• Development and Reproduction
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• v.25 no.1
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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.

• Development and Reproduction
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• v.4 no.1
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• pp.37-43
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• 2000

Spatiotemporal expression of two isoforms of zonular occludens-1 (ZO-1), tight junctional protein, was examined in mouse testis. By RT-PCR, transcripts encoding two isoforms of ZO-1; ZO-1$\alpha$+ and ZO-1$\alpha$- were detected in testis. Two different forms of ZO-1 antigens with Mr.225 and 2001 KDa were detected in western blot of extract of neonatal to adult testis, coinciding with the result of RT-PCR. The relative amount of ZO-1 $\alpha$- versus 20-1 $\alpha$＋ increased as the mice matured. In immunostaining using the pan antibody which detected both isoforms, ZO-1 was localized in the intercellular spaces in the Sertoli cell - Sertoli cell contacts in periphery of seminiferous tubule as well as Sertoli cell - germ cells contacts within the seminiferous tubule. The expression of ZO-1 was ubiquitous in both junctional area and cytoplasm of seminiferous tubule components. However, more intense signals were found in Sertoli cell junctional areas according to sexual maturation. The changes in the relative amount of both isoforms and spatial distribution of ZO-1 at the periphery of seminiferous tubule might be important for functional appearance of blood testis barrier and spermatogenesis.

• Korean Journal of Environmental Biology
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• v.17 no.3
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• pp.285-292
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• 1999

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer known as one of endocrine disruptors. The present study was carried out to investigate the alterations of function and ultrastructure in rat testes after oral intubation of DEHP in dosages of 1g/kg/day, 2 g/kg/day or 3 g/kg/day in 0.5 ml of corn oil for 15 days. DEHP reduced the growth of body and testes, inhibited apermatogenesis and induced structural changes on various cell types of the rat testis. Leydig cells, Sertoli cells and the developing germ cells seemed to be impaired their differentiations in terms of the structural changes of cell organelles. The increase of heterochromatin in amount were common features in all 3 cell types. In addition, the Leydig cells were characterized by the swelling of smooth endoplasmic reticulum and perinuclear space, the increases in number and size of Iysosomes. The Sertoli cells became irregular in nuclear envelope and the number of Iysosomes and vacuoles seemed to be increased. There were some indications of necrosis of the germ cells, such as vacuolized nucleus and segregated nucleolus. And also, DEHP lowered the level of testosterone in experimental rat serum. DEHP suppressed apermatogenesis decreasing developing germ cells and these effects of DEHP on the rat testis were dose dependent. The detrimental effect of DEHP on apermatogenesis and ultrastructure of rat testes seems to be derived from the decreased level of testosterone by Leydig cells, followed by the abnomalities of Sertoli cells and the germ cells.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2004.11a
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• pp.168-168
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• 2004

• Applied Microscopy
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• v.16 no.2
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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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• v.45 no.2
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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.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 1998.07a
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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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• v.63 no.5
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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.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.08a
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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.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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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.