyl)phthalate(DEHP)를 흰쥐에 15일 동안 구강 투여(1g/kg/day, 2g/kg/day, 3g/kg/day)한 후, 정자형성과정에 연관된 정소의 기능과 구조에 미치는 영향을 조사하였다. DEHP 처리군에서는 대조군에 비하여 체중 증가율이 감소하였을 뿐만 아니라 정소의 무게도 감소하였다. 또한 세정관의 직경이 고농도군으로 갈수록 작아지는 경향을 보였으며 세정관내의 세포층이 감소하는 현상이 나타났다. 미세구조의 변화를 관찰한 결과, 실험군은 세정관내 세포사이 공간이 증가하였으며 정원세포와 정모세포의 수가 감소하였고, 세포질내 공포가 증가하는 것이 관찰되었다. 또한 세정관내 Sertoli 세포의 전체 세포질 양이 감소하는 경향을 보였고, 정원세포의 경우 핵막 이중층이 분리되는 현상을 보였다. 특히 고농도군의 세정관내에서는 Sertoli 세포 이외의 세포는 거의 관찰되지 않았으며, 세포사이 공간과 공포들이 상당히 증가하였다. Sertoli 세포의 핵막은 심하게 함입된 형태를 나타냈으며 이질염색질이 증가하여 염색질의 덩어리를 이루고 있었고, Sertoli 세포들을 지지하는 기저판은 심하게 굴곡된 형태를 보였다. Leydig 세포의 미세구조에 있어서도 실험군은 대조군과 비교하여 현저한 차이를 보였다. 세포질내의 활면소포체와 핵막 이중층이 심하게 팽대되는 경향을 보였으며 핵내에 이질염색질이 상당히 증가하고 세포질내 리소솜이 증가하는 특징을 보였다.또한 혈청내 테스토스테론 함량에 있어서도 실험군은 현저히 낮은 수치를 나타냈다. 결론적으로, DEHP는 정소의 발달을 농도의존적으로 저해하고, Leydig 세포의 테스토스테론 합성기능을 저해하며, 이어서 세정관내부의 Sertoli 세포의 구조와 기능을 손상시켜 생식세포들의 괴사를 유도하는 과정을 통해 일련의 정자형성과정을 억제하는 것으로 사료된다.

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2004년도 KSOT/KEMS Fall Annual Meeting
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• pp.168-168
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• 2004

• Applied Microscopy
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• 제16권2호
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• pp.75-91
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• 1986

Differentiation of the rat testis was studied by light and electron microscope from the fetal stage up to the newborn or adult stage. The purpose of the present study is to investigate the ultrastructural changes of seminiferous tubules and interstitial tissue during the developmental process. The results were as follows: the seminiferous tubule diameter began to increase from birth and was fully developed at 30 to 40 days of age through intratubular cell proliferations. Basement membrane and myoid cells lining the seminiferous tubules were differentiated at 17 days gestation. At the fetal stage, seminiferous tubules were primarily composed of Sertoli cells and the differentiation of Sertoli and germ cells progressed from the newborn stage. Spermatids and immature spermatozoa are appeared at 40 days of age, so from this time, spermatogenesis occurred actively until the adult stage. Sertoli cells aided germ cell differentiation and phagocytosed the parts of the spermatid cytoplasm. Leydig ce]] development follows a biphasic pattern: a fetal phase and then an adult phase from 20 days of age. In conclusion, the rat testis is already developed to some extent by the fetal stage and is functional after 50 days of age. Therefore, these findings indicate that differentiation of Sertoli and Leydig cells precedes the onset of spermatogenesis.

• Clinical and Experimental Reproductive Medicine
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• 제45권2호
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• pp.75-81
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• 2018

Objective: Recapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium. Methods: Human WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at $36^{\circ}C$ for 3 more weeks. Results: The gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted. Conclusion: Human WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 1998년도 제4차 학술발표대회 및 정기총회
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• pp.43-44
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• 1998

Effect of somatic cell coculture on hatching of mouse blastocyst was examined. Mid-blastocysts were cocultured with granulosa cell primary culture or Sertoli cell line ($TM_{4}$) derived from mouse testis for 48 hr. Blastocysts cultured in medium (10% FBS) started to hatch more faster than cocultured embryos during 12 hr of coculture. After then blastocysts cocultured with somatic cell hatched faster than control. Degeneration of embryos was also greately reduced by coculture. This result suggested the potentiation of hatching as well as embryonic viability by coculture with somatic cell and Sertoli cell line can be used for embryo coculture.

• Journal of Animal Science and Technology
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• 제63권5호
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• pp.977-983
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• 2021

Closely correlated expression patterns between ubiquitin specific peptidase 9X-linked (USP9X) and adherens junction formation factor (Afadin) in mouse testis development suggests that Usp9x regulates the deubiquitination of Af-6 (also known as Afadin, AFDN), and subsequently, the cell adhesion dynamics during gametogenesis. However, this relationship has not yet been tested in other domestic animals. The study was examined the temporal and spatial expression patterns of porcine USP9X and AFDN from the pre-pubertal to adult stages using real time-PCR and immunohistochemistry. Furthermore, we detected the transcripts of USP9X and AFDN in the testis of 1-, 6- and 12-months old boar, respectively. USP9X and AFDN were found to have similar expressions patterns, with basal expression after 1 month followed by a significant up-regulation from 6 months (puberty) onwards. In addition, neither the AFDN or USP9X proteins were detected in spermatogenic cells but they were expressed in the leydig cells and sertoli cells. USP9X was detected around the basal lamina during pre-puberty, and predominantly expressed in the leydig cells at puberty. Finally, in adult testis, USP9X was increased at the sertoli cell-cell interface and the sertoli cell-spermatid interface. In summary, closely correlated expression patterns between USP9X and AFDN in boar testis supports the previous findings in mice. Furthermore, the junction connections between the sertoli cells may be regulated by the ubiquitination process mediated via USP9X.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2001년도 후기 제12차 학술대회 논문집
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• pp.38-38
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• 2001

TM4 Sertoli cell의 체외 관강형성 유도에 미치는 세포외기질 (ECM) 및 hepatocyte growth factor (HGF)의 역할과 세포분화 과정에서 MMP의 발현의 변화를 조사하였다. Matrigel bed(60%, v/v) 상에서 배양한 TM4 cell은 무혈청 조건하에서 chain 분화단계를 거쳐 cord의 구조로 분화하였다. 그러나 이후의 분화는 일어나지 않았다. TM4 cell에서 c-MET (HGF receptor)의 발현을 확인하였으며 HGF를 첨가한 배양액에서 분화가 촉진되었으며, cord에서 tubule로의 분화가 유도되었다. 또한 TM4 cell의 분화는 MMP-2 및 MMP-9의 발현이 증가를 수반하였으며 HGF는 MMPs의 발현을 증가시켰다. GFR-Matrigel과 성장인자인 HGF는 무혈청 배지에서 TM4 cell의 체외에서 관강형성에 필요한 환경을 제공하며, MMP-2 및 -9은 TM4 cell의 체외분화 과정에서 조절역할을 수행하는 것으로 사료된다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.63-63
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• 2003

In an effort to uncover the spermatogenic impairment by the polychlorinated biphenyls (PCBs), the expression of tight junctions (TJs) genes important for the formation of the blood testis barrier (BTB) were examined following the 3,3',4,4',5-pentachloro biphenyl (PCB126) treatment in cultured neonatal testis in mice. At 4 days (D4) after 10 and 100 nM PCB126 treatment the expression of claudin-11 was significantly increased when compared with vehicle control. In contrast no difference in occludin and claudin-1 expression was found among the experimental group. On D8, 100 nM PCB126 significantly increased the expression of claudin-11 but not occludin and claudin-1. 1 uM PCB126 treatment significantly decreased expressions of occludin and ciaudin -1, suggesting the general toxic effect on the Sertoli cell. Because PCB126 does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, it is likely that increase in the expression of claudin-11 by low dose of PCB126 may attribute to the alteration of the Sertoli cells differentiation in testis. It also emphasized that PCB126 might have differentially affected the transcription of TJ genes in Sertoli cells. In conclusion, this result suggests that the structure of TJ may be targeted by PCB126 in neonatal testis in mice and that co-PCB is potentially harmful to spermatogenesis by alteration of the development of BTB.