baburski2016

 Circadian rhythm of the leydig cells endocrine function is attenuated during
aging
Aleksandar Z. Baburski, Srdjan J. Sokanovic, Maja M. Bjelic, Sava M.
Radovic, Silvana A. Andric, Tatjana S. Kostic
PII:
DOI:
Reference:
S0531-5565(15)30080-2
doi: 10.1016/j.exger.2015.11.002
EXG 9729
To appear in:
Experimental Gerontology
Received date:
Revised date:
Accepted date:
23 May 2015
11 October 2015
3 November 2015
Please cite this article as: Baburski, Aleksandar Z., Sokanovic, Srdjan J., Bjelic, Maja M.,
Radovic, Sava M., Andric, Silvana A., Kostic, Tatjana S., Circadian rhythm of the leydig
cells endocrine function is attenuated during aging, Experimental Gerontology (2015), doi:
10.1016/j.exger.2015.11.002
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Title:
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Circadian rhythm of the Leydig cells endocrine function is attenuated during aging
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Aleksandar Z Baburski, Srdjan J Sokanovic, Maja M Bjelic, Sava M Radovic, Silvana A Andric, Tatjana S Kostic
LaRES, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Novi Sad, Serbia
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Running title: Aging & Leydig cell circadian rhythm
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Key words: Aging, Leydig cell, clock, testosterone, 24-hour rhythm
Tatjana S. Kostic,
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All correspondence to:
Department of Biology and Ecology
Faculty of Sciences,
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University of Novi Sad,
Dositeja Obradovica Sq 2
21000 Novi Sad
Serbia
Fax: +381 21 450 620
E-mail: [email protected]
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ABBREVIATIONS:
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Bmal1, brain-muscle Arnt-like protein 1;
BSA, bovine serum albumin;
cAMP, cyclic adenosine monophosphate;
Ck1, gene for casein kinase 1;
Clock/CLOCK, gene/protein for circadian locomotor output cycles kaput;
CRE, cAMP-responsive element;
Creb/CREB, gene/protein for cAMP response element-binding protein;
Cry/CRY, gene/protein for cryptochrome;
Cyp11a/CYP11a, gene/protein for cytochrome P450 side chain cleavage enzyme;
Cyp17a/CYP17a, gene/protein for 17α-hydroxylase/C17-20 lyase;
DBP, D site of albumin promote bind protein;
DHT, dihydrotestosterone;
DMEM/F12, Dulbecco's modified eagle medium;
Gapdh, gene for glyceraldehyde 3-phosphate dehydrogenase;
HDL, high density lipoprotein;
Hmgcr, gene for 3-hydroxy-3-methyl-glutaryl-CoA reductase;
Hsd17b, gene for hydroxysteroid dehydrogenase 17β;
Hsd3b, gene for hydroxysteroid dehydrogenase 3β;
Insl3/INSL3, gene/protein for insulin-like 3;
LDL, low density lipoprotein;
LH, luteinizing hormone;
Lhr/LHR, gene/protein for luteinizing hormone receptor;
Lipe, gene for Hormone sensitive lipase (HSL);
mo, month;
Nampt/NAMPT, Nicotinamide phosphoribosyltransferase;
Npas2, gene for Neuronal PAS domain-containing protein 2;
Nur77, gene for nerve growth factor;
Per1, gene for period circadian protein 1;
Per2, gene for period circadian protein 2;
Per3, gene for period circadian protein 3;
PKA, cAMP dependent protein kinase;
RIA, radioimmunoassay;
Rev-erba/b / REV-ERBA/B, gene/protein for reverse viral erythroblastis oncogene product alpha/beta;
Ror/ROR, gene/protein for retinoid acid related orphan receptor;
RQ-PCR, relative quantification polymerase chain reaction;
Scarb1, gene for Scavenger Receptor Class B, Member 1;
Sf1, gene for steroidogenic factor 1;
Sirt1/SIRT1, gene/protein for NAD-dependent protein deacetylase sirtuin-1;
Soat1, gene for Sterol O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 1;
Soat2, gene for Sterol O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 2;
Star/StAR, gene/protein for steroidogenic acute regulatory protein;
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T, testosterone;
Tspo, gene for translocator protein;
VLDL, very low density lipoprotein ;
ZT, Zeitgeber (German: “time giver”) time.
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Abstract
Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian
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nature has not been analyzed. Here we describe changes in circadian behavior in Leydig cells isolated from
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adult (3-month) and aged (18- and 24-month) rats. The results showed reduced circadian pattern of
testosterone secretion in both groups of aged rats despite unchanged LH circadian secretion. Although
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arrhythmic, the expression of Insl3, another secretory product of Leydig cells, was decreased in both groups.
Intracellular cAMP and most important steroidogenic genes (Star, Cyp11a1 and Cyp17a1), together with
positive steroidogenic regulator (Nur77), showed preserved circadian rhythm in aging although rhythm
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robustness and expression level were attenuated in both aged groups. Aging compromised cholesterol
mobilization and uptake by Leydig cells: the oscillatory transcription pattern of genes encoding HDL-receptor
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(Scarb1), hormone sensitive lipase (Lipe, enzyme that converts cholesterol esters from lipid droplets into
free cholesterol) and protein responsible for forming the cholesterol esters (Soat2) were flattened in 24month group. The majority of examined clock genes displayed circadian behavior in expression but only a
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few of them (Bmal1, Per1, Per2, Per3 and Rev-Erba) were reduced in 24-month-old group. Furthermore,
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aging reduced oscillatory expression pattern of Sirt1 and Nampt, genes encoding key enzymes that connect
cellular metabolism and circadian network. Altogether circadian amplitude of Leydig cell`s endocrine function
decreased during aging. The results suggest that clock genes are more resistant to aging than genes involved
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during aging.
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in steroidogenesis supporting the hypothesis about peripheral clock involvement in rhythm maintenance
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1. Introduction
In mammals, most physiological processes undergo daily oscillations that are controlled by an
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endogenous circadian timing system composed of a central clock in the suprachiasmatic nucleus that
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coordinates the subsidiary oscillators in peripheral tissues (Reppert and Weaver, 2001; Schibler and Sassone-
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Corsi, 2002; Yamamoto et al., 2004). The molecular machinery of this timing system, located in almost every
cell, is composed of a conservative interlocked transcriptional-translational feedback loop involving multiple
clock genes (Clock, Bmal1, Pers, and Crys) and their protein products that are required for generating
endogenous circadian oscillations (Albrecht, 2012). Other accessory proteins including the orphan receptors
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ROR and REV-ERBs and DBP constitute additional loops that make circadian oscillators robust and tunable
(Mohawk et al., 2012; Albrecht, 2012). In addition to regulating each other to sustain circadian oscillations,
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the core clock proteins (CLOCK, BMAL1, ROR, REV-ERBs, and DBP) also entrain rhythmic expression of
numerous genes through binding to an E-box, ROR response element (RORE), and D-box at their promoters.
The circadian clock appears to play a role in the aging process (Kondratov et al., 2006). With advancing age,
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animals exhibit numerous circadian disruptions (Benloucif et al., 1997; Davidson et al., 2008; Li and Satinoff,
that contribute to poor health
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1995; Valentinuzzi et al., 1997; Weinert and Waterhouse, 1999)
consequences and hastened senescence. For reasons yet unknown, there is a concomitant decline in
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circadian rhythms and the overall homeostasis of the organism. Reproductive function, in particular, is
profoundly influenced by aging. A number of studies showed a slow decline in serum total testosterone level
with aging even in the absence of disease (Harman et al., 2001; Mohr et al., 2005). Decline in serum
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testosterone is a consequence of the reduced ability of Leydig cells to produce testosterone in presence of
unchanged or elevated serum luteinizing hormone (LH) levels (Chen et al., 2002; Tajar et al., 2010). In
humans age-related decline in testosterone production Is routinely classified into those with secondary
hypogonadism (hypothalamic-pituitary failure) with low testosterone and low or normal gonadotropins or
primary hypogonadism (testicular failure) characterized by low testosterone and elevated gonadotropins
levels (Tajar et al., 2010; Surampudi et al., 2012; Zirkin and Tenover, 2012). In addition, clinical investigations
support a multisite impairment in aging, which includes decreased GnRH and LH pulse amplitude causing
decreased testosterone, and hence increased GnRH pulse frequency due to less negative feedback to the
hypothalamus ( Liu et al., 2005; Veldhuis et al., 2009).
A good parameter in assessing the functionality of Leydig cells is insulin-like factor 3 (Insl3/INSL3)
expression which appears to be down-regulated with age (Anand-Ivell et al., 2009; Sokanovic et al., 2014).
Leydig cells from aged rats have reduced LH-receptors number per cell, a reduced cAMP response to LH
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application (Chen et al., 2002; Lin et al., 1983), and compromised cholesterol transport mechanisms and
steroidogenic pathways (Culty et al., 2002).
In addition, testosterone secretion from Leydig cells has long been recognized to have a low-
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amplitude of circadian rhythm (Mock et al., 1978; Chacon et al., 2004) suggesting that Leydig cells could
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contain cell-autonomous circadian clock that measures 24 hours. It is shown that clock genes, Clock and
Bmal1, strongly influence reproductive competency (Alvarez et al., 2008; Liang et al., 2013; Hodžid et al.,
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2013) and that the circadian clock may control the timing and amplitude of testosterone synthesis in Leydig
cells (Alvarez et al., 2008). Mice deficient in the Bmal1 gene show early signs of aging and age-related
pathologies including reduced fertility (Khapre et al., 2011). Male Bmal1 KO mice had low testosterone and
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high LH serum concentrations, suggesting a defect in testicular Leydig cells (Alvarez et al., 2008).
The SIRT1, a NAD-dependent deacetylase, has been shown to be important for sustaining clock
network, cellular energy metabolism and aging (Bordone and Guarente, 2005). SIRT1 is implicated in
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circadian gene regulation by enhancing Bmal1 and Clock transcription (Fonseca Costa and Ripperger, 2015).
On the other hand SIRT1 could be interesting in study of Leydig cells since it is shown that male Sirt1-/- mice
level (Kolthur-Seetharam et al., 2009).
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has reduced expression of steroidogenic genes resulting in strongly reduced intratesticular testosterone
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However, circadian clock function and its way of regulation in testis are still not clear. Evidence on
rhythmic steroidogenic activity of Leydig cells and molecular mediators involved in their clock network
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during lifespan is missing. Here we analyzed rhythmic expression of clock and steroidogenesis-related genes
including the genes involved in cholesterol mobilization and trafficking in Leydig cells of aged rats. Agerelated alterations of analyzed genes were correlated with 24-h changes in testosterone production. The
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results support decline in circadian expression of both clock- and steroidogenesis-related genes while genes
involved in cholesterol mobilization and uptake lost circadian rhythm in old age. However, age-related
changes in steroidogenesis occurred earlier suggesting that this system is more susceptible to changes in
aging.
2. Material and methods
2.1. Ethical approval
All the experimental protocols were approved (I-2011-02) by the Ethics Committee on Animal Care
and Use at the University of Novi Sad, operating under the rules of National Council for Animal Welfare and
following statements of National Law for Animal Welfare (copyright March 2009). All our experiments were
performed and conducted in accordance with the National Research Council (NRC) publication Guide for the
Care and Use of Laboratory Animals (National Academy of Sciences, Washington DC, 1996) and NIH Guide
for the Care and Use of Laboratory Animals (NIH Publications, No. 80 23, revised, 7th ed., 1996). All the
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experiments were carried out in the Laboratory for Reproductive Endocrinology and Signaling, DBE, Faculty
of Sciences at University of Novi Sad.
2.2. Animals
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Male Wistar rats bred and raised in the animal facility of the Faculty of Sciences, University of Novi
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Sad (Novi Sad, Serbia) were used for the experiments. Animals were raised in controlled environmental
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conditions (22±2°C; 12 h light - 12 h dark cycle, lights on at 6:00 AM) and provided with food and water ad
libitum. Three groups were formed: (1) adult 3-month-old rats (18 animals); (2) aged 18-month-old rats (18
animals); (3) aged 24-month-old rats (18 animals). Animals were sacrificed in six time points during the 24
hours (ZT0, ZT5, ZT11, ZT16, ZT20 and ZT24, ZT0 - moment when light was turned on), 3 animals per time
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point for each group and experiments were repeated 3 times. Rats were quickly decapitated without
anesthesia (to avoid the effect of anesthesia on serum hormone levels), and the trunk blood was collected.
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Individual serum samples were stored at -80°C until hormone assay was performed. In vivo experiments
were repeated three times.
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2.3. Hormone and cyclic AMP measurements
Androgen level was measured by RIA and it was referred to testosterone + dihydro-testosterone (T +
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DHT) because the anti-testosterone serum No. 250 showed 100% cross-reactivity with DHT (Andric et al.,
2007; Kostic et al., 2010). All samples were measured in duplicate in one assay (sensitivity: 6 pg per tube;
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intra-assay coefficient of variation 5–8%). Androgen level in serum was assayed in 100 µL by direct RIA
(without extraction). Serum LH level was measured by RIA according to the manufacturer's protocol (Rat LH
RIA Kit; ALPCO Diagnostics); the minimum detectable concentration has been assayed at 0.14 ng/ml (intra-
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and interassay coefficients of variation, 8-12% and 7-22%, respectively)(Andric et al., 2010). Cyclic AMP was
extracted from cell content by ethanol using a procedure described previously (Kostic et al., 2001). Level of
intracellular cAMP was measured from Leydig cell’s content by the cAMP EIA Kit (Cayman, Ann Arbor, MI)
with a limit of quantification of 0.1 pmol/mL for acetylated samples.
2.4. Assay of lipid profile in serum
Profile of cholesterol, total cholesterol, HDL-cholesterol, nonHDL-cholesterol, triglyceride, LDL and VLDL,
were measured using commercial kits (Roche Diagnostic Basel, Switzerland) by analyzers (Roche
COBASIntegra®400 Plus System).
2.5. Leydig cells purification
For gene expression and cAMP level analyses we used LCs purified from testes obtained from 3mo,
18mo and 24mo rats prepared as described previously by our group (Andric et al., 2007; Kostic et al., 2010).
Briefly, testes were quickly removed, decapsulated, placed in medium 199 containing 0.25 mg/ml
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collagenase, 1.5% BSA, 20 mM HEPES, sodium bicarbonate and antibiotics and incubated in a shaking water
bath oscillating at 120 cycles/min at 34°C for 15 min. The dissociated cells were filtered through mesh no.
100 (Sigma) and the cell suspension was centrifuged at 160xg for 5 min at room temperature. The crude
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suspension of interstitial cells was applied to a Percoll gradient consisting of four 2-ml layers with densities
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of 1.090, 1.080, 1.065, and 1.045 g/ml and centrifuged at 1100xg for 28 min at room temperature. Leydig
cells were collected from 1.080/1.065 g/ml and 1.065/1.045 g/ml interfaces, washed and centrifuged at
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500xg for 7 min. Samples with LC’s pellet were stored at -80°C. The proportion of Leydig cells was
determined by staining for HSD3B activity and was found to be 92.5 ± 7.8%. The viability was tested by using
the 0.2% Trypan blue dye exclusion test (Sigma Inc.) which determined total cell counts and ensured that
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greater than 90% of the cells were viable.
2.6. RNA isolation and cDNA synthesis
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Total RNA from purified Leydig cells was isolated using the Rneasy kit reagent following the protocol
recommended by the manufacturer (QIAGEN, Valencia, California). After DNase I treatment, first-strand
cDNA was synthesized according to the manufacturer’s instructions (Invitrogen, Carlsbad, California).
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Negative controls consisting of nonreverse transcribed samples were included in each set of reactions.
group (Kostic et al., 2010).
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Quality of RNA and DNA integrity were determined using primers for Gapdh as described previously by our
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2.7. Real-time PCR and relative quantification
Relative gene expression was quantified by RQ-PCR using SYBR Green-based chemistry (Applied
Biosystems, Foster City, California) in the presence of a 5µL aliquot of the reverse transcription reaction
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product (25 ng RNA calculated on the starting RNA) and specific primers. The primer sequences used for RQPCR analysis and GenBank accession codes for full gene sequences (www.ncbi.nlm.nih.gov/sites/entrez) are
provided in Supplemental Table 2. Gapdh was also measured in the same samples and used to correct
variations in RNA content. The relative quantification of each gene was performed in duplicate and repeated
3 times for each gene.
2.8. Protein extraction and western blot analysis
Leydig cell’s pellets were washed twice with ice-cold PBS, lysed and western blot analysis was
performed (Andric et al., 2007; Kostic et al., 2010). The antisera for StAR protein was donation from
professor Douglas Stocco (dilution 1 : 1000, Clark et al., 1994); HSD3B was detected with antibody kindly
provided by Professor Ian Masson (dilution 1 : 1000; Bain et al., 1991); anti-BMAL1 purchased from Abcam,
(Cambridge, UK, #ab49421, dilution 1:800); anti-REVERBA from Santa Cruz Biotechnology, (Heidelberg,
Germany, #sc-135241, dilution 1 : 1000) and anti-ACTB from Santa Cruz Biotechnology, (Heidelberg,
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Germany, #sc-1616, dilution 1 : 1000). The integrated optical density was used to quantify both the area and
the intensity of the immunoreactive bands (Image J version 1.32).
2.7. Statistical and rhythm analysis
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The results represent group mean ± SEM values of three independent in vivo experiments. Rhythm
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parameters (p, Robustness, MESOR, Amplitude and Acrophase) were obtained by cosinor method using
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Cosinor program using 24h fitted period (http://www.circadian.org/softwar.html). Statistical significance
(p<0.05) between treated groups and control within the same time point was analyzed using Mann Whitney
test. Kruskal–Wallis one-way analysis of variance was used for determination of statistically significant
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differences between control (3-mo) and experimental (18-, 24-mo) groups.
3. Results
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Experiments were performed with 3-, 18- and 24-month-old Wistar rats. Although it has been shown
that aging of Wistar rats is often associated with spontaneous neoplasms mostly in endocrine and
integumentary system (Poteracki and Walsh, 1998), our results revealed that 12% of male aged 18 months
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and 35% of males aged 24 months had variable tumors. Most of these tumors were testicular and tumor-
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bearing animals were not included in the statistical analysis of in vivo experiments and were not used for in
vitro studies. In order to estimate rhythm of Leydig cells function in rats aged 3, 18 and 24 months we
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compared hormone secretion and transcription of genes in different time points during 24h.
3.1. Leydig cells endocrine rhythm was attenuated with aging
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Serum testosterone levels in all tested age groups showed a low-amplitude of diurnal rhythm.
However, aging reduced amplitude of testosterone oscillation (Fig. 1A) and gradually decreased rhythm
robustness of testosterone secretion (in 3-mo it was 57.4%, in 18-mo 36.0% and in 24-mo old rats 27.7%). In
all groups testosterone level reached peak around ZT11 and nadir around ZT23 (Fig. 1A, for properties of
circadian rhythm see Supplemental Table 1).
Serum LH levels in tested age groups also displayed 24-h oscillatory pattern. However, amplitude of
LH oscillation was unchanged (Fig. 1B, Supplemental Table 1).
Considering cAMP as a main stimulator of Leydig cells steroidogenic activity and potential
synchronizer of Leydig cell`s oscillator, the 24-h fluctuation of its content was measured. Similar to
testosterone, amount of cAMP in Leydig cells from all three groups showed 24-h oscillation but lower
amplitude in Leydig cells from 18- and 24-mo rats was registered (Fig. 1C). The acrophase/peak of cAMP
oscillation in Leydig cells happened a few hours before peak of testosterone secretion (Fig. 1C, Supplemental
Table 1).
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The transcription of Insl3, another endocrine product of Leydig cells, did not show 24-h variations
but was also attenuated in both aged groups (Fig. 1D). The reduced testosterone production along with
reduced Insl3 expression reflects dysfunction of aged Leydig cells.
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3.2. Aging attenuated the expression rhythm of the steroidogenesis-related genes in Leydig cells
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The effect of aging on 24-h oscillation in expression of genes involved in steroidogenesis was
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monitored by collecting RNA/protein samples using Leydig cells from all experimental groups at the
indicated time points and by performing RQ-PCR and western blot.
In all tested groups, the transcripts of key steroidogenic genes, Star, Cyp11a1 and Cyp17a1 showed
24-h oscillations with peak approximately at the middle of light phase (Fig. 2, Supplemental Table 1), i.e. a
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few hours before the peak of serum testosterone. At the age of 18- and 24-months the expression amplitude
and rhythm robustness of Star, Cyp11a, and Cyp17a in Leydig cells was reduced (Fig. 2, Supplemental Table
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1). Although, results did not support circadian oscillatory pattern in transcription of Lhr, Hsd3b1/2 and
Hsd17b4, aging significantly reduced their expression in Leydig cells of 18- and 24-mo old rats (Fig. 2,
Supplemental Table 1). However, at the protein level, StAR and HSD3B, showed circadian variation but with
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smaller amplitude in 24-mo-old rats comparing to 3-mo-old animals (Fig 4). Differently from the other
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examined genes, Tspo displayed higher transcription in 24-mo group comparing to the adult group (Fig. 2).
Further, the 24-h transcription patterns of main steroidogenic regulators were examined. Obtained
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results revealed that in Leydig cells of all tested groups steroidogenic transcriptional activator Nur77 showed
oscillatory profile with peak at ZT7 and bottom at ZT19 (Fig. 2, Supplemental Table 1). No significant
rhythmicity of main steroidogenic transcriptional activators, Sf1 (Fig. 2, Supplemental Table 1) and Creb1
(not shown) were noted. Aging reduced amplitude of rhythmic Nur77 and arrhythmic expression of Sf1 in 18-
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and 24-mo rats (Fig. 2, Supplemental Table 1).
3.3. Aging attenuated the expression of clock genes in Leydig cells
In order to follow effect of aging on peripheral clock in Leydig cells, analysis of canonical clock gene
expression was done (Fig. 3, Supplemental Table 1).
Majority of the examined clock genes (Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erba, Rev-erbb and
Rorb) had rhythmic expression in rat Leydig cells. In contrast Clock, Npas2, Rora (Fig. 3, Supplemental Table
1), Ck1e and Ck1d transcripts did not follow any significant 24-h pattern (Supplemental Table 1). Bmal1
expression showed rhythm with peak at the beginning of light phase (ZT3). Peak of Periods and
Cryptochromes (BMAL1-ClOCK negative regulators) was at the beginning of dark phase along with Bmal1
bottom. Negative regulators from secondary loop, Rev-erba, and Rev-erbb, had their peak at the end of the
light phase (Fig. 3, Supplemental Table 1).
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Only in Leydig cells isolated from 24-mo rats a decreased expression of clock genes (Bmal1, Per1,
Per2, and Rev-ebra) was registered but oscillatory pattern was preserved (Fig. 3, Supplemental Table 1).
Decreased protein level of BMAL1 and REVERBA in Leydig cells from 24-mo rats was confirmed by western
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blot analysis (Fig. 4). The aging did not affect Clock, Npas2, Rev-erbb, Cry1, Cry2, Rora and Rorb expression in
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Leydig cells (Fig. 3, Supplemental Table 1).
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3.4. Aging changed serum lipid profile and expression of genes involved in cholesterol metabolism in
Leydig cells
In attempt to better understand the age-related changes in testosterone production, some
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parameters of lipid and cholesterol metabolism in serum were analyzed. The results showed that serum
concentration of total cholesterol, very-low-density lipoprotein (VLDL) and triglycerides was quite stable
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during aging (Table 1). However, aging induces opposite changes in concentrations of high-density
lipoprotein (HDL) and low-density lipoprotein (LDL) in serum: HDL cholesterol was significantly decreased in
24-month-old group while LDL cholesterol was increased in 18- and 24-month old rats (Table 1). Among all
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analyzed lipid parameters in serum, only LDL cholesterol showed circadian oscillatory pattern (peak around
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ZT3 and bottom around ZT16) and this oscillation was preserved in aged groups (Table 1).
Further, we studied transcription level of genes involved in cholesterol metabolism in Leydig cells
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from rats of different age groups. Gene encoding a hormone sensitive lipase (Lipe), enzyme that converts
cholesterol esters from lipid droplets into free cholesterol, showed circadian rhythmicity in adult and 18-mo
group but that rhythmicity was lost in 24-mo group (Fig. 5, Supplemental Table 1). Leydig cells also expressed
transcripts of genes encoding the sterol O-acyltransferase 1 and 2 (Soat1 and Soat2) enzymes with HSL
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opposite activity. Transcription of Soat2 in adults showed circadian pattern but it was flattened and lost in
aged groups (Fig. 5, Supplemental Table 1). The Soat1 transcription was not circadian with no differences
during aging (Fig. 5, Supplemental Table 1). Gene encoding a HDL receptor, Scarb1, demonstrated circadian
transcription in adult group with peak around ZT12 while aging flattened and significantly decreased its
transcription (Fig. 5, Supplemental Table 1). Expression of the most important gene for de novo cholesterol
synthesis, Hmgcr, showed no changes through the process of aging (Fig. 5).
3.5. Expression of Sirt1 and Nampt was attenuated with aging
Considering SIRT1 as a key regulator that connects metabolism, circadian gene network and aging
we monitored its gene expression in Leydig cells from aged rats. Because function of SIRT1 absolutely
depends on NAD+ level we also monitored transcription of Nampt, encoding the enzyme that represents the
rate limiting enzymatic step in NAD+ salvage pathway (Nakahata et al., 2009; Ramsey et al., 2009). Results
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showed circadian expression of Sirt1 and Nampt and that rhythm of both genes were preserved during
aging. Both genes reached peak around ZT14. However, the mesor of Sirt1 transcription was lowered in 24-
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mo group while Nampt transcription was declined in 18- and 24-mo rats (Fig. 6).
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4. Discussion
Rat and human Leydig cells become hypofunctional and produce less testosterone during aging. This
is associated with reduced cAMP response to gonadotropin application (Chen et al., 2002; Lin et al., 1983;
Sokanovic et al., 2014), compromised cholesterol transport mechanisms illustrated by decreased StAR
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expression (Culty et al., 2002; Sokanovic et al., 2014) and reduced expression and activities of the
steroidogenic enzymes CYP11A, HSD3B, CYP17A, and HSD17B (Luo et al., 1996, 2005; Sokanovic et al., 2014).
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The first signs of compromised steroidogenic function of Leydig cells typically occurred at midway of life,
starting in rats from 12-mo and are further progressed and maintained at a low level from 15- to 24-mo
(Sokanovic et al., 2014).
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Although age-related hypofunction of Leydig cells is well illustrated across species, its circadian
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nature has not been analyzed. Here we describe circadian changes in the expression of genes involved in
steroidogenesis and clock genes in Leydig cells from adult and aged rats.
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The obtained results illustrate circadian organization of processes involved in Leydig cell
steroidogenesis: the peak of intracellular cAMP oscillation happened before the peak in transcription of key
steroidogenic elements (Star, Cyp11a1, Cyp17a1) and a few hours later testosterone secretion reached a
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peak. Aging affected this circadian organization by decreasing: rhythmic testosterone secretion together
with fluctuations of intracellular cAMP level; rhythm robustness and expression amplitude of main
steroidogenic elements Star, Cyp11a1, and Cyp17a1, and one of the main steroidogenic stimulator, Nur77.
These changes were associated with, although arrhythmic, reduced expression of Lhr, Hsd3b, Hsd17b and
most important transcriptional factor involved in regulation of steroidogenesis, Sf1 (Lavoie and King, 2009;
Sokanovic et al., 2014). Although Creb1 gene did not display any circadian pattern or changes in expression
level between the groups, it is very likely that the activity of the protein is under circadian control through its
phosphorylation (Gau et al., 2002), especially since cAMP displayed regular circadian rhythm in Leydig cells.
The circadian organization of Leydig cells` steroidogenesis was not significantly changed, although aging
reduced amplitude and/or messor of the expression of genes involved in cholesterol transformation to
testosterone.
Another question is whether the aging is also associated with dampened cholesterol metabolism
in other organs that limit the access to Leydig cells through the blood and/or with changed mobilization and
uptake of cholesterol by Leydig cells. In attempt to address this question we monitored (1) the level of
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transcripts for the key proteins involved in cholesterol metabolism of Leydig cells and (2) the lipid profile in
serum of control and aged rats during 24h. The results showed loss of circadian rhythmicity of Lipe (gene
encoding HSL), in Leydig cells from 24-mo group comparing to the control, suggesting that release of free
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cholesterol from cholesterol esters in lipid droplets is affected. Also, aging flattened and significantly
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decreased transcription of Soat2 (gene encoding protein responsible for forming the cholesterol esters and
VLDL) and Scarb1 (gene encoding a HDL receptor) in Leydig cells, suggesting that cholesterol metabolism and
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uptake are significantly dampen in Leydig cells from aged rats. In contrast to human steroidogenic cells
which mostly take up circulating LDL by receptor-mediated endocytosis directing the cholesterol to
endosomes, the rodent steroidogenic cells preferentially bind HDL via SCARB1, a multifunctional receptor
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that promotes the selective uptake of cholesteryl esters (Rone et al., 2009; Miller and Bose, 2011). SCARB1 is
not only essential for HDL cholesterol homeostasis and atherosclerosis susceptibility (Lorbek et al., 2015),
but also for maintaining lifespan of erythrocytes (Meurs et al., 2005). Accordingly, beside disturbed HDL
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cholesterol homeostasis, decreased Scarb1 transcription could eventually affect Leydig cells lifespan. On the
other hand, the analysis of lipid parameters in serum showed that aging induced opposite profile of HDL and
LDL with a significant decrease of HDL in serum from 24-month-old group and a significant increase of LDL in
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serum form 18- and 24-month old rats. This is in line with findings showing significant increase of LDL in
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serum of 22-month old rat (Helmy, 2012).
In addition to reduced testosterone production, aging reduced the expression of Insl3, another
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important marker of Leydig cell functionality. Even though testosterone and INSL3 are shown to be positively
regulated by LH, drop of LH or changes of rhythm parameters in aging were not detected. This observation is
in accordance with the findings that in rats LH secretion was not changed during aging (Midzak et al., 2009;
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Sokanovic et al., 2014).
Rhythmic activity of Leydig cells could be due to rhythmic activities of reproductive axis, but also the
consequences of clockwork in Leydig cells enabled by circadian expression of clock genes. Alvarez and
colleagues (2008) provide the evidence that the Bmal1 is necessary for proper testosterone production in
Leydig cells. In adrenocortical cells, an another type of steroidogenic cells, StAR transcription is regulated by
the BMAL1-CLOCK complex via the distal E box elements, and hence StAR can be regarded as a clock
controlled gene (Son et al., 2008). However, it is still unknown how peripheral clock in Leydig cells could
influence steroid production during Leydig cell aging. Our results showed that expression of Bmal1, Per1,
Per2 and Rev-Erba were reduced in 24-mo old rats but not in 18-mo old rats. This is in accordance with
findings that expression of core clock genes in peripheral tissues is preserved or slightly changed during
aging (Bonaconsa et al., 2014). The reduction in Bmal1 expression is associated with changes in Per1, Per2
and Rev-erba amplitude in the Leydig cells from aged rats, indicating downstream targets of Bmal1
responsible for circadian diminishing with advanced age. Consequently, the dampening of Per1, Per2 and
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Rev-erba expression could affect the rhythmic transcription of clock output genes. In addition, it is
documented that BMAL1 is important for the regulation of oxidative stress and DNA damage responses,
while deregulation of these processes upon BMAL1 deficiency leads to development of stress induced
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senescence in vivo (Khapre et al., 2011).
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DNA-binding activity of BMAL1-CLOCK is strongly influenced by the SIRT1 deacetylation (Nakahata et
al., 2008) which sense cellular metabolism using NAD+ as a coenzyme. The transcription of Nampt gene,
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encoding the rate limiting enzyme in NAD+ salvage pathway, is circadian, indicating circadian regulation of
NAD+ intracellular level of (Nakahata et al., 2009; Ramsey et al., 2009). That suggests the presence of
enzymatic feedback loop in which BMAL1-CLOCK could control its own activity by directing the oscillatory
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levels of NAD+ and recruiting a SIRT1 (Nakahata et al., 2009; Ramsey et al., 2009). In Leydig cells from aged
rats the expression of Sirt1 and Nampt decreased indicating possible involvement in expressional changes in
clock genes. On the other side, a reduced ratio of NAD+/NADH cofactors in aged Leydig cell could affect
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activity/expression of steroidogenic enzymes, especially HSD3B (Ohba et al., 1981).
In conclusion, intracellular cAMP and most important steroidogenic genes (Star, Cyp11 and Cyp17)
as well as clock genes showed circadian rhythmicity that is not cancelled in aging although robustness and
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expression level are attenuated. However, as age-related decrease of steroidogenic gene expression
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occurred earlier than clock genes, it seems that clock gene network is less affected by aging in Leydig cells.
The reduced rhythmic expression of Sirt1 and Nampt pointed to decreased cellular metabolism that, in turn,
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could affect clock network. Perhaps peripheral clock in Leydig cells could be at least partly responsible, along
with LH, for rhythm sustainability of steroidogenic genes despite lowered input signal.
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ACKNOWLEDGMENTS: We are very grateful to Professors Douglas Stocco (Texas Tech University) for
generous and continuous donation of StAR antiserum, prof Professor Ian Masson (University of Edinburgh)
for kind donation of HSD3B antisera and Gordon Niswender (Colorado State University) for supplying
antibodies for radioimmunoassay analysis. Also, we are thankful to Ljiljana Knezevic, PhD, for English
language editing and Ms Marica Jovic for technical assistance.
GRANTS: Supported by the Serbian Ministry of Science grant No. 173057 and the Autonomic Province of
Vojvodina grant No. 0970.
DISCLOSURE: The authors have nothing to disclose.
1. Albrecht U. (2012) Timing to perfection: the biology of central and peripheral circadian clocks.
Neuron. 74(2):246-60.
2. Alvarez JD, Hansen A, Ord T, Bebas P, Chappell PE, Giebultowicz JM, Williams C, Moss S, Sehgal A.
(2008) The circadian clock protein BMAL1 is necessary for fertility and proper testosterone
production in mice. J Biol Rhythms. 23(1):26-36.
14
ACCEPTED MANUSCRIPT
3. Anand-Ivell R, Heng K, Hafen B, Setchell B, Ivell R. (2009) Dynamics of INSL3 peptide expression in
the rodent testis. Biol Reprod. 81(3):480-7.
4. Andric SA, Janjic MM, Stojkov NJ, Kostic TS. (2007) Protein kinase G-mediated stimulation of basal
Leydig cell steroidogenesis. Am J Physiol Endocrinol Metab 293(5):E1399-408.
IP
T
5. Andric SA, Janjic MM, Stojkov NJ, Kostic TS. (2010) Testosterone-induced modulation of nitric oxidecGMP signaling pathway and androgenesis in the rat Leydig cells. Biol Reprod. 83(3):434-42.
SC
R
6. Bain PA, Yoo M, Clarke T, Hammond SH, Payne AH. (1991) Multiple forms of mouse 3 betahydroxysteroid dehydrogenase/delta 5-delta 4 isomerase and differential expression in gonads,
adrenal glands, liver, and kidney of both sexes. Proc Natl Acad Sci U S A. 88(20):8870-4.
7. Benloucif S, Masana MI, Dubocovich ML. (1997) Light-induced phase shifts of circadian activity
rhythms and immediate early gene expression in the suprachiasmatic nucleus are attenuated in old
C3H/HeN mice. Brain Res. 747(1):34-42.
NU
8. Bonaconsa M, Malpeli G, Montaruli A, Carandente F, Grassi-Zucconi G, Bentivoglio M. (2014)
Differential modulation of clock gene expression in the suprachiasmatic nucleus, liver and heart of
aged mice. Exp Gerontol. 55:70-9.
MA
9. Bordone L, Guarente L. (2005) Calorie restriction, SIRT1 and metabolism: understanding longevity.
Nat Rev Mol Cell Biol. (4):298-305.
D
10. Chacon F, Cano P, Jimenez V, Cardinali DP, Marcos A, Esquifino AI. (2004) 24-hour changes in
circulating prolactin, follicle-stimulating hormone, luteinizing hormone, and testosterone in young
male rats subjected to calorie restriction. Chronobiol Int. 21(3):393-404.
TE
11. Chen H, Hardy MP, Zirkin BR. (2002) Age-related decreases in Leydig cell testosterone production are
not restored by exposure to LH in vitro. Endocrinology. 143(5):1637–1642.
CE
P
12. Clark BJ, Wells J, King SR, Stocco DM. (1994) The purification, cloning, and expression of a novel
luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells.
Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem. 269(45):2831422.
AC
13. Culty M, Luo L, Yao ZX, Chen H, Papadopoulos V, Zirkin BR. (2002) Cholesterol transport, peripheral
benzodiazepine receptor, and steroidogenesis in aging Leydig cells. J Androl. 23(3):439–447.
14. Davidson AJ, Yamazaki S, Arble DM, Menaker M, Block GD. (2008) Resetting of central and peripheral
circadian oscillators in aged rats. Neurobiol Aging. 29(3):471-7.
15. Fonseca Costa SS and Ripperger JA. (2015) Impact of the Circadian Clock on the Aging Process. Front
Neurol. 2015; 6: 43.
16. Gau D, Lemberger T, von Gall C, Kretz O, Le Minh N, Gass P, Schmid W, Schibler U, Korf HW, Schütz
G. (2002) Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock.
Neuron. 34(2):245-53.
17. Harman SM, Metter EJ, Tobin JD, Pearson J, Blackman MR; Baltimore Longitudinal Study of Aging.
(2001) Longitudinal effects of aging on serum total and free testosterone levels in healthy men.
Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab. 86(2):724-31.
18. Helmy MM. (2012) Potential hepato-protective effect of a-tocopherol or simvastatin in aged rats.
Pharmacol Rep. 64(3):698-705.
19. Hodžid A, Ristanovid M, Zorn B, Tulid C, Maver A, Novakovid I, Peterlin B. (2013) Genetic variation in
circadian rhythm genes CLOCK and ARNTL as risk factor for male infertility. PLoS One. 8(3):e59220.
20. Khapre RV, Kondratova AA, Susova O, Kondratov RV. (2011) Circadian clock protein BMAL1 regulates
cellular senescence in vivo. Cell Cycle. 10(23):4162-9.
15
ACCEPTED MANUSCRIPT
21. Kolthur-Seetharam U, Teerds K, de Rooij DG, Wendling O, McBurney M, Sassone-Corsi P, Davidson I.
(2009) The histone deacetylase SIRT1 controls male fertility in mice through regulation of
hypothalamic-pituitary gonadotropin signaling. Biol Reprod. 80(2):384-91.
T
22. Kondratov RV, Kondratova AA, Gorbacheva VY, Vykhovanets OV, Antoch MP. (2006) Early aging and
age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock.
Genes Dev. 20(14):1868-73.
IP
23. Kostic TS, Andric SA, Stojilkovic SS. (2001) Spontaneous and receptor-controlled soluble guanylyl
cyclase activity in anterior pituitary cells. Mol Endocrinol. 15(6):1010-22.
Lavoie HA and King SR. (2009) Transcriptional regulation of steroidogenic genes: STARD1, CYP11A1
and HSD3B. Exp Biol Med (Maywood) 234:880-907.
NU
25.
SC
R
24. Kostic TS, Stojkov NJ, Janjic MM, Andric SA. (2010) Structural complexity of the testis and PKG I /
StAR interaction regulate the Leydig cell adaptive response to repeated immobilization stress. Int J
Androl. 33(5): 717-729.
MA
26. Li H and Satinoff E. (1995) Changes in circadian rhythms of body temperature and sleep in old rats.
Am Physiol Soc R208 –R214.
27. Liang X, Cheng S, and Jiang X, et al. (2013) The noncircadian function of the circadian Clock gene in
the regulation of male fertility. J Biol Rhythms 28(3):208-217.
28. Lin T, Vinson NE, Murono EP, Osterman J, Nankin HR. (1983) The aging Leydig cell. VIII. Protein
kinase activity. J Androl. 4(5):324-330.
TE
D
29. Liu PY, Liu PY, Takahashi PY, Roebuck PD, Iranmanesh A, Veldhuis JD. (2005) Age-specific changes in
the regulation of LH-dependent testosterone secretion: assessing responsiveness to varying
endogenous gonadotropin output in normal men. Am J Physiol Regul Integr Comp Physiol.
289:R721–728.
CE
P
30. Lorbek G, Perše M, Jeruc J, Juvan P, Gutierrez-Mariscal FM, Lewinska M, Gebhardt R, Keber R, Horvat
S, Bjӧrkhem I, Rozman D. (2015) Lessons from hepatocyte-specific Cyp51 knockout mice: impaired
cholesterol synthesis leads to oval cell-driven liver injury. Sci Rep. 5:8777 doi:10.1038/srep08777.
Luo L, Chen H, Zirkin BR. (1996) Are Leydig cell steroidogenic enzymes differentially regulated with
aging? J Androl. 17(5):509-515.
32.
Luo L, Chen H, Zirkin BR. (2005) Temporal relationships among testosterone production,
steroidogenic acute regulatory protein (StAR), and P450 side-chain cleavage enzyme (P450scc)
during Leydig cell aging. J Androl. 26(1):25-31.
33.
Meurs I, Hoekstra M, van Wanrooij EJ, Hildebrand RB, Kuiper J, Kuipers F, Hardeman MR, Van
Berkel TJ, Van Eck M. (2005) HDL cholesterol levels are an important factor for determining the
lifespan of erythrocytes. Exp Hematol. 33(11):1309-19.
AC
31.
34. Midzak AS, Chen H, Papadopoulos V, Zirkin BR. (2009) Leydig cell aging and the mechanisms of
reduced testosterone synthesis. Mol Cell Endocrinol. 299(1):23-31.
35. Miller WL and Bose, HS. (2011) Early steps in steroidogenesis: intracellular cholesterol trafficking. J
Lipid Res. 52(12):2111-35.
36. Mock EJ, Frankel AI. (1978) A shifting circannual rhythm in serum testosterone concentration in male
laboratory rats. Biol Reprod. 19(4):927-30.
37. Mohawk JA, Green CB, Takahashi JS. (2012) Central and peripheral circadian clocks in mammals.
Annu Rev Neurosci. 35:445-62.
16
ACCEPTED MANUSCRIPT
38. Mohr BA, Guay AT, O'Donnell AB, McKinlay JB. (2005) Normal, bound and nonbound testosterone
levels in normally ageing men: results from the Massachusetts Male Ageing Study. Clin Endocrinol
(Oxf). 62(1):64-73.
T
39. Nakahata Y, Kaluzova M, Grimaldi B, Sahar S, Hirayama J, Chen D, Guarente LP, Sassone-Corsi P.
(2008) The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling
and circadian control. Cell. 134(2):329-40.
IP
40. Nakahata Y, Sahar S, Astarita G, Kaluzova M, Sassone-Corsi P. (2009) Circadian control of the NAD+
salvage pathway by CLOCK-SIRT1. Science. 324(5927):654-7.
SC
R
41. Ohba H, Inano H, Tamaoki B. (1981) Contribution of microsomal cytochrome b5 electron transport
system coupled with delta 5-3 beta-hydroxysteroid dehydrogenase to androgen synthesis from
progesterone. Biochem Biophys Res Commun. 103(4):1273-80.
NU
42. Poteracki J and Walsh KM (1998) Spontaneous neoplasms in control Wistar rats: A comparison of
reviews. Toxicological Sciences. 45:1-8.
MA
43. Ramsey KM, Yoshino J, Brace CS, Abrassart D, Kobayashi Y, Marcheva B, Hong HK, Chong JL, Buhr ED,
Lee C, Takahashi JS, Imai S, Bass J. (2009) Circadian clock feedback cycle through NAMPT-mediated
NAD+ biosynthesis. Science. 324(5927):651-4.
44. Reppert SM and Weaver DR. (2001) Molecular analysis of mammalian circadian rhythms. Annu Rev
Physiol. 63:647-76.
TE
D
45. Rone MB, Fan J, Papadopoulos V. (2009) Cholesterol transport in steroid biosynthesis: role of
protein-protein interactions and implications in disease states. Biochem Biophys Acta. 1791(7):64658.
46. Schibler U and Sassone-Corsi P. (2002) A web of circadian pacemakers. Cell. 111(7):919-22.
Sok
anovic SJ, Janjic MM, Stojkov NJ, Baburski AZ, Bjelic MM, Andric SA, Kostic TS. (2014) Age related
changes of cAMP and MAPK signaling in Leydig cells of Wistar rats. Exp Gerontol. 58:19-29.
CE
P
47.
AC
48. Son GH, Chung S, and Choe HK, et al. (2008) Adrenal peripheral clock controls the autonomous
circadian rhythm of glucocorticoid by causing rhythmic steroid production. Proc Natl Acad Sci U S A
105(52):20970-20975.
49. Surampudi PN, Wang C, Swerdloff R. (2012) Hypogonadism in the aging male diagnosis, potential
benefits, and risks of testosterone replacement therapy. Int J Endocrinol. 2012:625434. doi:
10.1155/2012/625434.
50. Tajar A, Forti G, O'Neill TW, et al. (2010) Characteristics of secondary, primary, and compensated
hypogonadism in aging men: evidence from the European Male Ageing Study. J Clin Endocrinol
Metab. 95(4):1810-8.
51. Valentinuzzi VS, Scarbrough K, Takahashi JS, Turek FW. (1997) Effects of aging on the circadian
rhythm of wheel-running activity in C57BL/6 mice. Am J Physiol. 273(6 Pt 2):R1957-64.
52. Veldhuis JD, Keenan DM, Liu PY, Iranmanesh A, Takahashi PY, Nehra AX. (2009) The aging male
hypothalamic-pituitary-gonadal axis: Pulsatility and feedback. Mol Cell Endocrinol. 299(1):14-22.
53. Weinert D, Waterhouse J. (1999) Daily activity and body temperature rhythms do not change
simultaneously with age in laboratory mice. Physiol Behav. 66(4):605-12.
54. Yamamoto T, Nakahata Y, Soma H, Akashi M, Mamine T, Takumi T. (2004) Transcriptional oscillation
of canonical clock genes in mouse peripheral tissues. BMC Mol Biol. 5:18.
55. Zirkin BR and Tenover JL. (2012) Aging and declining testosterone: past, present, and hopes for the
future. J Androl. 33(6):1111-1118.
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Figure legends
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Figure 1. Aging influenced circadian rhythm of Leydig cells endocrine function. 3-, 18- and 24-month-old
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Wistar rats were euthanized in six time points during the 24h (ZT0, ZT5, ZT11, ZT16, ZT20 and ZT24) and
serum was collected for determination of androgen (T+DHT) (A) and LH levels (B). Leydig cells were purified
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and intracellular content was collected for cAMP measurements (C) as well as used as a source of mRNA for
RQ-PCR in presence of primers specific for Insl3 (D). Data points are group mean ± SEM values of 9 rats (for
A, B, C) and n=3 (for D). In this and following figures, the best fitting curves are composed by Cosinor
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method. For rhythm parameters (Robustness, MESOR, Amplitude and Acrophase) please see Suppl. Table 1.
* Statistical significance between 3- and 18-month old rat groups for the same time point (p < 0.05). #
Statistical significance between 3- and 24-month old rat groups for the same time point (p < 0.05). ZT in this
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and all other figures is Zeitgeber time.
Figure 2. Aging changed mesor and/or amplitude of rhythmic transcription of some steroidogenic related
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genes. Leydig cells obtained from 3-, 18- and 24-month-old rats were isolated at different time points and
used as a source of mRNA for RQ-PCR in presence of primers specific for Lhr and the most important
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steroidogenic genes: Star, Tspo, Cyp11a1, Hsd3b1/2, Cyp17a1, Hsd17b4 as well as steroidogenic regulators
Nur77 and Sf1. Data points are group mean ± SEM values of three independent experiments, n=3. For
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rhythm parameters (Robustness, MESOR, Amplitude and Acrophase) please see Suppl. Table 1. * Statistical
significance between 3- and 18-month old rat groups for the same time point (p < 0.05). # Statistical
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significance between 3- and 24-month old rat groups for the same time point (p < 0.05).
Figure 3. Aging changed mesor and/or amplitude of rhythmic transcription of some core clock genes.
Leydig cells obtained from 3-, 18- and 24-month-old rats were isolated at different time points and used as a
source of mRNA for RQ-PCR in presence of primers specific for Bmal1, Clock, Npas2, Per1, Per2, Per3, Cry1,
Cry2, Rev-Erba, Rev-Erbb, Rora and Rorb. Data points are group mean ± SEM values of three independent
experiments, n=3. For rhythm parameters (Robustness, MESOR, Amplitude and Acrophase) please see Suppl.
Table 1. # Statistical significance between 3- and 24-month old rat groups for the same time point (p < 0.05).
Figure 4. Aging changed expression pattern for StAR, HSD3B, BMAL1 and REVERBA in Leydig cells.
Protein level of StAR, HSD3B, BMAL1 and REVERBA were determined by western blot. Representative blots
are shown. Bars above blots represent mean ± SEM values from pooled data using scanning densitometry
values normalized on ACTB. # Statistical significance between 3-month and 24-month old rats for the same
time point (p < 0.05).
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Figure 5. Aging changed expression of genes involved in cholesterol metabolism in Leydig cells
Leydig cells obtained from 3-, 18- and 24-month-old rats were isolated at different time points and used as a
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source of mRNA for RQ-PCR in presence of primers specific for genes involved in cholesterol metabolism:
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Lipe, Soat1, Soat2, Scarb1 and Hmgcr. Data points are group mean ± SEM values of three independent
experiments, n=3. For rhythm parameters (Robustness, MESOR, Amplitude and Acrophase) please see Suppl.
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Table 1. * Statistical significance between 3- and 18-month old rat groups for the same time point (p < 0.05).
# Statistical significance between 3- and 24-month old rat groups for the same time point (p < 0.05).
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Figure 6. Aging changed mesor and/or amplitude of Sirt1 and Nampt rhythmic transcription. Leydig cells
obtained from 3-, 18- and 24-month-old rats were isolated at different time points and used as a source of
mRNA for RQ-PCR in presence of primers specific for Sirt1 and Nampt. Data points are group mean ± SEM
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values of three independent experiments, n=3. For rhythm parameters (Robustness, MESOR, Amplitude and
Acrophase) please see Suppl. Table 1. # Statistical significance between 3- and 24-month old rat groups for
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the same time point (p < 0.05).
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Table 1. Age related changes in serum lipid status
24 month
old rats
TRIGLYC
(mmol/l)
LDL
Cholesterol
(mmol/l)
VLDL
Cholesterol
(mmol/l)
0.84 ± 0.17
0.87 ± 0.15
0.83 ± 0.18
0.70 ± 0.10
0.77 ± 0.15
0.87 ± 0.17
0.86 ± 0.16
0.91 ± 0.20
0.81 ± 0.15
0.81 ± 0.17
0.94 ± 0.18
1.01 ± 0.18
1.05 ± 0.19
0.94 ± 0.16
0.81 ± 0.15
0.62 ± 0.16
0.81 ± 0.14
0.82 ± 0.15
0.47 ± 0.08
0.42 ± 0.07
0.46 ± 0.09
0.44 ± 0.08
0.50 ± 0.11
0.45 ± 0.08
0.41 ± 0.07
0.46 ± 0.08
0.41 ± 0.07
0.48 ± 0.08
0.54 ± 0.09
0.55 ± 0.10
0.21 ± 0.07#
0.22 ± 0.06#
0.14 ± 0.03#
0.14 ± 0.04#
0.19 ± 0.05#
0.14 ± 0.03#
0.37 ± 0.06
0.45 ± 0.08
0.37 ± 0.06
0.27 ± 0.05*
0.27 ± 0.04*
0.42 ± 0.08
0.46 ± 0.08
0.45 ± 0.08
0.40 ± 0.09
0.33 ± 0.01*
0.40 ± 0.07
0.45 ± 0.08
0.73 ± 0.13#
0.72 ± 0.10#
0.66 ± 0.09#
0.46 ± 0.08#*
0.61 ± 0.09#
0.68 ± 0.08#
0.58 ± 0.11
0.59 ± 0.11
0.70 ± 0.14
0.53 ± 0.10
0.76 ± 0.12
0.82 ± 0.15
0.52 ± 0.09
0.39 ± 0.08
0.45 ± 0.08
0.55 ± 0.11
0.59 ± 0.13
0.53 ± 0.15
0.66 ± 0.10
0.70 ± 0.14
0.61 ± 0.12
0.41 ± 0.11*
0.52 ± 0.11
0.66 ± 0.13
0.11 ± 0.02
0.18 ± 0.03
0.05 ± 0.01
0.03 ± 0.01*
0.04 ± 0.01
0.09 ± 0.01
0.22 ± 0.04#
0.27 ± 0.05#
0.20 ± 0.05#
0.08 ± 0.02#*
0.13 ± 0.04#
0.21 ± 0.05#
0.43 ± 0.08#
0.40 ± 0.07#
0.38 ± 0.07#
0.28 ± 0.05#*
0.37 ± 0.08#
0.40 ± 0.09#
0.26 ± 0.05
0.27 ± 0.05
0.32 ± 0.06
0.24 ± 0.05
0.35 ± 0.08
0.37 ± 0.08
0.24 ± 0.05
0.18 ± 0.04
0.20 ± 0.05
0.25 ± 0.06
0.27 ± 0.06
0.24 ± 0.05
0.30 ± 0.06
0.32 ± 0.07
0.28 ± 0.05
0.19 ± 0.04*
0.24 ± 0.05
0.30 ± 0.05
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* p<0.05 vs. corresponding ZT0; # * p<0.05 vs. 3 month old rats.
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non HDL
(mmol/l)
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18 month
old rats
HDL
Cholesterol
(mmol/l)
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3 month
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ZT0
ZT5
ZT11
ZT16
ZT20
ZT24
ZT0
ZT5
ZT11
ZT16
ZT20
ZT24
ZT0
ZT5
ZT11
ZT16
ZT20
ZT24
CHOLEST
(mmol/l)
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Highlights
Aging lowered circadian serum testosterone variations
Aging decline circadian expression of key steroidogenic and clock genes
Cholesterol metabolism and uptake are dampened in Leydig cells from aged rats
During aging the changes in steroidogenesis precede changes in clock network
Reduced circadian expression of Nampt/Sirt1 pointed decreased cellular metabolism
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