scholarly journals Generation of a hTERT-Immortalized Human Sertoli Cell Model to Study Transporter Dynamics at the Blood-Testis Barrier

Pharmaceutics ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1005
Author(s):  
Raymond K. Hau ◽  
Siennah R. Miller ◽  
Stephen H. Wright ◽  
Nathan J. Cherrington
Keyword(s):  
Mrna Expression ◽  
Cell Model ◽  
In Vitro Models ◽  
Replication Capacity ◽  
Seminiferous Tubules ◽  
Difference Threshold ◽  
Transport Pathways ◽  
Equilibrative Nucleoside Transporters ◽  
Blood Testis Barrier

The blood-testis barrier (BTB) formed by adjacent Sertoli cells (SCs) limits the entry of many chemicals into seminiferous tubules. Differences in rodent and human substrate-transporter selectivity or kinetics can misrepresent conclusions drawn using rodent in vitro models. Therefore, human in vitro models are preferable when studying transporter dynamics at the BTB. This study describes a hTERT-immortalized human SC line (hT-SerC) with significantly increased replication capacity and minor phenotypic alterations compared to primary human SCs. Notably, hT-SerCs retained similar morphology and minimal changes to mRNA expression of several common SC genes, including AR and FSHR. The mRNA expression of most xenobiotic transporters was within the 2-fold difference threshold in RT-qPCR analysis with some exceptions (OAT3, OCT3, OCTN1, OATP3A1, OATP4A1, ENT1, and ENT2). Functional analysis of the equilibrative nucleoside transporters (ENTs) revealed that primary human SCs and hT-SerCs predominantly express ENT1 with minimal ENT2 expression at the plasma membrane. ENT1-mediated uptake of [3H] uridine was linear over 10 min and inhibited by NBMPR with an IC50 value of 1.35 ± 0.37 nM. These results demonstrate that hT-SerCs can functionally model elements of transport across the human BTB, potentially leading to identification of other transport pathways for xenobiotics, and will guide drug discovery efforts in developing effective BTB-permeable compounds.

scholarly journals Benzo(a)pyrene (B[a]P) and its adverse effects on male testis

2018 ◽  
Author(s):  
Wei Liu ◽  
Aihua Gu
Keyword(s):  
Body Weight ◽  
Sertoli Cells ◽  
Molecular Mechanisms ◽  
Treated Group ◽  
Male Reproduction ◽  
Adverse Impact ◽  
Phase Cell ◽  
Seminiferous Tubules ◽  
Blood Testis Barrier

ABSTRACTIt has been proved that Benzo(a)pyrene (B[a]P) is mutagenic in somatic cells, whereas the adverse effect of BaP on male reproduction remains unclear. To investigate whether it can pass through the blood-testis barrier (BTB) and its potential reproductive toxicology and molecular mechanisms, mice were exposed to B[a]P (there are two doses, that is 13mg/kg body weight and 26 mg/kg body weight; three times per week) during 6 weeks and sacrificed 6 weeks after the final exposure to obtain B[a]P-exposed testis, blood and others. Electron microscopy analysis was performed to confirm whether the integrity of BTB and the ultra-structure changes in testes of B[a]P treated mice, which showed that the integrity of the BTB was disrupted, accompanied with the structure of sertoli cells seriously damaged, including the integrity of the nuclear membrane of the sertoli cells impaired and the basement membrane of the seminiferous tubules disrupted. X-ray imaging in vitro told us that BaP can overgo the BTB and gathered in the testis of mice. We found the significantly decreased expression of ZO-1, occludin, N-cadherin, vimentin and claudin-1 in the testes of B[a]P treated group by immunofluorescence detection. B[a]P induced BTB component protein decreased were also found in TM4 cells exposed to 5μmol/L B[a]P for 24h. We found a significantly decrease of testosterone level and a significantly increase of estrogen level in the serum of treated groups comparing with the control one by radioimmunoassay. TM4 cells, MLTC-1 cells and GC-2 cells was cultured with medium contains B[a]P. MTT Cell Proliferation and Cytotoxicity Assay, cell apoptosis analysis, FACScan analyzer, We observed apparent increase of TM4 and GC-2 cells apoptosis after expose to B[a]P for 24h. B[a]P induced TM4 cell, GC-2 cell and MLTC-1 cell G2/M phase cell arrest. In conclusion, these results suggested that BaP has an adverse impact on male reproduction, it can cross the blood-testis barrier and damage it, the component proteins of the BTB significantly decreased, it can also produce adverse impact on male germ cells.

scholarly journals Curcumin Alleviates the Senescence of Canine Bone Marrow Mesenchymal Stem Cells during In Vitro Expansion by Activating the Autophagy Pathway

2021 ◽  
Vol 22 (21) ◽  
pp. 11356
Author(s):  
Jiaqiang Deng ◽  
Ping Ouyang ◽  
Weiyao Li ◽  
Lijun Zhong ◽  
Congwei Gu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Mrna Expression ◽  
Light Chain ◽  
Therapeutic Potential ◽  
Cell Model ◽  
Biological Aging ◽  
Aging Effects

Senescence in mesenchymal stem cells (MSCs) not only hinders the application of MSCs in regenerative medicine but is also closely correlated with biological aging and the development of degenerative diseases. In this study, we investigated the anti-aging effects of curcumin (Cur) on canine bone marrow-derived MSCs (cBMSCs), and further elucidated the potential mechanism of action based on the modulation of autophagy. cBMSCs were expanded in vitro with standard procedures to construct a cell model of premature senescence. Our evidence indicates that compared with the third passage of cBMSCs, many typical senescence-associated phenotypes were observed in the sixth passage of cBMSCs. Cur treatment can improve cBMSC survival and retard cBMSC senescence according to observations that Cur (1 μM) treatment can improve the colony-forming unit-fibroblasts (CFU-Fs) efficiency and upregulated the mRNA expression of pluripotent transcription factors (SOX-2 and Nanog), as well as inhibiting the senescence-associated beta-galactosidase (SA-β-gal) activities and mRNA expression of the senescence-related markers (p16 and p21) and pro-inflammatory molecules (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)). Furthermore, Cur (0.1 μM~10 μM) was observed to increase autophagic activity, as identified by upregulation of microtubule-associated protein 1 light chain 3 (LC3), unc51-like autophagy-activating kinase-1 (ULK1), autophagy-related gene (Atg) 7 and Atg12, and the generation of type II of light chain 3 (LC3-II), thereby increasing autophagic vacuoles and acidic vesicular organelles, as well as causing a significant decrease in the p62 protein level. Moreover, the autophagy activator rapamycin (RAP) and Cur were found to partially ameliorate the senescent features of cBMSCs, while the autophagy inhibitor 3-methyladenine (3-MA) was shown to aggravate cBMSCs senescence and Cur treatment was able to restore the suppressed autophagy and counteract 3-MA-induced cBMSC senescence. Hence, our study highlights the important role of Cur-induced autophagy and its effects for ameliorating cBMSC senescence and provides new insight for delaying senescence and improving the therapeutic potential of MSCs.

scholarly journals IVIVC Assessment of Two Mouse Brain Endothelial Cell Models for Drug Screening

Pharmaceutics ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 587 ◽  
Author(s):  
Ina Puscas ◽  
Florian Bernard-Patrzynski ◽  
Martin Jutras ◽  
Marc-André Lécuyer ◽  
Lyne Bourbonnière ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Mrna Expression ◽  
Mouse Brain ◽  
Drug Screening ◽  
Cell Model ◽  
Drug Permeability ◽  
Mouse Brain Endothelial Cells ◽  
Primary Model

Since most preclinical drug permeability assays across the blood-brain barrier (BBB) are still evaluated in rodents, we compared an in vitro mouse primary endothelial cell model to the mouse b.End3 and the acellular parallel artificial membrane permeability assay (PAMPA) models for drug screening purposes. The mRNA expression of key feature membrane proteins of primary and bEnd.3 mouse brain endothelial cells were compared. Transwell® monolayer models were further characterized in terms of tightness and integrity. The in vitro in vivo correlation (IVIVC) was obtained by the correlation of the in vitro permeability data with log BB values obtained in mice for seven drugs. The mouse primary model showed higher monolayer integrity and levels of mRNA expression of BBB tight junction (TJ) proteins and membrane transporters (MBRT), especially for the efflux transporter Pgp. The IVIVC and drug ranking underlined the superiority of the primary model (r2 = 0.765) when compared to the PAMPA-BBB (r2 = 0.391) and bEnd.3 cell line (r2 = 0.019) models. The primary monolayer mouse model came out as a simple and reliable candidate for the prediction of drug permeability across the BBB. This model encompasses a rapid set-up, a fair reproduction of BBB tissue characteristics, and an accurate drug screening.

scholarly journals Claudin-11 expression and localisation is regulated by androgens in rat Sertoli cells in vitro

Reproduction ◽  
2007 ◽  
Vol 133 (6) ◽  
pp. 1169-1179 ◽  
Author(s):  
Tu’uhevaha J Kaitu’u-Lino ◽  
Pavel Sluka ◽  
Caroline F H Foo ◽  
Peter G Stanton
Keyword(s):  
Mrna Expression ◽  
Sertoli Cell ◽  
Sertoli Cells ◽  
Hormonal Regulation ◽  
Cell Contacts ◽  
Barrier Formation ◽  
Protein Components ◽  
Blood Testis Barrier

Claudin-11 and occludin are protein components in tight junctions (TJs) between Sertoli cells which are important for the maintenance of the blood–testis barrier. Barrier formation occurs during puberty, with evidence suggesting hormonal regulation of both claudin-11 and occludin. This study aimed to investigate the regulation of claudin-11 and occludin mRNA expression by testosterone (T) and FSH and their immunolocalisation at rat Sertoli cell TJsin vitro, and to correlate any steroid regulation with the functional capacity of TJs. Sertoli cells formed functional TJs within 3 days as assessed by transepithelial electrical resistance (TER). Both T and dihydrotestosterone significantly (P< 0.01) increased TER twofold and claudin-11 mRNA two- to threefold within 3 days. FSH partially stimulated TER and claudin-11 mRNA, but estradiol had no effect. T also promoted claudin-11 localisation into extensive intercellular contacts. In contrast to claudin-11, Tand FSH did not change occludin mRNA expression, however, T promoted localisation of occludin at cell contacts in a similar manner to claudin-11. Addition of flutamide to T-stimulated cells caused a twofold decrease in both TER and claudin-11 mRNA expression, and resulted in the loss of both proteins from cell contacts. This effect was reversible following flutamide removal. It is concluded that androgens i) co-regulate claudin-11 mRNA expression and TER, implicating claudin-11 in TJ formation and ii) promote the localisation of claudin-11 and occludin at Sertoli cell contacts. Hence, the ability of androgens to maintain spermatogenesisin vivois partly via their effects on TJ proteins and regulation of the blood–testis barrier.

scholarly journals Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression

2014 ◽  
Vol 28 (1) ◽  
pp. 40-52 ◽  
Author(s):  
Dimiter Avtanski ◽  
Horacio J. Novaira ◽  
Sheng Wu ◽  
Christopher J. Romero ◽  
Rhonda Kineman ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Mrna Expression ◽  
Transcriptional Control ◽  
Estrogen Receptor Α ◽  
In Vitro Models ◽  
Mrna Levels ◽  
Ovariectomized Mice ◽  
Serum Gh

Abstract Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α– and ERβ-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERβ mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERβ in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

scholarly journals The expression and regulation of Bcl-2-related ovarian killer (Bok) mRNA in the developing and adult rat testis

2001 ◽  
pp. 771-778 ◽  
Author(s):  
JS Suominen ◽  
W Yan ◽  
J Toppari ◽  
A Kaipia
Keyword(s):  
Mrna Expression ◽  
Northern Hybridization ◽  
Seminiferous Tubules ◽  
Testicular Development ◽  
Rat Testis ◽  
Adult Rat ◽  
Before And After ◽  
Hybridization Intensity ◽  
Pachytene Spermatocytes

OBJECTIVE: To study the role of Bcl-2-related ovarian killer (Bok) in the regulation of apoptosis in the testis of developing and adult rat. METHODS: Bok mRNA expression was analyzed by Northern hybridization before and after culturing rat seminiferous tubules in vitro. Seminiferous tubules were cultured with different hormones and growth factors. Changes in the expression level of Bok mRNA during testicular development was analyzed by Northern hybridization. Localization of Bok mRNA was verified by in situ hybridization. RESULTS: Bok mRNA was highly expressed in the rat testis, varying during development. Highest expression levels were found in immature rats. Highest hybridization intensity appeared to be in spermatogonia, pachytene spermatocytes and Sertoli cells. Treatment with FSH was able to inhibit spontaneous increase of Bok mRNA expression that occurred in the defined stages of the rat seminiferous epithelium. CONCLUSIONS: FSH protects germ cells from apoptosis and this protective effect may at least partly be due to the inhibition of Bok gene expression. The amount of apoptosis varies during testicular development and highest expression of Bok mRNA occurs at the time of apoptosis, suggesting a possible role for Bok in its regulation.

An in vitro screening method for probiotics with antidepressant-like effect using the enterochromaffin cell model

2021 ◽  
Author(s):  
Peijun Tian ◽  
Huiyue Zhu ◽  
Renying Zou ◽  
Qinming Kong ◽  
Mengshu Xu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Model ◽  
Screening Method ◽  
In Vitro Screening ◽  
Enterochromaffin Cell

Screening the probiotics with antidepressant-like effects through measuring the bacteria stimulated Tph1 mRNA expression and 5-HTP/5-HT secretion in the enterochromaffin cell model RIN14B.

scholarly journals Rv3737 is required for Mycobacterium tuberculosis growth in vitro and in vivo and correlates with bacterial load and disease severity in human tuberculosis

2021 ◽  
Author(s):  
Qing Li ◽  
Zhangli Peng ◽  
Xuefeng Fu ◽  
Hong Wang ◽  
Zhaoliang Zhao ◽  
...  
Keyword(s):  
Growth Rate ◽  
Mycobacterium Tuberculosis ◽  
Mrna Expression ◽  
Disease Severity ◽  
Cell Model ◽  
Bacterial Load ◽  
Mrna Level ◽  
Expression Level

AbstractRv3737 is the sole homologue of multifunctional transporter ThrE in Mycobacterium tuberculosis (Mtb). In this study, we aimed to investigate whether this transporter participates in vitro and in vivo survival of Mtb. To characterize the role of Rv3737, we constructed and characterized an Mtb H37RvΔRv3737. This strain was evaluated for altered growth rate and macrophage survival using cell model of infection. In addition, the comparative analysis was conducted to determine the association between Rv3737 mRNA expression and disease severity in active pulmonary TB patients. The H37RvΔRv3737 strain exhibited significant slow growth rate compared to H37Rv-WT strain in standard culture medium. Additionally, the survival rate of H37Rv-WT strain in macrophages was 2 folds higher than that of H37RvΔRv3737 at 72 h. A significant higher level of TNF-α and IL-6 mRNA expression was observed in macrophages infected with H37RvΔRv3737 as compared to H37Rv-WT. Of note, Rv3737 expression was significantly increased in clinical Mtb isolates than H37Rv-WT. The relative expression level of Rv3737 was positively correlated with lung cavity number in TB patients. Similarly, the higher Rv3737 mRNA level resulted in lower C(t) value by Xpert MTB/RIF assay, demonstrating that a positive correlation between Rv3737 expression and bacterial load in TB patients. In conclusion, our data is the first to demonstrate that the transporter Rv3737 is required for in vitro growth and survival of bacteria inside macrophages. In addition, the expression level of Rv3737 is associated with bacterial load and disease severity in pulmonary tuberculosis patients.

Evaluation of renal in vitro models used in ochratoxin research

2016 ◽  
Vol 9 (3) ◽  
pp. 435-454
Author(s):  
A.H. Heussner ◽  
T. Paget
Keyword(s):  
Cell Model ◽  
Primary Cultures ◽  
In Vitro Models ◽  
Cellular Target ◽  
Proximal Tubular ◽  
A Cell ◽  
S3 Segment ◽  
Renal Proximal Tubular ◽  
Tissue Alterations

Ochratoxin A (OTA) induces renal carcinomas in rodents with a specific localisation in the S3 segment of proximal tubules and distinct early severe tissue alterations, which have been observed also in other species. Pronounced species- and sex-specific differences in toxicity occur and similar effects cannot be excluded in humans, however precise mechanism(s) remain elusive until today. In such cases, the use of in vitro models for mechanistic investigations can be very useful; in particular if a non-genotoxic mechanism of cancer formation is assumed which include cytotoxic effects. However, potential genotoxic mechanisms can also be investigated in vitro. A crucial issue of in vitro research is the choice of the appropriate cell model. Apparently, the cellular target of OTA is the renal proximal tubular cell; therefore cells from this tissue area are the most reasonable model. Furthermore, cells from affected species should be used and can be compared to cells of human origin. Another important parameter is whether to use primary cultures or to choose a cell line from the huge variety of cell lines available. In any case, important characteristics and quality controls need to be verified beforehand. Therefore, this review discusses the renal in vitro models that have been used for the investigation of renal ochratoxin toxicity. In particular, we discuss the choice of the models and the essential parameters making them suitable models for ochratoxin research together with exemplary results from this research. Furthermore, new promising models such as hTERT-immortalised cells and 3D-cultures are briefly discussed.

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