Assessment of estradiol-induced gene regulation and proliferation in an immortalized mouse immature Sertoli cell line

    Assessment of estradiol-induced gene regulation and proliferation in an immortalized mouse immature Sertoli cell line Narender Kumar, Swati Srivastava, Malgorzata Burek, Carola Y. Forster, Partha Roy PII: DOI: Reference:

S0024-3205(16)30027-3 doi: 10.1016/j.lfs.2016.01.027 LFS 14664

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

7 March 2015 14 January 2016 15 January 2016

Please cite this article as: Kumar Narender, Srivastava Swati, Burek Malgorzata, Forster Carola Y., Roy Partha, Assessment of estradiol-induced gene regulation and proliferation in an immortalized mouse immature Sertoli cell line, Life Sciences (2016), doi: 10.1016/j.lfs.2016.01.027

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Assessment of estradiol-induced gene regulation and proliferation in an immortalized mouse

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immature Sertoli cell line

Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, Uttarakhand, India University Wurzburg, Department of Anaesthesia and Critical Care, Oberduerrbacher Strasse 6, 97080 Wurzburg, Germany

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Roya*

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Narender Kumar,a Swati Srivastava,a Malgorzata Burek,b Carola Y. Forsterb and Partha

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*To whom correspondence should be addressed: Molecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, Uttarakhand, India. Phone: +91 1332 285686; Fax: +91 1332 273560; E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Aims: The number of Sertoli cells during proliferative phase determines the fate of the germ cells in male reproductive system. A well-characterized cell line may help better

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understanding of Sertoli cell biology. Hence, the present study assessed estradiol signaling in

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a mouse immature Sertoli cell line (MSC-1) as an alternative model in place of primary

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culture of Sertoli cells.

Main methods: In this study, we used MSC-1 cell line, derived from 10-day old mice. The cell cycle parameters were assessed, and the expression and regulation of Sertoli cell-specific

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secretory genes (ABP; androgen-binding protein) and tight junction genes (claudin-5,

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occluding, and vimentin) in response to estradiol was studied. Key findings: The results obtained suggested the presence of both estrogen receptors (ERα and ERβ) in MSC-1 cells. In vitro scratch assay and cell-cycle analysis suggested the

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proliferative effects of estradiol in both time- and dose-dependent manner. The gene

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expression profiles of ABP, claudin-5, and occludin - showed biphasic regulation at low and high doses of estradiol. Analysis of signaling pathways suggested the activation of

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extracellular signal-regulated kinase (ERK) pathway with significantly increased pERK/ERK ratio (p < 0.05). The results also suggested down regulation in the expression of mir-17

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family members (mir-17, mir20b, and mir-106a) (p < 0.05).

Significance: Considering the limited number of Sertoli cell lines and long-term survival inability of primary culture of Sertoli cells, MSC-1 cells could be a potential cell line for understanding the mechanisms of various cellular events in Sertoli cells.

Keywords: Estrogens; Sertoli cell proliferation; Estrogen receptors; MAPK pathway; microRNAs; epigenetic

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Introduction

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Sertoli cells are the somatic cells in male testicular tissues, which provide support and

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nourishment to germ cells. Androgen signaling is of prime importance in these cells, but

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estrogens contribute equally toward the development and function of male reproductive system (Lucas et al., 2011). Although primary culture of Sertoli cells is considered to be the

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best model for mechanistic studies, long-term survival inability after successive subculture is the major restriction in this model. Hence, importance of transformed cell lines taken into

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account in such studies. Sertoli cells have been reported to be present in pre-pubertal as well as adult mice or rat model (Mather, 1980; Hofmann et al., 1992; Boekelheide et al., 1993;

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Jiang et al., 1997; Roberts et al., 1995). In the present study, we used MSC-1 cells, a mouse

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Sertoli cell line from transgenic mice carrying a fusion gene composed of human anti-

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mullarian hormone (AMH) transcriptional regulatory sequences linked to SV-40 T-antigen (Eskola et al., 1998). These cells ultrastructurally resemble normal Sertoli cells and secrete peptides such as transferrin, sulphated glycoproteins (SGP 1 & 2), ABP, and inhibin-β

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proteins (McGuinness et al., 1994). Proliferation of immature Sertoli cells is an important phenomenon which determines the supportive capacity at adult stages during cell differentiation. The presence of ER, that is, both ERα and ERβ, has been confirmed in Sertoli cells from rats of different age (Bois et al., 2010). Estradiol signaling in Sertoli cells includes binding of estradiol to ER which in turn activates Src kinases. The activated Src tyrosine kinases further stimulates epidermal growth factor receptor (EGFR). These signals in turn control the proliferation and differentiation of Sertoli cells. Besides, compelling evidence has shown the activation of G protein-coupled receptor in the Sertoli cells of a 15-day old rat; binding of estradiol to this protein may lead to 3

ACCEPTED MANUSCRIPT signal transduction and regulation of the expression of genes associated with apoptotic functions (Lucas et al., 2011). Moreover, precise mechanism/s of estradiol regulation has

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been recently elucidated by Lucas et al. (2014). According to this study, estradiol acts on

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ERα through NF-κβ-mediated activation of CCND1 resulting in cell proliferation, while ERβ

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activation results in cell cycle exit and differentiation via increase of GATA-1, DMRT-1, and CDKN1B. Hence, based on the above-mentioned facts and the already existing information, it can be predicted that estradiol regulates the proliferation of immature Sertoli cell by both

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genomic and non-genomic mechanism of actions (Simoncini et al., 2004).

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Further, besides the regulation at the genetic level, the role of microRNAs (miRNAs) in the post-transcriptional regulation of gene expression has gained much importance.

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MiRNAs are small (17 - 22 nucleotides) non-coding RNAs (nc-RNAs) that can bind to the

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3’-UTR of the target gene mRNA and are involved in the regulation of mRNA translation. MiRNAs play regulatory roles by various pathways includeing mRNA degradation,

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translational repression, DNA methylation, and chromatin modification (Bartal, 2004; Krol et al., 2010). Among various classes of miRNAs, mir 17 - 92 cluster is one of the most

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extensively characterized group which consists of mir-17, 18, 19a, 19b, 20a, and 92 (chromosome 13); mir-106a, 19b, 363, and 92 (X-chromosome); and mir-106b, 93, and 25 (chromosome 7) (Trompeter et al., 2011). Further, miRNAs act as pro-proliferative (Cloonan et al., 2008), anti-proliferative (Yu et al., 2008), and cell cycle regulators (Ivanovska et al., 2008) in proliferative and differentiation processes. Hence, it was intriguing to study miRNA expression in Sertoli cells in response to estradiol which in turn may regulate target gene expressions. Overall, the present study is focused on the development of MSC-1 cell line as a model for understanding the role of estradiol in MSC-1 cell proliferation and inhibition.

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ACCEPTED MANUSCRIPT Material and Methods Reagents

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17β-estradiol (1, 3, 5 [10]-Estratrien-3, 17β-diol) and PD 98059 (MAPK/ ERK kinase

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inhibitor) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Tamoxifen citrate, Cat. No. # 579000 (ER antagonist) was purchased from Calbiochem (Darmstadt, Germany). Primary as well as secondary antibodies were purchased from Santa Cruz

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Biotechnology (Santa Cruz, CA, USA). RNAzol RT (Cat. No. # R4533), MiRNA isolation reagent, was obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Other molecular

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biology grade chemicals such as agarose, ethanol, and dimethyl sulfoxide (DMSO) were

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purchased from Himedia (Mumbai, India).

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Cell Culture

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MSC-1 cells (mouse Sertoli cell line) were provided by Professor Ilpo Huhtaniemi, Imperial College, London, UK. The cells were received in healthy conditions and were cultured in Dulbecco’s modified Eagle’s medium (DMEM low glucose) containing 10% heat-inactivated

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fetal bovine serum (FBS) (GIBCO, BRL, Inchinnan, UK) and 1X penicillin/streptomycin (Himedia, Mumbai, India) in a 5% CO2 incubator. All the experiments were carried out in charcoal-stripped serum with phenol red-free medium to avoid steroid contamination. Scratch assay In order to evaluate the proliferative effects of estradiol on Sertoli cells, the MSC-1 cells were plated on 6-cm2 tissue culture dishes. A scratch was made in the thus-formed confluent monolayer using sterile micropipette- tip. The cells were then treated with vehicle (ethanol 0.1%) and estradiol (0.1, 1, and 10 nM), respectively. The cell proliferation over the scratched area was observed and documented by phase contrast microscopy at 0-, 24- and 485

ACCEPTED MANUSCRIPT h time intervals, and the percentage of the area covered by the proliferative cells was calculated by Image-J software (NIH, USA).

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Cell cycle distribution analysis using fluorescence-activated cell sorter (FACS)

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The MSC-1 cells were treated with increasing concentrations of estradiol (0.1 to 10 nM) for 48 h in medium containing charcoal-stripped serum. On completion of treatment, the cells were collected, washed, and fixed in cold 70% ethanol (MB grade, Himedia, India). The

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ethanol-fixed cells were then treated with RNase-A (50 µg/ml) (Himedia, Mumbai, India) and propidium iodide (50 µg/ml) (Sigma, MO, USA) in phosphate-buffered saline (PBS)

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containing 0.1% triton X-100. The cells were then incubated for 30 min in dark. The cellcycle analysis was performed using FACS (BD FACS-verse, BD Biosciences, San Jose, CA,

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USA).

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MiRNA-specific stem-loop RT-qPCR and real-time PCR for miRNA expression Mature miRNA sequences for mouse mir-17 family (i.e., mir-17, 20a, 20b, 93, and 106) were obtained from www.mirbase.org, and stem-loop RT, forward, and universal reverse primers

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were designed according to the methods provided by Czimmerer et al, (2013). After treatment of MSC-1 cells with varying concentrations of estradiol, small RNA isolation was carried out using RNAzol-RT kit according to the manufacturer’s instructions. Small RNAs were reverse-transcribed to cDNA by stem-loop pulsed methods as described by VarkonyiGasic et al. (2007) with slight modifications. Briefly, 500 - 1000 ng of small RNA was mixed with 0.5 µl of d-NTP mixture (10 mM stock), and the volume was made up to 12 µl with nuclease-free water. The mixture was then heated to 65ºC for 5 min and incubated on ice for 2 min. After cooling, 0.25 µl of M-MLV reverse transcriptase enzyme (New England Biolabs Inc., USA) (200 U/µl) was added along with 4 µl of 5X buffer, 2 µl of 0.1 M DTT, 0.1 µl 6

ACCEPTED MANUSCRIPT RNase (40 U/µl), and 1 µl (1 µM) of an appropriate stem-loop primer. RT reaction was performed in a thermocyclar with an initial temperature of 16ºC for 30 min followed by

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pulsed RT of 60 cycles at 30°C for 30 s, 42°C for 30 s, and 50°C for 1 s. Finally, the enzyme

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was deactivated by heating at 85ºC for 5 min. The sequences for stem-loop, forward and

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reverse primers are presented in Table 1 (Supplementary data).

In the next step, an equal amount of cDNA from the control and treated groups was mixed with 12 µl of QuantiTech®SYBR® Green (Qiagen, Valencia, USA) and 1 µl of each

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primer (forward and reverse) (0.8 µM each), and the volumes were made up to 25 µl. The

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PCR was performed on a Smart Cycler (Cepheid, Sunnyvale, CA, USA) with an initial denaturation for 15 min at 95°C followed by 40 cycles at 94ºC for 15 s and 60ºC for 30 s,

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and final extension of 60°C for 30 s (SYBR Green protocol 1). For a comparative evaluation,

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PCR was performed following protocol 2 comprising an initial denaturation of 15 min at 95°C followed by 45 cycles at 95°C for 5 s and 60°C for 10 s, and final extension of 72°C for

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1 s. However, since protocol 1 gave the best optimized results, it was considered for this study. The CT (threshold cycle) values from all RT-PCR reactions in triplicates were

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analyzed to detect target mRNA gene expression and the final reaction was also visualized on a 3% agarose gel. The sequences for mature murine miRNA are presented in Table 2 (Supplementary data). Transient transfection studies CHO and MSC-1 cells were seeded in 48-well tissue culture plates at density of 3×104 cells/well. All the treatments were carried out in media with charcoal-stripped serum to avoid steroid contamination. Briefly, after attaining the desired confluency, the CHO cells were transfected with 50 ng of pGL3-Claudin-5 luciferase promoter ( -1020/+111), 15 ng of pSG5ERα, and 20 ng of pSG5-β-gal in one set and 50 ng of pGL3-Claudin-5 luciferase promoter ( -1020/+111), 15 ng of pSG5-ERβ, and 20 ng of pSG5-β-gal in another set to investigate 7

ACCEPTED MANUSCRIPT regulation of Claudin-5 promoter by both ERα and ERβ respectively. On the other hand, MSC-1 cells due to endogenous presence of ERα and ERβ, were transfected only with 50 ng

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of pGL3-Claudin-5 luciferase promoter ( -1020/+111) and 20 ng of pSG5-β-gal.

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Transfections were performed using polyfect transfection reagent (Qiagen, Valencia, USA)

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according to the manufacturer’s instructions. Following 24 h post transfection, the medium was changed and the cells were stimulated with different concentrations of estradiol for another 24 h. After incubation, the cells were lysed and the luciferase activity was determined

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using Fluostar optima microplate reader (BMG Labtech, Germany) as per manufacturer’s

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instructions (Promega, USA). β-Galactosidase activities were also assayed. In order to determine transfection efficiency the luciferase activity was normalized to β-galactosidase

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activity. Each experiment was repeated three times in sets of triplicates.

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Semi-quantitative RT-PCR for gene expression The total RNA was extracted from MSC-1 cells on completion of respective treatments

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according to the method described earlier. The RNA was then quantified, and equal amounts of RNA obtained from the individual treatments were transcribed using the RT-PCR kit

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according to the manufacturer’s instructions (New England Biolabs Inc., USA). In order to examine the possibility of genomic DNA contamination, a negative control RT reaction was performed devoid of reverse transcriptase enzyme. PCR was performed with an initial denaturation at 94ᵒC for 35 s, and annealing at various temperatures (depending on the primer pairs used) for 35 s and extension at 72ᵒC for 45 s with different number of amplification cycles. The PCR products were then resolved in 1% agarose gel and visualized in a gel documentation system (Bio Rad, USA). The intensity of the bands on gels was converted into a digital image with a gel analyzer. The β-actin PCR products were used as internal standards and each of the RT-PCR was carried out three times. Primer sequence, product size,

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ACCEPTED MANUSCRIPT annealing temperature, and number of cycles used for all primers are presented in Table 3 (Supplementary data).

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Western blotting

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Total cell lysates from control and estradiol-treated groups were prepared with lysis buffer [20mM Tris pH 7.2, 5 mM EGTA, 5 mM EDTA, 0.65 % sodium dodecyl sulphate (SDS), and 1X protease inhibitor cocktail (Sigma, MO, USA)]. After cell lysis, the lysates were

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centrifuged at 12,000 rpm for 10 min at 4ºC. The supernatant containing protein was quantified using BCA Quanti-Pro protein estimation kit (Sigma Aldrich, USA). Total (40 µg)

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protein samples were separated on a 10% polyacrylamide gel and were transferred to a polyvinylidene fluoride (PVDF) membrane (MDI, NY, USA). The proteins transferred to the

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PVDF membrane were blocked in 1X TBST (Tris-buffered saline with Tween 20) containing

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5% BSA (bovine serum albumin). They were then washed with TBST and probed with the

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respective primary antibodies (Santa Cruz Biotechnology, USA) diluted in TBST buffer. Following overnight incubation at 4oC, blots were again washed and incubated with HRP (horseradish peroxidise )-conjugated anti-rabbit/ anti-mouse secondary antibodies at 1:10,000

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dilutions. The blots were then developed using ECL detection system (GE Healthcare, UK) as per manufacturer’s instructions. They were then subjected to denstinometric analysis, and mean values were calculated from three independent experiments. β-actin was used as the internal control. Statistical analysis Values are expressed as mean ±S.E.M. The results shown are representative of three independent experiments. The statistical significance was evaluated by one-way ANOVA at p < 0.05 level of significance. Dunnett’s post hoc test was used to compare the statistical 9

ACCEPTED MANUSCRIPT significance between various group means and the control mean. Statistical software packages such as Origin 6.1 software (Origin Lab Corporation, USA) and GraphPad Prism

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5.04 (GraphPad Software, San Diego, CA) were used.

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Results Effect of estradiol on MSC-1 cell viability and proliferation

Cellular proliferation in MSC-1 cells was evaluated by measuring the migration potential of

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cells towards the scratched area. As shown in Fig. 1, the percentage of the uncovered

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scratched area after cell migration and proliferation decreased significantly at 24 and 48 h of estradiol treatment, respectively. Further, estradiol enhanced the cellular proliferation in a

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time- and dose- dependent manner. As shown in Fig. 1, after 24 h of estradiol treatment (10

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nM), almost 23.98% of the scratched area remained uncovered as compared to the untreated control (45.37%). A similar trend was also observed after 48 h of treatment suggesting that

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estradiol treatment leads to enhanced cellular proliferation in MSC-1 cells.

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FACS assay for cell cycle analysis FACS assay is a useful tool to study the synthesis, replication, and growth of cells. In each phase, the cells have variable in DNA content. Proliferative index was calculated by the percentage of the cells entering into G2/M phase in the presence of different concentrations of estradiol. As depicted in Fig. 2, the percentages of cells in G2/M phase increased from 13.3% (control) to 20.62% (10 nM estradiol), respectively (p < 0.05). These data were in accordance with our previous scratch assay results and confirmed that more number of cells entered the proliferative phase, thus establishing the role of estradiol in the proliferation of MSC-1 cells. Immunodetection of ERs in MSC-1 cells 10

ACCEPTED MANUSCRIPT ERs play a differential role in the proliferation and differentiation of Sertoli cells. These receptors mediate estrogen activity (Lucas et al., 2014). Hence, it was important to assess the

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expression of both types of receptors (ERα and ERβ) in this cell line. As shown in Fig. 3B,

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the basal level of protein expression of both ERα and ERβ was detected in MSC-1 cells (left

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lane), which was even marginally up-regulated by estradiol treatment (5 nM) (right lane). However, a statistically significant increase in the receptor protein expression by estradiol was observed only in the case of ERα (p < 0.05) (Fig. 3C). Further, to verify the localization

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of both subtypes of receptors, confocal laser microscopic studies were also conducted. As

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shown in Fig. 3A, a prominent immunolocalization was observed for both the receptors suggesting the endogenous presence of both ERs in MSC-1 cells.

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Transcriptional expression of some important Sertoli cell-specific genes in response to

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estradiol treatment in MSC-1 cells

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Sertoli cells are the somatic cells which provide both support and nourishment to germ cells. Among various molecules, ABP is of utmost importance because it binds to free androgen and thus enables spermatogenesis. Similarly, occludin and claudin are important components

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of the tight junction complex, which form the blood-testis barrier (BTB). Estradiol treatment at lower doses (0.1 nM) leads to a significant increase in ABP expression, but no effects were observed for occludin at the same concentration (Fig. 4). However, at higher doses of estradiol (10 nM), a sharp decrease in the mRNA expression was observed in both ABP (77.5%) and occludin (26.8%) genes (p < 0.05). No changes were observed in the vimentin mRNA expression. Estrogens primarily regulate the target gene expression via ER and some of the prime tight junction genes including claudin-5 can be regulated by either ERα or ERβ subtypes. In order to investigate the potential interaction and regulation of claudin-5 by ER subtypes in 11

ACCEPTED MANUSCRIPT MSC-1 cells, CHO cells (which are naive for any ER) were transfected with both ER subtypes along with claudin-5 promoter- luciferase construct in one set. In another set of

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experiment, MSC-1 cells, which have endogenous ERs, were transfected with only a claudin-

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5 promoter- luciferase construct. As shown in Fig. 5A, estradiol treatment to CHO cells

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resulted in decreased claudin-5 promoter activities in cells co-transfected with ERα at all the doses tested, resulting in about 40% decrease in response to 10 nM estradiol. On the other hand, estradiol treatment (10 nM) in CHO cells co-transfected with ER-β and claudin-5

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promoter resulted in a significant (p < 0.05) increase in luciferase activity (34%) (Fig. 5A).

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Interestingly, in MSC-1 cell line, with an endogenous presence of both ERα and ERβ, a significant increase in claudin-5 promoter functions was observed in response to estradiol

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(about 69% increase at 10 nM concentration) (Fig. 5B) (p < 0.05).

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Estradiol induced signalling pathways in MSC-1 cells

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To elucidate the pathways involved in the proliferation of Sertoli cells in response to estradiol, immunoblot analysis of some probable signaling molecules was performed with varying concentrations of estradiol. As shown in Fig. 6, the increase in phosphorylation of

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both AKT and ERK occurred in a dose-dependent manner in response to estradiol. More than 50% increase in the phosphorylation of both AKT (Fig. 6A) and ERK (Fig. 6B) was observed at 10 nM concentration of estradiol as compared to control (p < 0.05). Similarly, increased expression of phosphorylated NF-κB was observed albeit at lower doses (Fig. 6C). Overall, this dataset suggested the prominent role of MAPK signaling in estradiol-induced proliferation of MSC-1 cell line. MiRNA expression analysis in MSC-1 cells in response to estradiol treatment

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ACCEPTED MANUSCRIPT Genomic studies have revealed that excessive transcription in certain cases may lead to the formation of nc-RNAs along with coding RNAs. Some of these nc-RNAs including miRNAs

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regulate the gene expression at post transcriptional level. Considering the prominent role of

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mir-17 family in cell differentiation, progression, and proliferation, it was intriguing to study

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the effects of estrogens on the expression of mir-17 family genes in MSC-1 cells. The expression of mir-17 family was optimized using both gel-based semi-quantitative PCR and real-time PCR systems. As shown in Fig. 7, both the methods showed specific amplifications

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of the desired miRNA without non-specific amplifications. SYBR Green protocol 1 as shown

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in Fig. 7 (Lane 4) was best optimized and was taken into consideration for further study (details of all protocols are provided in the methodology section). As shown in Fig. 8A,

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among mir-17 family members, the expression of mir-20b and mir-106a was down regulated

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at higher concentrations of estradiol. Maximum decrease in expression was observed at 10 nM concentration of estradiol in mir - 20b (12%) and mir - 106a (36%) as compared to

Discussion

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control (p < 0.05) (Fig. 8B).

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The importance of studying Sertoli cell signaling lies in the fact that the number of Sertoli cells determines the testicular size and daily sperm production. Only immature Sertoli cells have the ability to proliferate, and thus the final Sertoli cell number is predetermined before adulthood. Various hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone are mentioned in literature, which act as positive regulator for Sertoli cell proliferation. However, among these hormones, FSH is of prime importance and reported to increase Sertoli cell proliferation by up to 40% (Sharpe et al., 2003). Studies on the role of estrogen in male reproductive development came into light when diethylstilbestrol (DES) exposure in pregnant women led to cryptorchidism and 13

ACCEPTED MANUSCRIPT epidydymal malfunctions in male offspring and ERα-knockout mice. This further suggests the significance of estrogen signaling in male fertility (Hess et al., 2003). The present study

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investigated estrogen signaling in Sertoli cell line (MSC-1) in order to develop a model cell

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line to study Sertoli cell functions. Results of in vitro scratch assay suggested that estrogen

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treatment at physiological concentrations enhanced the proliferation of MSC-1 cells. Positive effects of estradiol on MSC-1 cell proliferation were further confirmed by cell-cycle analysis. Estradiol treatment at gradient concentrations leads to more number of cells in the G2/M

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phase as compared to vehicle control cells indicating the presence of more number of

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dividing cells. Moreover, it could also be related to increased expression of cell cycle regulator proteins including cyclinD1 and D3. CyclinD1 was previously reported to be

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involved in the progression of cell cycle from G0/G1 to S phase and the presence of NF-κB

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transcription factor binding sites in the promoter region of cyclinD1 suggested the putative mechanisms of proliferation (Hinz et al., 1999).

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In general, estrogen or estrogen-like molecules perpetuate the signals through the cognate receptors present on the cells. Once bound to the ligand, ER dimerizes and is

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separated from chaperone proteins, present in active form; it then binds to target DNA through estrogen response elements (EREs) or indirectly through other transcription factors such as activator proteins (AP-1), NF-κB, and specificity protein (SP-1) to regulate target gene expression. The presence of both the ER subtypes has been reported in Sertoli cells, and the functionality is associated with cell proliferation and differentiation (Lucas et al., 2011). Hence, it is important to examine the pattern of ER expression in MSC-1 cells. The expression of both ERα and ERβ subtypes was observed within the cells. Besides activation through canonical ERs, estrogen also mediate its biological effects on Sertoli cells via the activation of GPCRs namely G-protein coupled ER (GPER) (Lucas et al., 2014). It has been 14

ACCEPTED MANUSCRIPT reported that GPER employs a Gβγ-subunit protein-dependent mechanism to promote estrogen-mediated transactivation of the epidermal growth factor receptor (EGFR)-to-Erk

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signaling axis (Gaudet et al., 2015). Hence ER and GPER may mediate the signals essential

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for Sertoli cell function and homeostasis. However, further detailed studies are needed to

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understand the crosstalk between those two receptor types for regulating Sertoli cell functions.

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Further, Sertoli cells have a unique characteristic feature of forming inter-Sertoli cell tight junction known as BTB. BTB controls the para-cellular movement between the basal

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and apical regions of seminiferous epithelium. Various studies have demonstrated the significant role of some adhesion molecules such as occludin, claudin-5, zona occludin-1, N-

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cadherin, and β-catenins in the inter-Sertoli cell tight junctions. It has also been reported that

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hormones (testosterone and estradiol) and cytokines are involved in their regulation (Cheng and Mruk, 2012). In the present study, estradiol treatment to MSC-1 cells at lower doses (0.1

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and 1 nM) marginally up-regulated occludin gene transcription, while at higher concentrations the effects were reversed. The biphasic nature of estradiol regulation of

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occludin can be explained on the basis of a previous study where estrogen exposure to vaginal- ectocervical epithelial cells resulted in the release of a 50-kDa truncated occluding, which was facilitated by MMP-7 activity (Ye et al., 2003; Zeng et al., 2004; Gorodeski, 2007). Similarly, it was reported that claudin-5, another important member of tight junction proteins, is a novel target for estrogens in brain and heart endothelium (Burek et al., 2010). Hence, it was intriguing to study its regulation by estradiol in MSC-1 cells. MSC-1 cells transiently transfected with mouse claudin-5 promoter (tagged with luciferase reporter gene) followed by treatment with increasing concentrations of estradiol resulted in increased luciferase activities, indicating positive regulation of claudin-5 in MSC-1 cells in response to 15

ACCEPTED MANUSCRIPT estradiol (Fig. 5B). Further, one of the critical findings of the claudin-5 promoter activity was that ER subtypes (ERα and ERβ) showed differential regulation towards claudin-5 promoter

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activation with positive regulation by ERβ and negative regulation by ERα. One of the

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probable mechanisms of differential gene regulation by ERs includes the formation of a

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homodimer or heterodimer upon binding of ligand. Monroe et al. (2005) based on their study suggested that expression of ERα/β heterodimer in osteoblast cells controls the regulation of a distinct set of genes as compared to the genes regulated by homodimer formation, and the

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possible mechanism may include the differential interaction or recruitment of the associated

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transcription factors.

Besides the formation of tight junction-associated molecules, Sertoli cells secrete

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ABP that maintains the circulatory level of androgen. Estrogen exposure at a higher

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concentration (10 nM) reduced ABP gene expression in MSC-1 cells (Fig. 4). Although it is difficult to interpret the probable reason of this effect by the in vitro data available, in vivo

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experiments conducted earlier attributed this to the decreased depressed levels of gonadotrophin secretion and testosterone biosynthesis (Smith et al., 1982). This result was

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also congruent with the study conducted by Danzo et al. (1990), where they showed that among various hormones, only estradiol has the ability to reduce ABP secretion in the serum of adult rats.

The role of ERK 1/2 and protein kinase-A (PKA) in response to 17β-estradiol in boar Sertoli cell proliferation has already been reported previously (Xian-zhong et al., 2014). In order to ascertain the most active pathway, immunoblot expression study was conducted for some of the key signaling molecules. Experiments carried out with MSC-1 cells showed that both PKA and ERK 1/2 pathways were activated in response to estradiol. However, a significant increase in ERK phosphorylation was observed as compared to AKT 16

ACCEPTED MANUSCRIPT phosphorylation, thus suggesting the dominant role of ERK pathways in MSC-1 cell proliferation. Further, increased phosphorylation of NF-κB /p65 was observed at 0.1 and 1

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nM concentration of estradiol. NF-κB is another important transcription factor involved in

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immune response, inflammatory response, and cell adhesion and proliferation (Oeckinghaus

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and Ghosh, 2009). Canonical pathways include the activation of IKK (IκB kinase complex) by signals from an upstream member followed by the destruction of IκB/NFκB complex. Released NF-κB migrates into the nucleus where it regulates the expression of target genes.

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For example, c-myc is one of the target genes involved in cell proliferation and regulated by

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NF-κB (Romashkova and Makarov, 1999). Similarly, increased expression of NFκB/p65 is correlated with an increase in CCND 1 gene expression in Sertoli cells (Royer et al., 2012).

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CCND 1 belongs to the cyclin protein family which regulates cell cycle and proliferation, and

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the presence of NF-κB binding sites on the CCND 1 promoter has already been reported in literature (Hinz et al., 1999). The present study has alos reported on the correlation between

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increased expression of NF-κB and the progression of cell cycle. In the new era of gene regulation, miRNAs are of prime importance and regulate gene

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expression by binding to the 3’-UTR of the target gene. In the next phase, we tried to elucidate the effects of estradiol on miRNA expression of mir-17 family. It is reported that mir-17 family (mir-17, mir-20b, and mir-106a) regulates MAPK inversely and the inhibition of mir-17 family leads to an increased expression of MAPK in keratinocyte differentiation. Mir-17 may also act as an intermediate bridge between the signaling molecules (MAPK) and the important regulatory proteins including p63 which enhance proliferation or differentiation (Wu et al., 2012) and similar downregulation of miR-17 family induced neuronal differentiation (Beveridge et al., 2009). Various self renewal- and differentiation-associated proteins including LIF, STAT3, MYC, Bmp2, Smad5, and Smad 7 are reported to be the 17

ACCEPTED MANUSCRIPT targets of mir-17 family in embryonic stem cells (Foshay and Gallicano, 2009). In this case, the prominent role of pERK1/2 as compared to other signaling molecules encouraged us to

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study the expression of mir-17 family. Significant downregulation in miRNA expression of

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mir-17 family members (mir-17, mir-20b, and mir-106a) strongly validates the results that

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mir-17 family may play an important role in estradiol-induced MSC-1 cell proliferation by directly acting as a regulator of MAPK or may also act as an intermediate between signaling and target genes responsible for proliferation. However, the exact relationship between

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miRNA regulation and signaling kinase expression in Sertoli cells demands future detailed

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studies. Conclusions

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The present study highlights the estradiol-induced functions in MSC-1 cells via both genetic

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and epigenetic mechanisms. Differential role of ER subtypes in certain gene regulations

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provided some interesting insight of its functions. Further, the role of miR-17 family in MSC1 cell proliferation provides novel pathways besides genomic and non-genomic mechanism of action (Fig. 9). Hence, considering the problems associated with Sertoli cell lines and

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primary culture of Sertoli cells, MSC-1 cells could be one of the potential cell lines that can be used for understanding various cellular events in Sertoli cells. Further, based on the current findings, similar experiments need to be conducted in primary culture of Sertoli cells and in vivo for better understanding the role of gene regulatory molecules like miRNA in Sertoli cells proliferation and differentiation, which is however the limitation of the present study. That would allow better addressing the mechanisms as discussed in this manuscript. Conflict of interest The authors do not have any conflict of interest to disclose. 18

ACCEPTED MANUSCRIPT Funding information This work was supported by the Department of Biotechnology (No. BT / PR 14836 / AAQ /

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01 / 455 / 2010) and Council of Scientific and Industrial Research (No. 37(1402)/10/EMR II),

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Government of India as funded projects to PR. NK thanks Department of Biotechnology,

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Government of India for fellowship and research support (Reference no. DBT-JRF/200910/481).

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Acknowledgements

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The authors would like to convey their sincere thanks to Professor Ilpo T. Huhtaniemi (Imperial College London, UK) for providing MSC-1 cell line as well as estrogen receptor

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(ERs) plasmids (detail mentioned in the methodology section) used for transfection study.

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The authors would also like to thank Dr. Naveen Kumar Navani, Dr. Ranjana Pathania, and Dr. Rajnikant Sharma (Department of Biotechnology, Indian Institute of Technology

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ACCEPTED MANUSCRIPT Legend of the figures Figure 1. In vitro scratch assay for cell proliferation study. MSC-1 cells were seeded in 6-

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well tissue culture plates and grown to 80% confluency. A scratch was made with sterile (0.2

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ml) pipette tip and the cells were cultured for 24 h and 48 h in the presence of estradiol at 0.1,

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1 and 10 nM concentrations. Histogram in the lower panel shows the percentage of the uncovered scratched area. *** indicates significant level of difference from 0 h treatment at p < 0.001 and # indicates differences at 24 and 48 h from their respective controls at p < 0.05.

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Values are ± SEM of three individual experiments. Es, estradiol. Image-J software was used

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to measure the percentage of the uncovered area after estradiol treatment. Figure 2. Effects of estradiol on the regulation of cell cycle in cultured MSC-1 cells.

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MSC-1 cells cultured with estradiol for 48 h were post fixed in 70% ethanol and analyzed for

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cell cycle distribution. The number of cells in the G0/G1, S and G2/M phases was calculated by gating the cells according to the protocol provided in FACSverseTM machine (BD

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Biosciences, USA). Each experiment was carried out in set of triplicates and the percentage of cells in different phases of cell cycle was evaluated in the final attempt when the

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experimental parameters were best optimized. Es, estradiol. Figure 3. Analyzing the expression levels of estrogen in MSC-1 cells. (A) Confocal laser microscopy for immunolocalization of ERα and ERβ proteins in MSC-1 cells. Magnification 60x. (B) Representative immunoblot data showing the expression of ER subtypes in MSC-1 cells in the absence and presence of estradiol (5 nM). (C) Histogram shows mean ± SEM of arbitrary pixel intensities of three individual experiments as shown in B. * indicates the significant level of difference at p < 0.05 with respect to the control (vehicle treated) cells. Es, estradiol; ns, non-significant.

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ACCEPTED MANUSCRIPT Figure 4. Effects of estradiol on the expression of some major secretory and tight junction-associated genes in MSC-1 cells. The figure is the representative transcriptional

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analysis as determined by RT-PCR. Histogram in the lower panel indicates the mean ± SEM

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of arbitrary pixel intensities of PCR products of three independent experiments. * indicates

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significant difference from the respective control group at p < 0.05.

Figure 5. Interaction of ER isoforms with claudin-5 promoter in response to estradiol.

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Transient transfection of (A) CHO cells with ERα, ERβ and mouse claudin-5 promoterluciferase constructs and (B) MSC-1 cells with only claudin-5 promoter- lucifearse constructs

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followed by estimation of luciferase activities. Details of the experiment are mentioned in the methodology section. Data represented here show relative luciferase activities as percent

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change over vehicle-treated control which was given a value of 100. Values are mean ± SEM

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of three independent experiments. *, ** and *** indicate the significant level of difference at

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p < 0.05 , p < 0.01 and p < 0.001 respectively as compared to the respective vehicle-treated control cells. Es, estradiol.

Figure 6. Effect of estradiol in the regulation of various signalling molecules in MSC-1

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cells. Representative immunoblot analysis for (A) pAKT/AKT, (B) pERK/ ERK and (C) pNFκB and pIκB in MSC-1 cells treated with increasing concentrations of estradiol. Histograms in the respective right panels of each figure show mean ± SEM of arbitrary pixel intensities of three individual experiments. *, ** and *** indicate the significant level of difference at p < 0.05, p < 0.01 and p < 0.001 respectively, as compared to the respective control (vehicle treated) cells. Es, estradiol. Figure 7. Optimization of real-time stem loop-based PCR method for miRNA expression. Total small RNA (< 200 base pairs) was isolated from MSC-1 cells and reverse transcribed to c-DNA. Amplification was performed using miRNA-specific forward primer 26

ACCEPTED MANUSCRIPT and universal reverse primer with two different protocols (SYBR Green protocol 1 & 2 as described in methodology section). Maximum amplification was observed in protocol 1 as

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shown in lane 4 of agarose gel (left panel) which was verified by real-time PCR (right

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Figure 8. The expressions of miRNAs (mir-17 family) in response to estradiol in MSC-1 cells. (A) Representative data showing the relative miRNA expressions in response to

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varying doses of estradiol. (B) Histograms on the right panel show the relative changes in the expressions of various miRNAs as compared to the respective vehicle-treated control group.

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Data represented here are the mean ± SEM of three independent experiments. * and ** indicate the significant level of differences at p < 0.05 and p < 0.01 respectively, as compared

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to the respective control cells. Sno 202 was used as normalizer. Es, estradiol.

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Figure 9. Schematic representation of the probable mechanism of action of estradiol in

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MSC-1 cells. Firstly, estradiol binds to ER or GPER in cytoplasm and cell membrane, respectively. The estrogen-bound cytoplasmic receptors (homodimer / heterodimer partners) translocate to the nucleus and bind to DNA of target genes. The membrane-bound GPER in

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response to estradiol activates the PI3/AKT pathway and indirectly regulates target gene transcription. Secondly, another mechanism involves the activation of SRC that eventually phosphorylates epidermal growth factor receptor and activates mitogen-activated protein kinases (MAPK). Thirdly, regulation via mir-17 family is a significant phenomenon. Activation of MAPK by estradiol may lead to the inhibition of mir-17 family miRNA expression which further affects Sertoli cell proliferation and differentiation. GPER, Gprotein-coupled estrogen receptor; E2, estradiol; ER, estrogen receptor; SRC, steroid receptor coactivators; EGFR, epidermal growth factor receptor.

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