activity in TM4 Sertoli cells

Accepted Manuscript Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells Soledad P. Rossi, Stefanie Windschuettl, María E. Matzkin, Verónica Rey-Ares, Claudio Terradas, Roberto Ponzio, Elisa Puigdomenech, Oscar Levalle, Ricardo S. Calandra, Artur Mayerhofer, Mónica B. Frungieri PII:

S0303-7207(16)30223-4

DOI:

10.1016/j.mce.2016.06.021

Reference:

MCE 9545

To appear in:

Molecular and Cellular Endocrinology

Received Date: 26 February 2016 Revised Date:

17 June 2016

Accepted Date: 17 June 2016

Please cite this article as: Rossi, S.P., Windschuettl, S., Matzkin, M.E., Rey-Ares, V., Terradas, C., Ponzio, R., Puigdomenech, E., Levalle, O., Calandra, R.S., Mayerhofer, A., Frungieri, M.B., Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells, Molecular and Cellular Endocrinology (2016), doi: 10.1016/j.mce.2016.06.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

1

Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2)

2

regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells

3 Soledad P. Rossia,b,*, Stefanie Windschuettlc, María E. Matzkina,b, Verónica Rey-Aresc,

5

Claudio Terradasd, Roberto Ponzioe, Elisa Puigdomenechf, Oscar Levalled, Ricardo S.

6

Calandraa, Artur Mayerhoferc, Mónica B. Frungieria,b

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7 a

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Científicas y Técnicas, Vuelta de Obligado 2490 (1428), Ciudad de Buenos Aires,

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Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones

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Argentina

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b

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Aires, Paraguay 2155 (1121), Ciudad de Buenos Aires, Argentina

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c

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Anatomy III, Großhaderner Str. 9 (D-82152), Planegg, Germany

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d

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Aires, Díaz Vélez 5044 (1405), Ciudad de Buenos Aires, Argentina

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e

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Buenos Aires, Paraguay 2155 (1121), Ciudad de Buenos Aires, Argentina

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f

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Argentina

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BioMedizinisches Centrum (BMC), Ludwig-Maximilians-University (LMU), Cell Biology,

División Endocrinología, Hospital Durand, Facultad de Medicina, Universidad de Buenos

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Instituto de Investigaciones en Reproducción, Facultad de Medicina, Universidad de

Instituto Médico PREFER, Güemes 2348 (1650), San Martín, Provincia de Buenos Aires,

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Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos

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Abstract

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Reactive oxygen species (ROS) regulate testicular function in health and disease. We

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previously described a prostaglandin D2 (PGD2) system in Sertoli cells. Now, we found that

25

PGD2 increases ROS and hydrogen peroxide (H2O2) generation in murine TM4 Sertoli 1

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cells, and also induces antioxidant enzymes expression suggesting that defense systems

2

are triggered as an adaptive stress mechanism that guarantees cell survival. ROS and

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specially H2O2 may act as second messengers regulating signal transduction pathways

4

and gene expression. We describe a stimulatory effect of PGD2 on lactate dehydrogenase

5

(LDH) expression via DP1/DP2 receptors, which is prevented by the antioxidant N-acetyl-

6

L-cysteine and the PI3K/Akt pathway inhibitor LY 294002. PGD2 also enhances Akt and

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CREB/ATF-1 phosphorylation.

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Our results provide evidence for a role of PGD2 in the regulation of the oxidant/antioxidant

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status in Sertoli cells and, more importantly, in the modulation of LDH expression which

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takes place through ROS generation and the Akt-CREB/ATF-1 pathway.

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Keywords: Sertoli cell, prostaglandin D2, reactive oxygen species, H2O2, lactate

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dehydrogenase, testis.

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Abbreviations: AR, androgen receptor; AMH, antimüllerian hormone; Cat, catalase; CLU,

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clusterin; CRE, cyclic-AMP-responsive element; CREB, CRE binding protein; COX2,

17

cyclooxygenase 2; GDNF, glial cell line derived neurotrophic factor; Gpx, glutathione

18

peroxidase; H-PGDS, hematopoietic PGD synthase; H2O2, hydrogen peroxide; LDH,

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lactate dehydrogenase; L-PGDS, lipocalin PGD synthase; NAC, N-acetyl-L-cysteine;

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NR5A1, nuclear receptor subfamily 5, group A, member 1; PDHA1, pyruvate

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dehydrogenase E1 alpha subunit; PGD2, prostaglandin D2; PGs, prostaglandins; PI3K,

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phosphatidylinositol 3-kinase; Pxr1, peroxiredoxin 1; ROS, reactive oxygen species; SCO,

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Sertoli cell only; SHBG, sex hormone binding globulin; Sod1, superoxide dismutase 1;

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SOX9, SRY-box 9.

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1 2

*

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Soledad P. Rossi

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Vuelta de Obligado 2490

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(1428) Buenos Aires, Argentina

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Telephone: +54 11 4783 2869, extension 209, Fax +54 11 4786 2564

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e-mail: [email protected]

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Corresponding author:

8 1. Introduction

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In spite of the large amount of studies in semen, there has been little research so far on

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the contribution of prostaglandins (PGs) to testicular physiology/pathology.

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Cyclooxygenase 2 (COX2), the inducible and rate-limiting isozyme of PG biosynthesis, is

13

expressed in different testicular cell populations including Sertoli cells (Frungieri et al.,

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2015). In fact, several authors reported the production of PGs and the existence of

15

prostanoid receptors in Sertoli cells (Ishikawa and Morris, 2006; Kristensen et al., 2011;

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Matzkin et al., 2012; Winnal et al., 2007). Sertoli cells transduce signals from FSH and

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testosterone into the synthesis of factors that are required to support spermatogenesis. In

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a recent study, our group demonstrated that FSH and testosterone exert a stimulatory

19

effect on COX2 expression and the production of some PGs (PGF2α and 15d-PGJ2) in

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Sertoli cells from sexually inactive adult hamsters kept under a short-day photoperiod,

21

through a mechanism that involves Erk1/2 phosphorylation (Matzkin et al., 2012). We also

22

found that PGF2α and 15d-PGJ2 via FP receptor and the nuclear PPARγ receptor,

23

respectively, inhibit FSH- and testosterone-induced uptake of [2,6-3H]-2-deoxy-D-glucose,

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a non-metabolizable glucose analogue in Sertoli cells from reproductively quiescent adult

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hamsters (Matzkin et al., 2012). The transport of glucose through the plasma membrane is

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the rate-limiting step in glucose metabolism and, consequently, in lactate production (Riera

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et al., 2001, 2009). The provision of adequate levels of lactate is one of the most important

3

Sertoli cell functions to guarantee germ cell development. The conversion of pyruvate to

4

lactate is catalyzed by the enzyme lactate dehydrogenase (LDH). LDH is encoded by the

5

Ldh a, Ldh b and Ldh c genes (Li, 1989). Combinations of these gene products result in

6

the tetrameric LDH isozymes LDH-1 (B4), LDH-2 (A1B3), LDH-3 (A2B2), LDH-4 (A3B1),

7

LDH-5 (A4) and LDH C4 (Gupta, 1999, 2012). Whereas LDH C4 isozyme is only present

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in spermatozoa of the mature testis (Gupta, 1999; Markert et al., 1975), the other

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isozymes have been found in variable proportions in different somatic cell populations

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including Sertoli cells (Santiemma et al., 1987).

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Sertoli cells also produce prostaglandin D2 (PGD2) and express DP receptors (Matzkin et

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al., 2012; Moniot et al., 2009; Samy et al., 2000). Although the involvement of PGD2 in the

13

activation of Sertoli-expressed genes that are required for germ cell development and

14

maintenance during organogenesis (i.e. Sox9, Notch1 and Cyp26B1) has already been

15

addressed (Moniot et al., 2009; Rossitto et al., 2015), the role of this eicosanoid in the

16

response of Sertoli cells to oxidative stress has not been explored so far. We have recently

17

reported a marked increase in the production of PGD2 in testes of short-lived mice

18

undergoing oxidative processes (Matzkin et al., 2016). In this regard, we have also found

19

that levels of reactive oxygen species (ROS) are higher in peritubular cells of patients with

20

impaired spermatogenesis and fibrosis than in peritubular cells from men with normal

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spermatogenesis (Kampfer et al., 2012). In the same infertility patients, we had previously

22

described the existence of a testicular PGD2 system (Schell et al., 2007) and the

23

participation of the PGD2-metabolite 15d-PGJ2 in the testicular generation of ROS (Schell

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et al., 2010). Catalase (Cat) levels are also significantly elevated in peritubular cells of

25

infertile patients suggesting that these cells present an improved ability to handle oxidative

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stress (Kampfer et al., 2012). In addition, we have seen that the testicular concentration of

2

the antioxidant melatonin positively correlated with the expression of the antioxidant

3

enzymes superoxide dismutase 1 (Sod1), peroxiredoxin 1 (Pxr1) and Cat in infertile

4

patients (Rossi et al., 2014).

5

Considering these preliminary data, we decided to investigate whether the PGD2 system

6

participates in the modulation of the oxidant-antioxidant status in Sertoli cells. Despite the

7

low oxygen tensions that characterize the testicular micro-environment, this tissue remains

8

vulnerable to oxidative stress due to the abundance of highly unsaturated fatty acids and

9

the presence of systems that generate ROS including the mitochondria and a variety of

10

enzymes such as the xanthine- and NADPH- oxidases and the cytochrome P450s (Aitken

11

and Roman, 2008). It has been well established that an excessive production of ROS is

12

harmful to the cell and consequently, counter-defense systems are activated (Aitken and

13

Roman, 2008). However, low and regulated ROS production may be relevant to Sertoli cell

14

activity under physiological conditions (Galardo et al., 2014; Valko et al., 2007). In this

15

context, ROS might act as secondary messengers responsible for signal transduction

16

pathways which control gene expression (Hossain et al., 2015). Thus, bearing in mind the

17

critical contribution of Sertoli cells to the supply of lactate into the spermatogenic

18

environment, the involvement of PGD2-dependent ROS generation in the physiological

19

regulation of Ldh a gene expression in these somatic cells of the tubular compartment was

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investigated.

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2. Materials and methods

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2.1. Cell culture

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The TM4 Sertoli cell line derived from immature mouse Sertoli cells [American Tissue

2

Culture Collection (ATCC® CRL1715™), Riversville, MD, USA] was cultured in RPMI1640

3

medium (Gibco, NY, USA) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich,

4

St. Louis, MO, USA). For all experiments, cells had been subcultured no more than 10

5

times. TM4 Sertoli cells were incubated in FCS-free medium for 24 h to synchronize the

6

cell cycle. To drive G0 cells into cell cycle, we treated serum-starved TM4 Sertoli cells with

7

medium containing FCS. After approximately 16-20 h, when cells entered into S phase,

8

they were incubated at 37°C under a humid atmospher e with 5% CO2, in the presence or

9

absence of the following chemicals: PGD2 (0.1nM, 10nM, 0.1µM and 1µM, Sigma-Aldrich),

10

N-acetyl-L-cysteine (NAC; 100µM, 1mM and 5mM, Sigma-Aldrich) and hydrogen peroxide

11

(H2O2; 1mM, Merck Millipore, KGaA, Darmstadt, Germany). In some experiments, TM4

12

Sertoli cells were pre-incubated for 1 h in the presence of phosphatidylinositol 3-kinase

13

(PI3K) inhibitor LY 294002 (10µM, Sigma-Aldrich), the DP1 receptor antagonist BW

14

A686C (1µM and 10µM, Cayman Chemical, Ann Arbor, MI, USA) or the DP2 receptor

15

antagonist CAY 10471 (1µM and 10µM, Cayman Chemical).

16

PGD2, LY 294002, BW A686C and CAY 10471 were dissolved in absolute ethanol (Merck

17

Millipore) and then further diluted in RPMI1640. An appropriate volume of ethanol (5-

18

10µl/ml medium RPMI1640) was added to control experiments to account for possible

19

effects of ethanol. NAC and H2O2 were diluted in RPMI1640, which was then used as

20

vehicle for control incubations.

21

After incubations, cells were used for RNA extraction followed by RT-qPCR, protein

22

extraction followed by immunoblotting, assessement of LDH activity or quantification of

23

intracellular PGD2 concentrations. Media were frozen at -70ºC until the levels of PGD2

24

secreted to the incubation media were determined by immunoassay.

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2.2. RT-PCR and RT-qPCR analyses

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Total RNA was isolated from confluent TM4 Sertoli cells using TRIzol reagent (Life

3

Technologies, Carlsbad, CA, USA) and from microdissected human Sertoli cells using the

4

Paradise Plus Reagent system (Applied Biosystems, Bedford, MA, USA) according to the

5

manufacturers’ instructions. RT reactions were performed using dN6 random primers as

6

previously described (Rossi et al., 2012).

7

PCR conditions were 95°C for 5 min, followed by 30- 40 cycles of 94°C for 1 min, 55-60°C

8

(annealing temperature) for 1 min, 72°C for 1 min, and a final incubation at 72°C for 5 min.

9

When required, a second PCR amplification using nested primers was also carried out.

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The following oligonucleotide primers were used for amplification of 18S rRNA (5´-

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ACACGGACAGGATTGACAGATT and 5´-CGTTCGTTATCGGAATTAACCA), mouse DP1

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receptor (5´-TGCAACCTGGGTGCCATG and 5´-GGCTTGGAGGTCTTCCGA), human

13

DP1

14

GCTCGGAGGTCTTCTGCT; hemi-nested set 5´-CAACCTCTATGCGATGCA and 5´-

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GCTCGGAGGTCTTCTGCT), mouse DP2 receptor (5´-CATGTGCTACTACAACTTGC and

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5´-GCAGACTGAAGATGTGGTAGG),

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TATGTGCTACTACAATGTGC and 5´-GCAGGCTGAACACGTGGTAGG; hemi-nested set

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5´-TATGTGCTACTACAATGTGC and 5´-GTGGCTCGAGGCGATGATCG), COX2 (5´-

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GCTTCAAACAGTTTCTCTACAA and 5´-CATTTCTTCCCCCAGCAAC), lipocalin PGD

20

synthase

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TCCTCAGGAAAAACCAGTGT),

hematopoietic

22

GAATAGAACAAGCTGACTGGC

and

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receptor subfamily 5, group A, member 1 (NR5A1; 5´-CCCTGTTGGATTACACCTTG and

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5´-GTTGCCAAATGCTTGTGGTA), glial cell line derived neurotrophic factor (GDNF; 5′-

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TTGCAGCGGTTCCTGTGAAT

(first

set

5´-TGCAACCTCGGCGCCATG

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receptor

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DP2

receptor

(first

5´-

set

5´-

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human

and

(L-PGDS;

5´-CTCCACCACTGACACGGAG

and

5´-

PGD

synthase

and

5´-

(H-PGDs;

AGCCAAATCTGTGTTTTTGG),

5´-

nuclear

5´-TCTTAGAATATGGTAAACCAGGTTGTCA), 7

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clusterin

5´

CCACGCCATGAAGATTCTCCTGC

and

5´-

2

CTCCCTGGACGGCGTTCTGA)

and

(SHBG;

5´-

3

GACATTCCCCAGCCTCATGCA and 5´-TGCCTCGGAAGACAGAACCACG).

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For mouse DP1 receptor, COX2, L-PGDS, CLU and SHBG oligonucleotide primers were

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designed as homologous to regions of different exons. PCR products were separated on

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2% agarose gels and visualized with ethidium bromide. The identity of the cDNA products

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was confirmed by sequencing, using a fluorescence-based dideoxy-sequencing reaction

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and an automated sequence analysis on an ABI 373A DNA sequencer.

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qPCR

sex

hormone

binding

globulin

were

performed

using

oligonucleotide

primers

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assays

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(CLU;

for

Sod1

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AAAGCGGTGCGTGCTGAA

and

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peroxidase

5´-CCTCAAGTACGTCCGACCTG

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CAATGTCGTTGCGGCACACC),

Pxr1

(5´-CACCCAAGAAACAAGGAGGA

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GAGATACCTTCATCAGCCTT),

Cat

(5´-CCGACCAGGGCATCAAAA

14

CATTGGCGATGGCATTGA), Ldh a (5´-CATTGTCAAGTACAGTCCACACT and 5´-

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TTCCAATTACTCGGTTTTTGGGA) and Gapdh (5´-GACGGCCGCATCTTCTTGT and 5´-

16

AACGACCTTCACCATTTTGTCT) which was selected as the housekeeping gene.

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Reactions were conducted using SYBR Green PCR Select Master Mix and the ABI PRISM

18

7500 sequence detector System (Applied Biosystems). Following the mathematical model

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of Pfaffl (2001), the relative levels of mRNA expression were determined for each sample

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as previously described (Rossi et al., 2012).

glutathione and

5´and

5´-

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5´-

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(Gpx;

5´-CAGGTCTCCAACATGCCTCT),

(5´-

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2.3. Immunoblotting

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TM4 Sertoli cell protein homogenates (5-50µg), prepared as previously described (Rossi et

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al., 2012) were loaded onto 15% tricine-SDS-polyacrylamide gels, electrophoretically

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separated, and blotted onto nitrocellulose membrane. Protein concentrations were 8

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measured by the method of Lowry et al. (1951). We performed immunoblots as described

2

(Rossi et al., 2012) using a mouse monoclonal anti-actin antibody (1:5000, Calbiochem, La

3

Jolla, CA, USA), a rabbit monoclonal anti-Cat antibody (1:1500, Epitomics Inc.,

4

Burlingame, CA, USA), a rabbit monoclonal anti-Pxr1 antibody (1:3000, Abcam, Milton,

5

Cambridge, UK), a rabbit polyclonal anti-phospho Akt kinase serum (p-Akt; 1:500, Cell

6

Signaling Technology Inc., Beverly MA, USA), a rabbit polyclonal anti-Akt kinase serum

7

(1:500, Cell Signaling Technology Inc.), a rabbit polyclonal anti-phospho extracellular

8

signal-regulated kinase (p-Erk) 44/42 (1/2) MAP kinase serum (1:500, Cell Signaling

9

Technology Inc.), a rabbit polyclonal anti-Erk1/2 kinase serum (1:500, Cell Signaling

10

Technology Inc.), a mouse monoclonal anti-phospho P38 MAP kinase antibody (p-P38;

11

1:500, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), a rabbit monoclonal anti-P38

12

kinase antibody (1:500, Cell Signaling Technology Inc.), a rabbit polyclonal anti-phospho

13

CRE binding protein (p-CREB) and anti-phospho ATF-1 (p-ATF-1) serum (1:500, Cell

14

Signaling Technology Inc.) or a rabbit polyclonal anti-phospho pyruvate dehydrogenase E1

15

alpha subunit (p-PDHA1) serum (1:1000, Novus Biological, Littleton, CO, USA).

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2.4. Immunocytochemical analyses

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Formaldehyde-fixed TM4 Sertoli cells were used for immunodetection of SRY-box 9

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(SOX9) and the antimüllerian hormone (AMH) as previously described (Matzkin et al.,

20

2012). Cells were incubated overnight at 4 °C with a rabbit polyclonal anti-SOX9 serum

21

(1:25, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) or a rabbit polyclonal anti-

22

AMH serum (1:100, for further information about the antiserum see Dutertre et al., 1997).

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For control purposes, the first antiserum was omitted or incubations were carried out with

24

normal non-immune sera.

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2.5. ROS measurement

2

Generation of ROS was determined with the cell-permeant fluorogenic probe 2´,7´-

3

dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Invitrogen, Darmstadt,

4

Germany) as described (Rossi et al., 2014). H2DCFDA diffuses into the cell, where it is

5

enzymatically converted by intracellular esterases and oxidized into the high fluorescence

6

compound DCF, which allows the determination of H2O2, peroxynitrite anions and peroxyl

7

radicals. Approximately 4 x 103 TM4 Sertoli cells per well were plated on white bottom 96-

8

well plates in extracellular fluid (140nM NaCl, 3mM KCl, 1mM CaCl2, 1mM MgCl2, 10mM

9

HEPES and 25mM glucose; pH 7.4). After 2 h incubation, cells were loaded with 10µM

10

H2DCFDA for 30 min at 37°C and 5% CO 2. Then, cells were treated with or without PGD2

11

(1µM) in the presence or absence of NAC (1mM) for 2 h at 37°C. DCF generation was

12

measured over time using a fluorometer (Fluostar BMG Labtech, Offenburg, Germany) at

13

492nm excitation and 520nm emission.

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14 2.6. H2O2 measurement

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The production of H2O2 was determined with the Amplex® Red reagent (10-acetyl-3,7-

17

dihydroxyphenoxazine; Molecular Probes). In the presence of horseradish peroxidase

18

(HPR) the Amplex® Red reagent reacts in a 1:1 stoichiometry to produce the red-

19

fluorescent oxidation product, resorufin. Consequently, resorufin generation allows the

20

detection of the H2O2 released from biological samples.

21

Approximately 2 x 103 TM4 Sertoli cells were plated on black bottom 96-well plates in

22

120µl of working solution containing 40µM Amplex® Red reagent and 0.1U/ml HPR, and

23

incubated in the presence or absence of PGD2 (0.1 and 1µM) for 2 h at 37°C. Resorufin

24

generation was measured over time using a fluorometer (Fluostar BMG Labtech) at 544nm

25

excitation and 590nm emission.

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1 2.7. Live cell Imaging studies

3

TM4 Sertoli cells were grown overnight in a glass bottomed cultured dish (µ-Dish, Ø 35

4

mm; Ibidi, Martinsried, Germany) under standard conditions. Then, cells were incubated in

5

the presence or absence of PGD2 (1µM) in RPMI1640 serum-free medium under a humid

6

atmosphere with 5% CO2 at 37°C for 24 h. Optimal incubation conditions (5% CO2, 37 °C,

7

95% relative humidity) were created by using an Ibidi heating system and an Ibidi gas

8

incubation system. Time-lapse series were generated by taking a picture every 20 min for

9

24 h with a ProgRes MF camera (Jenoptik, Jena, Germany), the Micro-Manager 1.3

10

Microscopy Software (Ron Vale’s laboratory at UCSF, San Francisco, CA, USA) and a

11

transmitted light microscope (Axiovert 135; Zeiss, Oberkochen, Germany).

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12 2.8. Proliferation assay

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Approximately 9 x 103 TM4 Sertoli cells were plated on 96-well plates and incubated for 24

15

h in the presence or absence of PGD2 (0.1nM, 10nM and 1µM). Cellular metabolic activity

16

was determined using the CellTiter 96 AQueous One Solution cell-proliferation assay

17

(Promega Corporation, Madison, WI, USA) as previously described (Rossi et al., 2014).

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2.9. LDH activity assay

20

Total LDH activity was determined using the LDH-P unitest kit (Wiener Laboratories,

21

Rosario, Argentina) which includes pyruvate and NADH as substrate and co-factor,

22

respectively, of the reaction catalyzed by the LDH enzyme.

23

Approximately 1 x 106 TM4 Sertoli cells were resuspended in 100µl 0.9% NaCl, disrupted

24

by ultrasonic irradiation and centrifuged at 15800 x g for 10 min at 4°C. Supernatants were

25

used to determine total LDH activity spectrophotometrically at 340nm following the

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manufacturer’s protocol. From each sample, an aliquot was preserved for protein

2

determinations. Results are expressed as µmol/min (IU) per mg protein.

3 2.10. Patients and human testicular samples

5

The study was designed in agreement with the Helsinki Declaration and its last

6

modification in Tokyo 2004 on human experimentation and was approved by the Ethics

7

Committee of the Instituto de Biología y Medicina Experimental (Buenos Aires, Argentina)

8

and the Ethics Committee of the Durand Hospital (Buenos Aires, Argentina). Informed

9

consent was obtained from all the patients included in the study.

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Twenty men (27-42 years old) with idiopathic infertility and non-obstructive azoospermia

11

without an infection process who presented a normal karyotype were registered in this

12

study. Patients with known etiology of infertility, such as genitourinary infections, mumps

13

orchitis, varicocele, hypogonadotropic hypogonadism, chromosome anomalies, obstruction

14

or agenesia of seminal ducts were excluded.

15

Patients were assessed and diagnosed by open testicular biopsy. A small piece of

16

testicular tissue was immersed in Bouin’s solution and submitted to histopathological

17

diagnosis. Biopsies were classified either as hypospermatogenesis or Sertoli cell only

18

(SCO) appearance according to McLachlan et al. (2007). The hypospermatogenesis group

19

included men in which the biopsy specimens contained less than 17 mature spermatids

20

per tubule but all stages of spermatogenesis are present. SCO syndrome term is used

21

when there are no tubules containing germ cells (Silber and Rodriguez-Rigau, 1981; Silber

22

et al., 1997).

23

A second piece of testicular tissue was preserved for molecular biology studies. Ethical

24

and legal considerations excluded the possibility of studying testicular samples from

25

healthy men.

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1 2.11. PGD2 immunoassay

3

PGD2 concentrations in TM4 Sertoli cells and human testicular biopsies were determined

4

using a commercial kit (Cayman Chemical) as previously described (Matzkin et al., 2012).

5

In order to prevent further PG chemical degradation samples were treated with

6

methoxylamine hydrochloride (MOX HCl). The levels of PGD2 secreted from TM4 Sertoli

7

cells to the incubation media were also quantified. After MOX HCl treatment and

8

acidification using 1M citrate buffer (pH 4.7), media were injected into C18 columns and

9

then eluted with ethyl acetate. Eluted fractions were evaporated to dryness under nitrogen

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stream and reconstituted in assay buffer.

11

The minimum detectable immunoassay concentration was 0.28 femtomole (fmol)/tube.

12

Intra-assay and inter-assay coefficients of variation were less than 10% and less than

13

15%, respectively. PGD2 levels were expressed as pmol per 106 cells and pmol per g

14

tissue.

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2.12. Immunohistochemical analysis followed by laser capture microdissection and RT-PCR

17

A rabbit polyclonal anti-androgen receptor (AR) serum (1:100, Santa Cruz Biotechnology

18

Inc.) was used to detect the immunological expression of ARs. Afterwards, laser capture

19

microdissection

20

Microdissected samples containing AR immunopositive Sertoli cells were used for RT-PCR

21

as described above.

was

performed

as

previously

described

(Rossi

et

al.,

2014).

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2.13. Statistical analyses

24

Data analyses were performed using GraphPad Prism® 5.03 (GraphPad Software, San

25

Diego, CA, USA). Statistical analyses were performed using one-way ANOVA followed by 13

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Student’s t test for two comparisons or Student–Newman–Keuls test for multiple

2

comparisons. Data are expressed as mean ± S.E.M.

3

For immunoblotting studies, bands were quantified by densitometry and normalized to the

4

housekeeping gene using SCION IMAGE (SCION Corporation, Frederick, MD, USA).

5 6

3. Results

7

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3.1. Expression of Sertoli cell markers in TM4 cells

9

Immunocytochemical assays showed cytoplasmic AMH and nuclear SOX9 expression in

10

TM4 Sertoli cells (Fig. 1A). Furthermore, the mRNA expression of NR5A1, GDNF, CLU

11

and SHBG was detected in TM4 Sertoli cells by RT-PCR studies (Fig. 1B).

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3.2. Identification of a PGD2 system in TM4 Sertoli cells involved in the induction of ROS

14

generation and antioxidant enzymes expression

15

The existence of a PGD2 system in TM4 Sertoli cells was investigated by RT-PCR assays.

16

Studies revealed the expression of the PGD2 receptors DP1 and DP2, as well as the PGD2

17

biosynthetic enzymes, COX2, L-PGDS and H-PGDs in TM4 Sertoli cells (Fig. 2A). In

18

addition, TM4 Sertoli cells produce and release PGD2 into the incubation media (Fig. 2B).

19

In order to analyze a potential effect of PGD2 on cellular oxidative status, ROS generation

20

was investigated. TM4 Sertoli cells were incubated in the presence or absence of 1µM

21

PGD2 with or without the antioxidant NAC (1mM). After 120 min incubation, the levels of

22

ROS were significantly higher than those detected under control conditions. This effect

23

was prevented by the antioxidant NAC (Fig. 2C and D).

24

To investigate whether antioxidant mechanisms leading to cell survival are activated after

25

PGD2 treatment, the expression of antioxidant enzymes was evaluated by RT-qPCR and

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immunoblotting. In addition, cell viability and morphology were assessed by a colorimetric

2

assay and live cell imaging studies, respectively.

3

PGD2 (1µM) induced a significant increase in Sod1, Gpx, Pxr1 and Cat mRNA expression

4

(Fig. 2E), as well as on Pxr1 and Cat protein expression (Fig. 2F).

5

After 24 h incubation in the presence or absence of PGD2 (1µM, 10nM and 0.1nM), no

6

significant differences were seen in cell viability between groups (data not shown).

7

Throughout 24 h incubation, live cell imaging showed no changes in the morphology of

8

TM4 Sertoli cells incubated in the presence or absence of 1µM PGD2 (data not shown).

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3.3. Identification of the main compounds of the signaling pathway triggered by PGD2 in

11

TM4 Sertoli cells

12

Taking into account that among all ROS and other oxygen-derived free radicals, H2O2 has

13

been recently suggested to act as a central player in signal transduction pathways

14

(Hossain et al., 2015), we evaluated H2O2 generation by a fluorometric assay in TM4

15

Sertoli cells incubated in the presence or absence of PGD2.

16

Incubations carried out with 1µM PGD2 saturated H2O2 measurement in seconds (data not

17

shown). Thus, experiments were repeated in the presence of 0.1µM PGD2 to evaluate

18

H2O2 production throughout 120 min incubation (Fig. 3A). H2O2 production was significantly

19

higher in TM4 Sertoli cells incubated with 0.1µM PGD2 than in cells kept under basal

20

conditions (Fig. 3A and B).

21

Since it has been reported that the mechanism by which ROS act as signaling molecules

22

involves several transduction pathways (Ji et al., 2010; Loh et al., 2009; Tai and Ascoli,

23

2011), immunoblotting experiments for detection of protein phosphorylation were

24

conducted. PGD2 significantly increased the phosphorylation levels of Akt in TM4 Sertoli

25

cells after 15 and 30 min incubation (Fig. 4A and B, respectively). This stimulatory effect of

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PGD2 on phospho-Akt was reverted by the antioxidant NAC after 15 and 30 min incubation

2

(Fig. 5A and B, respectively). In contrast, phospho-Erk1/2 and phospho-P38 levels were

3

not affected by PGD2 (Fig. 4A and B). When TM4 Sertoli cells were treated with 1mM

4

H2O2, phosphorylation levels of Erk1/2 and Akt were markedly increased, but phospho-

5

P38 levels remained unchanged (Fig. 4B).

6

After 15 and 30 min incubation, PGD2 also exerted a stimulatory effect on the

7

phosphorylation levels of CREB and ATF-1 (nuclear transcription factors associated to Akt

8

kinase) that was prevented by NAC (Fig. 5A and B).

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3.4.

of

the

signaling

pathway

by

which

PGD2

regulates

LDH

11

expression/activity

12

In order to investigate the participation of PGD2 in the regulation of LDH

13

expression/activity, TM4 Sertoli cells were incubated in the presence or absence of 1µM

14

PGD2. RT-qPCR and LDH activity assays showed a stimulatory effect of PGD2 on both

15

Ldh a mRNA expression and LDH activity (Fig. 6A-D).

16

Ldh a mRNA expression and LDH activity were also significantly increased by 1mM H2O2

17

(Fig. 6A and B).

18

To further evaluate the involvement of ROS as well as the participation of the PI3K/Akt

19

pathway in the PGD2-dependent regulation of LDH, the antioxidant NAC (100µM and

20

5mM) or the PI3K/Akt inhibitor LY 294002 (10µM) were added to the incubation media.

21

NAC and LY 294002 prevented the stimulatory effect of PGD2 on Ldh a mRNA expression

22

(Fig. 6A and C). Furthermore, NAC also blocked the positive regulation exerted by PGD2

23

on LDH activity, but LY 294002 had no effect (Fig. 6B and D, respectively).

24

Bearing in mind that increased LDH activity could result not only from higher Ldh a mRNA

25

levels but also from increased substrate availability (Spriet et al., 2000), we investigated

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the regulation of the pyruvate dehydrogenase complex (PDC). We found that PDHA1 is

2

phosphorylated by 10µM LY 294002 (Fig. 6E) and therefore its activity is inhibited.

3

Consequently, higher concentrations of pyruvate might remain available for its conversion

4

to lactate and explain why LY 294002 failed to block the positive modulation exerted by

5

PGD2 on LDH activity while abolishing the stimulatory effect of this PG on Ldh a gene

6

expression. In contrast, no significant changes were observed in phospho-PDHA1 when

7

TM4 Sertoli cells were incubated in the presence or absence of NAC (100µM and 5 mM)

8

or PGD2 (1 µM) (Fig. 6E).

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3.5. The stimulatory effect of PGD2 on Ldh a mRNA expression and LDH activity is

11

reverted by specific DP receptor antagonists

12

In order to analyze the participation of DP receptors in the stimulatory effect exerted by

13

PGD2 on Ldh a mRNA expression and LDH activity, TM4 Sertoli cells were incubated in

14

the presence or absence of 1µM PGD2 with or without BW A686C and CAY 10471 (1µM

15

and 10µM), specific antagonists of DP1 and DP2 receptors, respectively. The stimulatory

16

effect of PGD2 on the relative levels of Ldh a mRNA expression was fully blocked by CAY

17

10471 (Fig. 7B). After 30 min incubation, BW A686C reverted the positive modulation of

18

PGD2 on Ldh a mRNA expression but its effect was less profound after 60 min incubation

19

(Fig. 7A).

20

Both DP receptor antagonists reverted the PGD2-dependent stimulation of LDH activity

21

(Fig. 7C and D).

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3.6. Identification of the main components of the PGD2 system in the human testis

24

To examine whether this newly described system is present in human Sertoli cells, we

25

studied testicular biopsies from men presenting hypospermatogenesis or SCO syndrome. 17

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PGD2 testicular concentrations were determined. No significant differences were found

2

between the groups (Fig. 8A). By laser capture microdissection and RT-PCR studies,

3

expression of DP1 and DP2 receptors was detected in human Sertoli cells isolated from

4

the testis of patients revealing hypospermatogenesis (data not shown) or SCO syndrome

5

(Fig. 8B and C).

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6 4. Discussion

8

Sertoli cells are essential for testis formation and spermatogenesis (Griswold, 1998).

9

Hence, morphological and functional alterations of these cells have severe consequences

10

on male fertility. PGs are potential inducers of intracellular oxidative stress (Kondo et al.,

11

2001). Recently, we described the existence of a PGD2 system in Sertoli cells from

12

reproductively inactive adult hamsters (Matzkin et al., 2012), as well as a marked increase

13

in the production of PGD2 in testes of short-lived mice undergoing oxidative processes

14

(Matzkin et al., 2016). Consequently, in this study, our investigations attempted to

15

determine the potential impact of PGD2 on the oxidant-antioxidant status in Sertoli cells.

16

To our knowledge, functional studies in human Sertoli cells are not possible. In addition,

17

because of germ cell contamination, reports using Sertoli cells purified from reproductively

18

active rodents have been scarce. Therefore, prepubertal/early-pubertal mice and rats or

19

alternatively, adult hamsters with impaired spermatogenesis as a consequence of their

20

exposure to a short-day photoperiod are frequently used for Sertoli cell isolation (Matzkin

21

et al., 2012; Riera et al., 2001, 2009; Yang and Han, 2010). TM4, derived from immature

22

mouse Sertoli cells, is a non-transformed, non-tumorigenic and well characterized cell line

23

that retains the most important aspects of Sertoli cell physiology (Lan et al., 2013; Mather,

24

1980, 1982; Nakhla et al., 1984; Simon et al., 2007). In addition, the TM4 cell population

25

available in our laboratory expresses significant levels of the Sertoli cell markers AMH,

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SOX9, GDNF, SHBG, CLU and NR5A1. Therefore, in this study, the TM4 cell line was

2

chosen as our experimental model to investigate the participation of PGD2 in the

3

occurrence of oxidative events in Sertoli cells.

4

We initially found that TM4 Sertoli cells express key enzymes in the biosynthesis of PGD2

5

namely COX2 and the two distinct types of PGDS (L-PGDS and H-PGDS). In fact, TM4

6

Sertoli cells produce and secrete PGD2 and also express DP1 and DP2 receptors.

7

Recent published data from our group indicate that PGD2 concentrations in mouse testes

8

range between 3000 to 9000 pmol per g tissue (Matzkin et al., 2016). Bearing in mind the

9

testicular tissue density of 1.05 g/cm3 (Yang et al., 1990; Zhou et al., 2010), in this study

10

we used doses from 1 to 10µM PGD2 which can be considered as physiological

11

concentrations.

12

Two different effects on the oxidant-antioxidant status were observed when TM4 Sertoli

13

cells were incubated with physiological concentrations of PGD2. Firstly, PGD2 stimulated

14

overall ROS production and, specifically, H2O2 generation. Secondly, PGD2 increased the

15

expression levels of the antioxidant enzymes Sod1, Gpx, Pxr1 and Cat. Furthermore,

16

Sertoli cell viability and morphology were not negatively affected by PGD2. Depending on

17

the levels of ROS produced, signaling could lead to death program and apoptosis, a gross

18

structural/metabolic damage and necrosis, or to induction of antioxidant mechanisms and

19

cell survival. In this context, under normal physiological states, the antioxidant capacity of

20

the testicular tissue seems to be enough to avoid cellular damage (Turner and Lysiak,

21

2008). Thus, we conclude that TM4 Sertoli cells activate enzymatic defense mechanisms

22

and prevent a potential cell injury triggered by the PGD2-dependent ROS generation.

23

The combination of a long life span and a small size allows the H2O2 molecule to cross

24

through the internal membrane systems of the cell facilitating signaling functions (Hossain

25

et al., 2015). For this reason, among all ROS, H2O2 has been recently postulated to act as

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a central player in signal transduction pathways (Hossain et al., 2015). As mentioned,

2

PGD2 increased H2O2 generation in TM4 Sertoli cells. We also found that PGD2 induced

3

mRNA expression of Ldh a, a key enzyme in lactate production. This stimulatory action of

4

PGD2 on the mRNA expression levels of Ldh a was prevented by the antioxidant NAC.

5

Thus, H2O2 might act as transducer of the PGD2-triggered signaling pathway involved in

6

lactate production in TM4 Sertoli cells. Several hormones and factors through the protein

7

kinase A and C signal pathways and the transcription factor CREB contribute to the

8

regulation of LDH expression and/or activity in Sertoli cells (Boussouar and Benahmed,

9

1999; Jungmann et al., 1986, 1998; Nehar et al., 1997; Riera et al., 2001; Short et al.,

10

1994). When we analyzed the phosphorylation of various protein kinases and nuclear

11

factors, we found that Erk and P38 phosphorylation were unaffected by PGD2. In contrast,

12

this eicosanoid significantly increased the phosphorylation levels of Akt kinase and the

13

transcription factors ATF-1 and CREB. It is well known that CREB and ATF-1 are

14

regulatory targets for the protein kinase Akt (Du and Montminy, 1998; Fan et al., 2005; Jia

15

et al., 2006). Interestingly, the antioxidant NAC not only blocked the stimulatory action of

16

PGD2 on Ldh a gene expression in TM4 Sertoli cells but also the PGD2-dependent

17

phosphorylation of Akt, ATF-1 and CREB. Furthermore, incubations of TM4 Sertoli cells in

18

the presence of LY 294002, an inhibitor of PI3K-dependent Akt phosphorylation and

19

kinase activity, abolished the stimulatory effect of PGD2 on Ldh a gene expression. Taken

20

together, these results let us speculate that PGD2 increases ROS generation and

21

subsequently Akt phosphorylation. Then, this kinase might phosphorylate and activate the

22

transcription factors ATF-1 and CREB leading to up-regulation of Ldh a gene expression in

23

TM4 Sertoli cells. However, this hypothesis awaits further testing.

24

Behaving as expected, PGD2 also stimulated LDH activity in TM4 Sertoli cells and this

25

stimulatory effect was prevented by the antioxidant NAC. LY 294002 abolished the

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stimulatory effect of PGD2 on Ldh a gene expression but it failed to block the positive

2

modulation exerted by this PG on LDH activity. Subsequent experiments showed an

3

increased phosphorylation of PDHA1 by LY 294002 in TM4 Sertoli cells, leading to

4

inactivation of the PDC that converts pyruvate into acetyl-CoA. It has already been

5

established that LDH becomes more active under experimental and physiological

6

conditions which result in an increase of the substrates required for lactate production

7

(Spriet et al., 2000). Thus, our results support the idea that PGD2 via the Akt/PI3K pathway

8

not only induces Ldh a gene expression but also indirectly regulates LDH activity by

9

increasing substrate availability.

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Experiments using specific antagonists of DP1 and DP2 receptors, allowed us to conclude

11

that PGD2 actions on Ldh a gene expression and LDH activity in TM4 Sertoli cells are

12

exerted via a direct mechanism which involves PGD2 interacting with specific G-protein

13

coupled DP receptors. Both DP1 and DP2 receptors have previously been described in the

14

testis (Moniot et al., 2014; Rossitto et al., 2015).

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To our knowledge, this is the first time that PGD2 concentrations are quantified in the

17

human testis. Keeping in mind the tissular levels of PGD2 as well as the average weight,

18

volume and density of the human testis (Sośnik, 1988; Yang et al., 1990; Zhou et al.,

19

2010), concentrations used in this study ranging from 1 to 10µM PGD2 can be considered

20

as physiological concentrations.

21

Previously, we described the expression of DP1 receptors in the interstitial compartment of

22

the adult human testis (Schell et al., 2007). In this study, we detected the expression of

23

DP1 and DP2 receptors in Sertoli cells isolated from testicular biopsies of patients

24

suffering from hypospermatogenesis or SCO syndrome. However, PGD2 levels and the

25

expression of DP receptors in testes of fertile men remain unknown as a consequence of

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1

ethical and legal considerations. At least, a recent report showed a negative impact of non-

2

steroidal

3

spermatogenesis suggesting a physiological role of testicular PGs in adult men (Albert et

4

al., 2013). Thus, our studies performed in mouse TM4 Sertoli cells might be of relevance

5

to the human situation.

drugs

(NSAIDs)

in

human

testes

showing

normal

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anti-inflammatory

6

In summary, our data reveal the existence of a PGD2-dependent testicular source of ROS

8

generation. Furthermore, ROS not only act as antioxidants in Sertoli cells but also as

9

signaling molecules involved in the regulation of LDH expression/activity. Sertoli cell

10

lactate production is crucial for the support of germ cell development. Hence, the

11

modulation of LDH by free radicals including H2O2 supports the assumption of a

12

stimulatory role of PGD2 on spermatogenesis taking place through its binding to specific

13

DP receptors located in the Sertoli cell membrane and the subsequent local generation of

14

ROS.

15

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Acknowledgements

17

We are grateful to Dr. P. Pomata of the Institute of Biology and Experimental Medicine,

18

Buenos Aires, Argentina, for his expert technical assistance during the laser capture

19

microdissection procedure. We thank Prof. Dr. R. A. Rey (Centro de Investigaciones

20

Endocrinológicas Dr. César Bergadá –CEDIE-, CONICET-FEI, División de Endocrinología,

21

Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina) for providing the AMH

22

antiserum.

23

This study was supported by grants from the Consejo Nacional de Investigaciones

24

Científicas y Técnicas (CONICET PIP 2015-2017 11220150100267CO), Agencia Nacional

25

de Promoción Científica y Tecnológica (ANPCyT BID PICT 2012 1917, BID PICT 2014

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1200), Fundación Alberto J. Roemmers, Fundación René Barón, and Deutsche

2

Forschungsgemeinschaft (DFG MA1080/25-1).

3

5

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insulin

sensitivity.

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260-272.

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enhance

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Figure Legends

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14 Fig. 1. Identification of Sertoli cell markers expressed in murine TM4 cells.

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(A) Positive immunostaining for AMH and SOX9 was found in TM4 cells. No reaction was

17

observed when TM4 cells were incubated only with normal non-immune serum and the

18

conjugated antibody (Co, control). Bar, 50 µm. (B) mRNA expression of GDNF (171 bp),

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CLU (160 bp), SHBG (161 bp) and NR5A1 (134 bp) was detected by RT-PCR in TM4

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cells. Co: non-template control.

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Fig. 2. Identification of a PGD2 system in TM4 Sertoli cells: PGD2-induced ROS generation

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and antioxidant enzymes expression.

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(A) mRNA expression of the PGD2 receptors DP1 (275 bp) and DP2 (262 bp), as well as of

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the PGD2 biosynthetic enzymes COX2 (111 bp), L-PGDS (118 bp) and H-PGDS (136 bp) 31

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was detected by RT-PCR in TM4 Sertoli cells. (B) TM4 Sertoli cells were incubated in

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basal conditions for 3 h. Intracellular PGD2 concentrations and PGD2 levels secreted to the

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incubation media from TM4 Sertoli cells were determined by enzyme immunoassay. Bar

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plot graphs represent the mean ± SEM (n = 5). (C) ROS generation was determined over

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time by a fluorometric assay in TM4 Sertoli cells incubated in the presence or absence of

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PGD2 (1µM) with or without NAC (1mM). This graph is representative of three independent

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experiments performed in different cell preparations that showed comparable results. Co,

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cells incubated in basal conditions. (D) Bar plot graph illustrates the endpoint fluorescence

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values after 120 min incubation. This graph is representative of three independent

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experiments performed in different cell preparations that showed comparable results. Bar

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plot graph represents the mean ± SEM (n = 8 replicate wells per treatment). Different

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letters denote a statistically significant difference between groups. p < 0.05. Student-

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Newman–Keuls test. (E) TM4 Sertoli cells were incubated in the presence or absence of

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PGD2 (1µM) for 3 h. The relative levels of mRNA Sod1, Gpx, Pxr1 and Cat expression

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were determined by RT-qPCR using Gapdh as the housekeeping gene. The mRNA

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antioxidant enzymes expression levels obtained in three independent experiments were

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analyzed using the mathematical model of Pfaffl. Bar plot graph represents the mean ±

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SEM. Different letters denote a statistically significant difference between groups. p < 0.05.

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Student’s t test. (F) TM4 Sertoli cells were incubated in the presence or absence of PGD2

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(1µM) for 5 h. Pxr1 (22 kDa), Cat (60 kDa) and actin (42 kDa) protein levels were

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determined by immunoblotting. These immunoblots are representative of three

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experiments performed in different cell preparations that showed comparable results. Bar

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plot graph represents the mean ± SEM and depicts the quantification by densitometry of

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the bands obtained in three independent experiments. Results are expressed as fold

25

change relative to the control (cells incubated in basal condition) which was assigned a

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value of 1 and normalized to actin. Different letters denote a statistically significant

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difference between groups. p < 0.05. Student’s t test.

3 Fig. 3. Stimulatory effect of PGD2 on H2O2 production in TM4 Sertoli cells.

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(A) H2O2 production was determined over time by a fluorometric assay in TM4 Sertoli cells

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incubated in the presence or absence of PGD2 (0.1µM). This graph is representative of

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three independent experiments performed in different cell preparations that showed

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comparable results. (B) Bar plot graph illustrates the endpoint fluorescence values after

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120 min incubation. This graph is representative of three independent experiments

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performed in different cell preparations that showed comparable results. Bar plot graph

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represents the mean ± SEM (n = 8 replicate wells per treatment). Different letters denote a

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statistically significant difference between groups. p < 0.05. Student’s t test.

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Fig. 4. Participation of Akt in the signaling pathway triggered by PGD2 in TM4 Sertoli cells.

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(A) TM4 Sertoli cells were incubated in the presence or absence of PGD2 (1µM) for 15

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min. (B) TM4 Sertoli cells were incubated in the presence or absence of PGD2 (1µM) or

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H2O2 (1mM) for 30 min. Total and phosphorylated levels of Erk1/2 (44-42 kDa), P38 (38

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kDa), and Akt (60 kDa) were determined by immunoblotting. These immunoblots are

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representative of three experiments performed in different cell preparations that showed

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comparable results. Bar plot graphs represent the mean ± SEM and depict the

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quantification by densitometry of the bands obtained in three independent experiments.

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Results are expressed as fold change relative to the control (cells incubated in basal

23

condition), which was assigned a value of 1, and normalized to total Erk1/2, P38 or Akt,

24

respectively. Different letters denote a statistically significant difference between groups. p

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< 0.05. Student’s t test.

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1 Fig. 5. Inhibitory effect of the antioxidant NAC on the PGD2-dependent phosphorylation of

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Akt/CREB/ATF-1 in TM4 Sertoli cells.

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(A) and (B) TM4 Sertoli cells were incubated in the presence or absence of PGD2 (1µM)

5

for 15 min and 30 min, respectively, with or without NAC (5mM). The levels of total and

6

phosphorylated Akt (60 kDa), phospho-CREB (p-CREB; 43 kDa), phospho-ATF-1 (p-ATF-

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1; 38 kDa) and actin (42 kDa) were determined by immunoblotting. These immunoblots

8

are representative of three experiments performed in different cell preparations that

9

showed comparable results. Bar plot graphs represent the mean ± SEM and depict the

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quantification by densitometry of the bands obtained in three independent experiments.

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Results are expressed as fold change relative to the control (cells incubated in basal

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condition), which was assigned a value of 1, and normalized to total Akt or actin. Different

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letters denote a statistically significant difference between groups. p < 0.05; Student-

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Newman–Keuls test.

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Fig. 6. Stimulatory effect of PGD2 on LDH expression/activity in TM4 Sertoli cells:

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participation of ROS and the PI3K/Akt pathway.

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(A) and (B) TM4 Sertoli cells were incubated in the presence or absence of PGD2 (1µM)

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for 30 min and 60 min (left panel) as well as for 6 h (right panel) with or without NAC

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(100µM or 5mM). Cells were also incubated in the presence or absence of 1mM H2O2. Cell

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extracts were used for RT-qPCR assays and determination of LDH activity using the LDH-

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P unitest commercial assay.

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(C) and (D) TM4 Sertoli cells were incubated in the presence or absence of PGD2 (1µM)

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for 30 min and 60 min (left panel) as well as for 6 h (right panel) with or without the

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PI3K/Akt inhibitor LY 294002 (10µM). Cell extracts were used for RT-qPCR assays and

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determination of LDH activity using the LDH-P unitest commercial assay.

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The relative levels of mRNA Ldh a expression were determined using Gapdh as the

4

housekeeping gene in three independent experiments and analyzed by the mathematical

5

model of Pfaffl. Bar plot graphs represent the mean ± SEM. Different letters denote a

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statistically significant difference between groups. p < 0.05. Student-Newman–Keuls test.

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LDH activity graphs are representative of three independent experiments performed in

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different cell preparations that showed comparable results. Bar plot graphs represent the

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mean ± SEM. (n = 3 replicates per treatment). Different letters denote a statistically

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significant difference between groups. p < 0.05. Student-Newman–Keuls test.

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(E) TM4 Sertoli cells were incubated in the presence or absence of PI3K/Akt inhibitor LY

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294002 (10µM), NAC (100µM and 5mM) or PGD2 (1µM) for 90 min. Phospho-PDHA1 (p-

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PDHA1; 43 kDa) and actin (42 kDa) protein levels were determined by immunoblotting.

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These immunoblots are representative of three experiments performed in different cell

15

preparations that showed comparable results. Bar plot graph represents the mean ± SEM

16

and depicts the quantification by densitometry of the bands obtained in three independent

17

experiments. Results are expressed as fold change relative to the control (cells incubated

18

in basal condition), which was assigned a value of 1, and normalized to actin. Different

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letters denote a statistically significant difference between groups. p < 0.05; Student-

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Newman–Keuls test.

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Fig. 7. Participation of DP1 and DP2 receptors in the PGD2-triggered stimulation of LDH

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expression/activity in TM4 Sertoli cells.

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TM4 Sertoli cells were incubated in the presence or absence of PGD2 (1µM) with or

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without BW A686C (1µM and 10µM) and CAY 10471 (1µM and 10µM), specific

3

antagonists of DP1 and DP2 receptors, respectively.

4

(A) and (B) The relative levels of mRNA Ldh a expression were determined by RT-qPCR

5

using Gapdh as the housekeeping gene. The mRNA Ldh a expression levels obtained in

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three independent experiments were analyzed using the mathematical model of Pfaffl. Bar

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plot graphs represent the mean ± SEM. Different letters denote a statistically significant

8

difference between groups. p < 0.05. Student-Newman–Keuls test.

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(C) and (D) LDH activity was determined using the LDH-P unitest commercial assay. LDH

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activity graphs are representative of three independent experiments performed in different

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cell preparations that showed comparable results. These bar plot graphs represent the

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mean ± SEM. (n = 3 replicates per treatment). Different letters denote a statistically

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significant difference between groups. p < 0.05. Student-Newman–Keuls test.

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Fig. 8. Concentration of PGD2 and expression of DP receptors in human testes.

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(A)

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hypospermatogenesis (H) or Sertoli cell only syndrome (SCO) were determined by

18

enzyme immunoassay. Bar plot graph represents the mean ± SEM (n = 4-5 testicular

19

biopsies per group).

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(B) Using the laser capture microdissection technique, androgen receptor (AR)-

21

immunoreactive Sertoli cells were isolated from the testis of a patient with SCO syndrome

22

and subjected to RT-PCR studies. Bar 40µm. (C) mRNA expression of 18S rRNA (109 bp)

23

and the PGD2 receptors DP1 (345 bp) and DP2 (138 bp) was detected by RT-PCR. Similar

24

results were obtained when biopsies from patients suffering from hypospermatogenesis

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were analyzed (data not shown).

concentrations

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Reactive oxygen species (ROS) production triggered by prostaglandin D2 (PGD2) regulates lactate dehydrogenase (LDH) expression/activity in TM4 Sertoli cells

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Soledad P. Rossia,b, Stefanie Windschuettlc, María E. Matzkina,b, Verónica Rey-Aresc, Claudio Terradasd, Roberto Ponzioe, Elisa Puigdomenechf, Oscar Levalled, Ricardo S. Calandraa, Artur Mayerhoferc, Mónica B. Frungieria,b

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a

Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas

y Técnicas, Vuelta de Obligado 2490 (1428), Ciudad de Buenos Aires, Argentina

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Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires,

Paraguay 2155 (1121), Ciudad de Buenos Aires, Argentina c

BioMedizinisches Centrum (BMC), Ludwig-Maximilians-University (LMU), Cell Biology, Anatomy

III, Großhaderner Str. 9, D-82152 Planegg, Germany, d

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División Endocrinología, Hospital Durand, Facultad de Medicina, Universidad de Buenos Aires,

Díaz Vélez 5044, 1405 Buenos Aires, Argentina e

Instituto de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos

f

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Aires, Paraguay 2155, 1121 Buenos Aires, Argentina Instituto Médico PREFER, Güemes 2348, 1650 San Martín, Provincia de Buenos Aires,

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Argentina

Highlights

PGD2 is a local modulator of the oxidant/antioxidant status in Sertoli cells. PGD2 is a stimulatory factor of ROS and H2O2 production in Sertoli cells. ROS/H2O2 are mediators of PGD2 effect on LDH expression/activity in Sertoli cells. PGD2 up-regulates LDH expression/activity via DP1/DP2 receptors in Sertoli cells. ROS and the Akt-CREB/ATF-1 pathway are proposed to partake in PGD2 action on LDH.