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 DOI: 10.1177/1933719114522519

published online 10 February 2014Reproductive SciencesV. Kanimozhi, K. Palanivel, B. Kadalmani, Graciela Krikun and Hugh S. Taylor

Mechanism of Male InfertilityApolipoprotein E Induction in Syrian Hamster Testis Following Tributyltin Exposure: A Potential

  

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Original Article

Apolipoprotein E Induction in SyrianHamster Testis Following TributyltinExposure: A Potential Mechanismof Male Infertility

V. Kanimozhi, MPhil1, K. Palanivel, MSc1, B. Kadalmani, PhD1,Graciela Krikun, PhD2, and Hugh S. Taylor, MD2

AbstractTributyltin (TBT) is a common environmental contaminant used as the active ingredient in many products such as a biocides,wood preservatives, disinfecting agents, and antifouling paints. The TBT is a known endocrine disruptor. The aim of the currentinvestigation was to determine the toxicity of TBT in the reproductive tract of adult male Syrian hamsters and to ascertainwhether this compound results in untoward effects on apolipoprotein E (ApoE), a lipoprotein central to sex hormone synthesis.The TBT was administered orally to male Syrian hamsters at doses of 50, 100, and 150 ppm/kg for 65 days of treatment. Wedetermined body weight, testis weight, sperm count, sperm morphology, testis histology, ApoE expression, serum lipid profile,testosterone level, follicle-stimulating hormone receptor (FSHR), and steroid hormone receptor expression compared tovehicle-treated controls. High doses of TBT significantly affected each of these parameters in Syrian hamsters. Weight andmorphology of the testis were altered as well as sperm production. Real-time reverse-transcriptase polymerase chain reactionanalysis revealed that expression of ApoE messenger RNA was upregulated in testes from TBT-treated groups compared withcontrols while the expression of androgen receptor, FSHR, estrogen receptor a (ESR1), and estrogen receptor b (ESR2) wasdecreased. We posit that exposure to TBT hinders intracellular cholesterol transport resulting in abnormal sex steroid biosynth-esis and subsequent spermatogenic defects. Importantly, these effects may account for the decreased level of normal spermobserved in hamsters exposed to TBT.

Keywordstributyltin, apolipoprotein E, testosterone, FSHR, AR, spermatogenesis

Introduction

Tributyltin (TBT) is one of the most commonly used organotins

(organic compounds linked to Tin) in agriculture and industry

where it functions as a biocide, heat stabilizer, and chemical

catalyzer; however, its persistence in the environment has

raised concerns about adverse effects to human health.1

Xenobiotics such as TBT tend to accumulate slowly and may

cause long-term adverse effects in humans.2 There are many

reported biological effects of TBT on the male reproductive

system, which include inhibition of sex steroid metabolism,3-6

retarded sexual development,5,7,8 diminished size of the testis,

epididymis and coagulating gland,5 abnormal testicular histol-

ogy as well as decreased sperm count,9 sperm density, viability,

and morphology.10,11 Furthermore, TBT exposure blocks

leydig and Sertoli cell (SC) function through the downregulation

of estrogen receptor (ESR) and androgen receptor (AR).7-8

Leydig and SCs are responsible for testicular development

and normal spermatogenesis.12-14 In testis, spermatogenesis is

under the control of 2 gonadotropins, follicle-stimulating

hormone (FSH) and luteinizing hormone (LH).15-17 The FSH

acts directly on SCs promoting spermatogenesis while LH

induces testosterone production in Leydig cells. Sertoli cells

express both FSH receptor (FSHR) and AR, thus integrating

androgen and FSH signaling.18 The FSH concentrations

increase steadily after birth, which promote SC proliferation

and induce AR expression in these cells.18-20 Moreover, the

concentrations of FSH and testosterone are increased at puberty

and augment AR expression, which is essential for final matura-

tion of SCs.20 Upon hormone binding, AR translocates to the

1 Department of Animal Science, School of life Sciences, Bharathidasan

University, Tiruchirappalli, Tamil Nadu, India2 Department of Obstetrics, Gynecology & Reproductive Sciences, Yale

School of Medicine, Yale University, New Haven, CT, USA

Corresponding Author:

B. Kadalmani, Department of Animal Science, Bharathidasan University,

Tiruchirappalli 620024, Tamil Nadu, India.

Email: kadalmanibdu@rediffmail.com

Reproductive Sciences1-9ª The Author(s) 2014Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/1933719114522519rs.sagepub.com

at Yale University Library on February 17, 2014rsx.sagepub.comDownloaded from

nucleus where it regulates transcription of androgen-responsive

genes. The AR interacts with numerous coregulatory proteins that

modulate its function and activity.21-23 Testosterone synthesis in

testis requires lipoprotein transfer from the liver mediated by the

carrier apolipoprotein E (ApoE).24,25 Apolipoprotein E plays a

key role in determining the rate of adrenal steroidogenesis possi-

bly by regulating the availability of cholesterol to the mitochon-

dria or by modulating cholesterol side chain cleavage activity.

To date, no studies have been conducted to evaluate the

effects of TBT on ApoE induction and subsequent testicular

dysfunction. Herein, we hypothesized that TBT alters serum

lipid biochemical parameters by upregulating ApoE, which in

turn negatively affects the function of testis by altering lipids,

sex steroids, and sex steroid receptors.

Materials and Methods

Chemicals

Tributyltinchloride was purchased from Sigma Aldrich

(St Louis, Missouri; purity >97%). The TBT was suspended

in water to obtain final TBT concentrations of 50, 100, and

150 ppm. Testosterone, triglyceride, cholesterol, and acridine

orange (AO) were purchased from Sigma Aldrich. All other

chemicals were of analytical grade and were obtained from

Medox Biotech (India).

Animal Experimentation

Six- to seven-week-old male Syrian hamsters were used in the

current study. Animals were housed under 12-hour light/12-

hour dark cycle with controlled conditions (21 + 2�C, 51 +7% humidity) and were fed standard food and allowed water

ad libitum. Food and water consumption of the animals was

measured daily and body weights were recorded on day 0 and

at the end of the experimental period. The hamsters were ran-

domly divided into 4 groups, each containing 8 animals. Of the

4 groups, 3 were used as treatment groups and 1 as the control

group. Animals in the control group were fed standard food and

water ad libitum. The TBT was administered orally to the treat-

ment groups at 50, 100, or 150 ppm/kg/d doses for 65 days. At

the end of the TBT treatment, hamsters were sacrificed by

cervical dislocation and serum was separated from blood

samples for hormone measurement. Testis and all remaining

organs were collected and stored at �20�C.

Epididymal Sperm Count and Sperm Motility

The epididymal sperm suspension was prepared in 1 mL

phosphate-buffered saline (PBS) at pH 7.2. The sperm count was

determined in a hemocytometer. An aliquot from the suspension

(1 mL) was diluted 1:40 with PBS. A sample of the diluted sus-

pension was placed into a counting chamber (Neubauer chamber,

Marienfeld). The total sperm count in 8 separate squares (1 mm2

each) was determined and multiplied by 5� 104 to assess the total

count. Sperm motility was also determined in same 8 squares and

percentage of motile sperm was recorded.

Acridine Orange and Ethidium Bromide Stainingof Sperm

In order to find the viability of spermatozoa, fresh sperm were

stained with AO and ethidium bromide (EtBr). A suspension was

stained with 25 mL of AO-EtBr. About 1 drop of stained suspen-

sion was placed on a clean slide and allowed to dry. The prepara-

tions were examined by fluorescent microscopy. Images were

captured with a Sony DXC-151AP CCD camera (Tokyo, Japan).

Randomly selected spermatozoa from each slide were observed

and categorized as normal, head alone, or flagellar defect.

Histopathology

For histological light microscopic examinations, the testicular

tissues were dissected and the tissue samples were fixed in

Bouin fixative solution for 14 to 18 hours, processed in a series

of graded ethanol solutions, and embedded in paraffin. Paraffin

sections were cut with a microtome at 5-mm thickness and

stained with hematoxylin and eosin. The sections were viewed

and photographed on a light microscope (OlympusBX51,

Tokyo, Japan) with an attached camera (Olympus C-5050;

Olympus Optical Co Ltd, Japan).26

Determination of Lipid Biochemical Parameters

Serum lipid profile (total cholesterol, high-density lipoprotein

[HDL] cholesterol, triglyceride), low-density lipoprotein

(LDL), and very LDL (VLDL) were determined after 65 days

of TBT treatment using enzymatic colorimetric assay kits

(Sigma Aldrich). The concentration of LDL, VLDL, and

cholesterol was calculated as described previously.27

Quantitation of Testosterone

Serum testosterone levels were measured using enzyme-

linked immunosorbent assay kits per the manufacturer’s

Table 1. List of Primer Sequences Used for Real-Time RT-PCR.

Gene Symbol Sequences

AR Sense: 50-CTAGCGCGTGCCTTCCTTTACA-30

Antisense: 50-CCCACCTGCGGGAAGCT-30

FSHR Sense: 50-TTTACTTGCCTGGAAGCGACTAA-30

Antisense: 50-CCCAGGCTCCTCCACACA-30

ESR 1 Sense: 50-AATTCTGACAATCGACGCCAG-30

Antisense: 50-GTGCTTCAACATTCTCCCTCCTC-30

ESR 2 Sense: 50-GAAGCTGAACCACCCAATGT-30

Antisense: 50-CAGTCCCACCATTAGCACCT-30

ApoE Sense:50-TTGGTCCCATTGCTGACAG-30

Antisense: 50-ACCGTCAGTTCCTGTGTGAC-30

GAPDH Sense: 50-AGGTTGTCTCCTGCGACTTCA-30

Antisense: 50-GCATCAAAGGTGGAAGAGTGG-30

Abbreviations: ApoE, apolipoprotein E; AR, androgen receptor; FSHR, follicle-stimulating hormone receptor; GAPDH, glyceraldehyde 3-phosphate dehy-drogenase; RT-PCR, reverse-transcriptase polymerase chain reaction.

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instructions (United Biotek, Canada). Working hormone—

horseradish peroxidase conjugate reagent of 100mL, 50mL of rab-

bit antihormone antibody, and 25 mL of samples or standard were

added to each well and incubated at 37�C for 90 minutes. The

wells were rinsed 5 times with distilled water, followed by

the addition of 100 mL of 3,30,5,500-tetramethylbenzidine (and

incubation at room temperature). The reaction was then stopped

by 1 N HCl solution. Absorbance was measured at 450 nm.

Total RNA Isolation and Real-Time Polymerase ChainReaction Analysis

Total RNA was extracted from testis of untreated and treated

hamsters using RNeasy mini kit (Qiagen, Valencia, Califor-

nia) according to the manufacturer’s instructions. The con-

centration and integrity of total RNA were calculated by

measuring absorbance at 260 nm using a ultraviolet spectro-

photometer and agarose gel electrophoresis. Quantitative Real-

Time reverse-transcriptase polymerase chain reaction (PCR)

was conducted as previously described.28 Primer sequences

of AR, ESR1, ESR2, FSHR, ApoE, and glyceraldehyde

3-phosphate dehydrogenase (GAPDH) are given in Table 1.

The housekeeping gene, GAPDH, was used as an internal

control. Each sample was analyzed in triplicate and the fold

changes in the target gene expressions were calculated.

Statistical Analysis

Statistical analysis was performed using Student t test. Data

were expressed as mean + standard deviation. Statistical

significance is represented with 1 asterisk for P values <.05,

2 asterisks for P values <.01, and 3 asterisks for P values <.001.

Results

Effect of TBT on Body Weight, Epididymis, CoagulatingGland, and Ventral Prostate

Tributyltin was administered orally to hamsters at 50, 100, or

150 ppm/kg bw/d doses for 65 days, as reported previ-

ously.5,9,29,30 Similar to the previous reports, body weight, tes-

tis weight, and other reproductive organ weights (epididymis,

seminal vesicle, coagulating gland, and ventral prostate) were

significantly decreased following treatment with TBT at 50,

100, and 150 ppm/kg bw.5,9 In particular, higher doses of TBT

Table 2. Changes in the Body Weight, Daily Food, and Water Intake of Control and TBT-Treated Hamsters.a

Control 50 ppm/kg/d 100 ppm/kg/d 150 ppm/kg/d

Initial body weight, g 169.98 + 9.49 170.70 + 9.39 170.56 + 5.06 170.76 + 9.06Final body weight, g 355.86 + 8.87 313.49 + 8.03b 295.95 + 5.07c 284.44 + 9.77d

Body weight gain, g 185.88 + 8.95 142.78 + 7.23b 131.43 + 7.32c 113.68 + 15.46d

Food intake, g 33.17 + 1.64 32.75 + 0.74 31.22 + 2.51 28.1 + 0.98b

Water intake, mL 45.39 + 2.15 45.62 + 3.22 39.42 + 1.05b 33.89 + 1.90c

Abbreviations: SD, standard deviation; TBT, tributyltin.aValues are represented as mean + standard deviation of 8 hamsters in each group (mean + SD; n ¼ 8).bSignificant at P < .05.cSignificant at P < .01.dSignificant at P < .001.

Table 3. Changes in the Reproductive Organ Weights and Accessory Reproductive Organ Weights in TBT-Treated Hamsters.a

Control 50 ppm/kg/d 100 ppm/kg/d 150 ppm/kg/d

Testis wt (pair), g/100 g bw 2.18 + 0.03 2.06 + 0.12 1.89 + 0.52b 1.58 + 0.1b

Cauda epididymis wt, mg/100 g bw 201.38 + 14.82 197.27 + 3.92 182.92 + 18.92b 163.90 + 21.27b

Seminal vesicle wt, mg/100 g bw 173.93 + 15.93 170.93 + 0.51 162.10 + 9.35b 154.93 + 7.93b

Coagulating gland wt, mg/100 g bw 69.04 + 5.93 71.06 + 5.06 64.90 + 13.93b 56.03 + 11.93b

Ventral prostate wt, mg/100 g bw 228.23 + 32.94 207.37 + 21.85b 194.043 + 6.93b 181.23 + 14.94b

Abbreviations: SD, standard deviation; TBT, tributyltin; wt, weight.aValues are represented as mean + SD of 8 hamsters in each group (mean + SD; n ¼ 8).bsignificantly different from control group (P < .05).

Table 4. Effect of TBT on Sperm Motility and Sperm Count in MaleSyrian Hamsters.a

GroupsSperm Count,

million/mLSperm

Motility, %Sperm

Viability, %

Control 28.43 + 0.94 71.93 + 1.27 98.13 + 2.150 ppm/kg/d 27.05 + 1.30 72.40 + 2.27 92.07 + 0.83100 ppm/kg/d 24.68 + 0.75b 53.58 + 1.01b 79.21 + 1.46150 ppm/kg/d 22.11 + 1.17c 45.83 + 1.38c 64.58 + 1.57

Abbreviation: TBT, tributyltin.aData represented as mean + standard deviation (SD) of 8 hamsters in eachgroup (mean + SD; n ¼ 8).bSignificant at P < .05.cSignificant at P < .01.

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more significantly suppressed body weight gain (100 and 150

ppm/kg/d; P < .01 and P < .001) compared to control and 50

ppm group after 65 days of treatment.

Food and water consumption was abnormal throughout the

experimental treatment (Table 2). Significant reductions in tes-

ticular weights were observed among TBT-treated animals

compared with controls (Table 3). Treatment of hamsters with

50 ppm/kg/d did not result in a significant change in the weight

of the testes compared to that of the other treatment groups.

However, administration of 100 and 150 ppm/kg/d caused

severe loss in testis weight (P < .05 and P < .001, respectively).

Also, weights of accessory sex organs such as epididymis,

seminal vesicle, coagulating gland, and ventral prostate were

decreased significantly after 65 days of TBT treatment in

comparison to the control group.

Tributyltin-Induced Sperm Defects

Data on the epididymal sperm count, sperm motility, and live/

dead counts are presented in Table 4. The TBT treatment

decreased epididymal sperm motility and count significantly

compared to the control group. The most significant reduction

was observed at 150 ppm/kg/d on epididymal sperm count and

motility when compared to the control group (Table 4). There was

a significant dose-dependent increase in abnormal sperm mor-

phology that was assessed by AO/EtBr fluorescent staining. The

TBT 100 and 150 ppm/kg-treated hamsters showed high levels of

abnormal sperm morphology. The abnormalities were in the head,

neck, and tail region of the sperm, that is, banana and detached

heads were found; acrosomes were observed to be up, down, or

altogether absent. Curved neck and curved, bent, round, loop, and

folded tails were seen at the 2 higher TBT doses (Figure 1).

Effect of TBT on Testis Structure

Sections of control hamster testis were analyzed under light

microscopy (Figures 2 and 3). Control testis showed normal

architecture of seminiferous tubules filled with normal sperma-

tocytes, spermatogonia, spermatozoa, and SCs. The TBT treat-

ment resulted in abnormalities such as sloughing of epithelial

Figure 1. Acridine orange and ethidium bromide (AO/EtBr)-stained sperm of TBT-treated hamsters. A, A representative normal live sperm; (B)detached heads and the sperms are nonviable; (C) coiled sperm; and (D) granulation of sperm head indicated by orange fluorescence at TBT 50,100, and 150 ppm-treated groups. TBT indicates tributyltin.

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Figure 2. A, Photomicrograph of control hamsters showing normal morphological architecture with all the consecutive phases of spermatogen-esis, lumen filled with spermatozoa (��200) and (B) tributyltin (TBT) at 50 ppm/kg/d treated hamsters. Photomicrograph showing degeneratedsemniferous tubule having decreased number of spermatogenic rudiments (indicated by arrow). Lumen contains fewer spermatozoa (��200).

Figure 3. A, Tributyltin (TBT) at 100 ppm/kg/d treated hamsters. Photomicrograph of testis showing inhibited spermatogenesis. Lumen is filledwith few sperm along with debris (indicated by arrow). Interstitial cells (leydig cells) are disrupted (indicated by symbol asterisk; ��200);(B) TBT at 150 ppm/kg/d treated hamsters. Semniferous tubule showing complete spermatogenic obstruction-damaged germinal epitheliumand increased intertubular space. Lumen is filled with cellular debris (��200). TBT indicates tributyltin.

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cells, outer membrane shrinkage, presence of vacuoles, dead

cell debris, binucleated giant cells with enlarged nucleus, and

granulation of cells. The presence of these abnormalities was

largely found in groups treated at the 2 higher concentrations

(TBT 100 and 150 ppm; Figure 3). However, the maximum

number of distorted seminiferous epithelium, vacuoles, multi-

nucleated giant cells, and dead cell debris appeared at higher

dose TBT 100 and 150 ppm/kg (Figure 3).

Biochemical Findings

The lipid profile in serum of hamsters demonstrated significant

increases in total lipids, triglycerides, total cholesterol, VLDL,

and LDL cholesterol in TBT-treated groups compared to the

control group (Figure 4A and B). The content of HDL choles-

terol showed a significant decrease in 100 and 150 ppm TBT-

treated group compared to the control group (P < .01 and P <

.001, respectively). No significant difference (P > .05) was evi-

dent in the lipid profile of groups treated at 50 ppm/kg com-

pared to the control.

Changes in Serum Testosterone Concentration

The serum testosterone levels were significantly decreased

after TBT administration in hamsters (Figure 5). The TBT

100 and 150 ppm/kg-treated hamsters showed a significant

decrease in serum testosterone levels compared to control and

TBT 50 ppm-treated groups.

Effect of TBT on Testis FSHR, AR, ESR1, ESR2, and ApoEMessenger RNA

Gene expression analysis in the testis of TBT-treated hamsters

showed large changes in the messenger RNA (mRNA) expres-

sion of genes involved in normal testicular function and sper-

matogenesis. Figure 6A–F shows the real-time PCR results of

FSHR, AR, ESR1, and ESR2 mRNA in the control and 3

TBT-treated groups. There was a significant reduction in the

mRNA expression levels of AR, FSHR, ESR1, and ESR2 in

hamsters treated with TBT 100 and 150 ppm/kg. No significant

changes were observed in 50 ppm-treated groups. High doses

of TBT (100 and 150 ppm) induced overexpression of ApoE

mRNA while there were no significant changes in the 50

ppm-treated group compared to control group. These results

indicate that ApoE expression is correlated with SC functions,

testis development, and spermatogenesis.

Discussion

Organotins such as TBT are considered to be chronic contami-

nants, which accumulate slowly and may cause long-term

adverse health effects in humans.2,31 The bioaccumulation of

these compounds has been found in water and in the food chain

in many parts of the world.31 However, the mechanisms by

which exposure to these compounds result in abnormal repro-

ductive function are still not fully understood.

In the present study, TBT exposure in a Syrian hamster

model led to deleterious health effects. The TBT exposure

Figure 4. A and B, Effect of TBT on the levels of serum cholesterol, triglycerides, very LDL (VLDL), LDL, and HDL. Values are given as mean +standard deviation (SD). *, significantly different from control group (P < .05) and **, significantly different from control and TBT 50 ppm-treatedgroup (P < .01). HDL indicates high-density lipoprotein; LDL, low-density lipoprotein; TBT tributyltin.

Figure 5. Changes in the level of testosterone hormone following tri-butyltin (TBT) treatment in hamsters. Values are given as mean +standard deviation (SD; n ¼ 8). *, significantly different from controlgroup (P < .05). **, significantly different from control and TBT 50ppm-treated group (P < .01).

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resulted in altered expression of sex steroid receptors, changes

in body and organ weight accompanied by increased ApoE

expression, serum triglycerides, total cholesterol, LDL, and

decreased HDL. The TBT significantly decreased sperm counts

and increased sperm abnormalities. The increased frequencies

of sperm with morphological abnormalities observed in the

treated hamsters are consistent with previous reports showing

that TBT can cause spermatotoxic defects.3-6

Interestingly, conflicting data on the levels of testosterone

following TBT exposure have been reported. Studies in mice

and rats have reported either increased or decreased levels of

testosterone.9,31-33 In the present study using a Syrian hamster

model, we observed decreased levels of circulating testosterone

associated with TBT exposure.

Testosterone is necessary for the maintenance of normal

cellular arrangement in the seminiferous tubules. Intratesticular

testosterone acts via a paracrine mechanism on AR in the semini-

ferous tubules.19,34 In adulthood, the action of androgens on the

seminiferous tubules is essential for normal spermatogenesis and

fertility. The organization of the seminiferous tubules is disrupted

by TBT exposure, likely induced by decreased testosterone lev-

els.9,32,33 Serum testosterone regulates testicular steroidogenic

enzyme activity,32,33,35 maintains spermatogenesis, and inhibits

germ cell apoptosis.33 There was a dose-dependent decrease in

serum testosterone concentration of the hamsters administered

TBT at 100 and 150 ppm/kg. Testosterone biosynthesis requires

a continuous supply of cholesterol36 and the inhibition of choles-

terol biosynthesis pathway results in a decline in testosterone,

which may in turn lead to a lower sperm count.37

Apolipoprotein E has an essential role in the global transport

of lipids, including cholesterol, facilitating lipid import and

redistribution among cells within tissues, hepatic uptake of

chylomicron, and VLDL remnants38 as well as hepatic

secretion of VLDL, adiposity, insulin sensitivity, and glucose

metabolism.39 Both circulating and tissue pools of cholesterol

precursor are controlled by ApoE.35,40 We suggest that TBT

induced increases in ApoE expression and leads to altered lipid

parameters, which in turn lead to diminished sex steroid pro-

duction and testicular dysfunction. It was already reported that

ApoE mRNA expression is inversely correlated with the level

of adrenal steroidogenesis. For instance, ApoE overexpression

in adrenal cells shows reduced basal steroidogenesis. It is very

clear that elevated levels of ApoE expression limit steroido-

genic activity in adrenal cells.41 In the present study, the

inverse correlation between TBT-induced testis ApoE mRNA

expression and the level of steroidogenesis is consistent with

our assumption that ApoE may play a role in TBT-induced

testicular dysfunction in male Syrian hamster.

In conclusion, the current study suggests that ApoE

overexpression has a role in TBT-induced male infertility.

We propose a potentially important new mechanism for the

deleterious effects of TBT, namely, an increase in ApoE inhi-

bits cholesterol biosynthesis, which, in turn, alters sex steroid

production and leads to testicular dysfunction. Further studies

are essential to understand the functional role of ApoE in

TBT-induced testicular dysfunction in male Syrian hamsters

and ultimately in humans.

Acknowledgment

The authors thank the Instrumentation facility of the Department of

Animal Science, Bharathidasan University extended under UGC-

SAP and DST-FIST scheme.

Figure 6. A–E, Tributyltin-induced changes in the messenger RNA (mRNA) levels of steroidoenic receptors and ApoE. Total RNA was isolatedusing TRIzol reagent, 1 mg of RNA from TBT-treated and control hamsters was reverse transcribed into complementary DNA (cDNA), andquantitative real-time polymerase chain reaction (PCR) was performed as described in the Materials and Methods.

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Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to

the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the

research, authorship, and/or publication of this article: This study was

supported by Department of Science & Technology (DST), Govt. of

India through DST-Fast Track scheme for Young Investigators.

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