Role of AA-NAT and TPH 1 in the Ghrelin-Dependent Regulation of Melatonin Secretion in Sheep during Different Seasons : An In Vitro Study

Several studies suggests that ghrelin (GHRL) has neurobiological effects that extend beyond control of food intake. Our previous results confirmed that GHRL modulates the secretory activity of the pineal gland (PG) through nocturnal melatonin (MEL) secretion in sheep, the seasonally reproductive animals. Here we investigated the effects of GHRL (10 ng/ml) on the expression of enzymes limiting synthesis of MEL, including tryptophan 5-hydroxylase 1 (TPH1), serotonin N-acetyltransferase (AA-NAT) and its phosphorylated form p31T-AA-NAT in sheep PG explants (n = 72) during the 4-hour incubation in a gas-liquid interface, at a short (SD) and long (LD) photoperiods. After each hour of incubation selected explants were frozen in liquid nitrogen and stored at -80°C for subsequent analysis (real-time PCR, western-blotting, ELISA). Results show that GHRL regulates nightly MEL secretion in a TPH1-independent manner. The factor modulating GHRL activity was photoperiod. During SD photoperiod GHRL significantly reduced the expression of p31T-AA-NAT, AA-NAT and inhibited MEL secretion from PG explants. Whereas, during LD photoperiod no effect of GHRL on MEL secretion and expression of examined enzymes was noted. Studies indicate that GHRL directly affects PG under in vitro conditions and causes MEL secretion in animals which exhibit seasonality in reproductive and metabolic processes.


Introduction
Ghrelin (GHRL), which is mainly produced in the stomach, is one of the most important hormones engaged in maintaining the energy balance of the body at a constant level.A preprandial increase in GHRL secretion activates mechanisms that counter energy deficiencies, stimulating food searching and intake [1].The circadian system also plays a pivotal role in determining plasma GHRL levels by fine-tuning the timing of GHRL release throughout the 24hour cycle, as has been shown in humans and rodents [2].Studies with a GHRL analogue (GHRP-6) have confirmed the influence of GHRL on neuron activity in the mammalian "master clock" (suprachiasmatic nuclei, SCN) and period circadian clock 2 gene expression [3].Electron microscopic analysis has revealed that the SCN and intergeniculate leaflet efferents establish symmetrical synaptic contacts on GHRL-immunoreactive cell bodies and dendrites [4].In seasonal animals, such as sheep [5][6][7][8][9][10], the blue fox (Alopex lagopus) [11], the raccoon dog (Nyctereutes procyonoides) [12] and the golden-mantled ground squirrel (Spermophilus lateralis) [13], season-dependent changes in the secretion and endocrine activity of GHRL have also been observed.
It is a relatively recent finding that GHRL in sheep is involved in the regulation of pineal gland (PG) activity [6,8].The PG, together with the retina and the SCN, constitutes a part of the central system that controls the rhythms of physiological processes.The mammalian PG disseminates information about circadian time to peripheral tissues through the synthesis and release of melatonin (MEL), which is restricted to nighttime.The initiation of MEL synthesis begins within the well-known sympathetic pathways [14].MEL is formed by a four-stage reaction.The first two stages are associated with the conversion of tryptophan into serotonin (5-HT), which undergoes acetylation by serotonin N-acetyltransferase (AA-NAT) into N-acetylserotonin (NAS).Then, NAS is methylated by hydroxyindole O-methyltransferase (HIOMT) to generate the final biologically functional N-acetyl-5-methoxytryptamine [14].
Intracerebroventricular infusions of GHRL have been shown to significantly alter nightly MEL secretion in sheep depending on the season [8].In vitro studies of PG sheep explants [6] and rat pinealocytes [15] have confirmed the direct effect of GHRL or its analogues on the PG and the GHRL receptor (growth hormone secretagogue receptor type 1a; GHSR-1a) has been identified in goldfish pinealocytes [16].However, the mechanism through which GHRL acts on the biological clock of seasonal mammals, such as sheep, including its effects on the regulation of MEL biosynthesis and secretion, has not been well explained.
In studies on sheep [17] and rat [18] PGs, in vitro MEL synthesis was found to require de novo 5-HT synthesis, suggesting an essential role of tryptophan 5-hydroxylase 1 (TPH1) in that process.Therefore, the first objective of our study was to evaluate the action of GHRL on TPH1 mRNA expression and protein concentration in ovine PG explants.The second objective was to determine the role of GHRL in the nightly changes in phosphorylated AA-NAT (p31T-AA-NAT) protein expression and concentration in the PG of sheep.The posttranscriptional control of this enzyme plays a dominant role in regulating MEL synthesis in sheep [19,20], cattle [21,22] and primates [23].Phosphorylated AA-NAT binds to 14-3-3 proteins to maintain its catalytic activity.The last objective of the study was to resolve how GHRL-induced changes in TPH1 and AA-NAT expression levels affect the nighttime profile of MEL secretion from ovine PG explants.Considering the seasonal dependence of changes in the concentration and activity of GHRL and MEL in sheep [5,[8][9][10]24], all experiments were carried out during the short-day (SD) and long-day (LD) photoperiods testing the hypothesis that the influence of GHRL on the PG is related to the time of year.The results of the present study will improve our understanding of the PG as a potential site to integrate metabolic signals sparked by feeding with light information, producing the most favorable circadian rhythm and circannual physiological and behavioral activities under prevalent environmental conditions.

GHRL increases TPH1 mRNA expression during the SD season but has no effect on TPH1 protein concentration in ovine PG explants
To clarify the potential mechanisms of action of GHRL on PG activity at night, we investigated whether GHRL affects TPH1 mRNA expression and protein concentration in ovine PG explants collected during different seasons.We performed real-time PCR and enzyme-linked immunosorbent assays (ELISAs) to determine how TPH1 transcript and protein levels in PG explants incubated with GHRL change at various time intervals (1, 2, 3 and 4 hrs).As shown in Figure 1A, the effect of GHRL on TPH1 mRNA expression in ovine PG explants was dependent on the season.During the SD season, TPH1 transcript levels were significantly higher in the GHRL-treated PGs than in the control PG explants (P <0.01).During the LD season, TPH1 mRNA levels were similar in both control explants and samples incubated in the presence of GHRL (Figure 1A).However, after analyzing the time profile of the experiment, the effect of GHRL was found to be dependent on the duration of incubation.During the SD season, we noticed a stimulatory effect of GHRL on TPH1 mRNA expression levels in explants after 2 and 4 hrs of incubation (Figure 1B).During the LD season, GHRL was observed to initially, at 1 hr of incubation, significantly reduce TPH1 mRNA expression (P <0.001).However, after this time point, we did not observe any significant differences in TPH1 transcript levels relative to the control samples (Figure 1C).There were no significant differences in the basal endogenous TPH1 expression levels of PGs isolated during the SD and LD seasons (Figure 1D).
As shown in Figure 2A, there were no significant (P > 0.05) effects of GHRL on TPH1 protein concentrations compared with concentrations in the control group in both seasons.
However, within the GHRL group, the mean TPH1 concentration was greater (P < 0.05) for PG explants collected from ewes during the SD season than for those collected from ewes during the LD season (Figure 2A).The time charts (Figures 2B and 2C) show a decrease (P< 0.05) in TPH1 protein concentration in comparison with concentrations in the control groups after incubating PG explants with GHRL for 1 hr.This effect was similar during the SD (Figure 2B) and LD (Figure 2C) seasons.For the remaining incubation time intervals, there was no effect of GHRL (P > 0.05) on the levels of examined proteins (Figures 2B and 2C).

2.2.The effect of GHRL on p31T-AA-NAT protein expression and AA-NAT protein concentration in ovine PG explants is dependent on the season
To further understand the molecular mechanism of nocturnal MEL synthesis, we analyzed p31T-AA-NAT protein expression via western blotting and measured AA-NAT protein concentrations via ELISA in ovine PGs collected during different photoperiods and incubated with GHRL for 1, 2, 3 and 4 hrs.The immunoblot analysis demonstrated that p31T-AA-NAT protein levels in the PG explants treated with GHRL for 3 and 4 hrs were lower (P < 0.05 and P < 0,01, respectively) than those in the control PG explants collected from sheep during the SD season (Figure 3A).Compared with expression in the controls, a decrease (P < 0.05) in p31T-AA-NAT protein expression was observed after 4 hrs of GHRL exposure for PG explants obtained from ewes in the LD season (Figure 3B).Within the control group, the mean AA-NAT concentration was greater (P < 0.01) for PG explants collected during the SD season than for those collected during the LD season (Figure 4A).GHRL treatments decreased (P < 0.01) AA-NAT concentrations in the PG explants obtained from sheep in the SD season compared with concentrations in the untreated controls (Figure 4A).For PGs collected from sheep during the SD season, AA-NAT protein concentrations in PG explants treated with GHRL for 1, 2, and 3 hrs were decreased (P < 0.01) compared with concentrations in the controls (Figure 4B).In contrast, there was no effect of GHRL (P > 0.05) on AA-NAT concentrations in the PG explants collected from ewes during the LD season compared with concentrations in the untreated controls (Figure 4C).

2.3.GHRL decreases MEL concentration in the culture medium during the SD season but has no effect on MEL secretion during the LD season
To determine whether the exogenous GHRL is involved in the nightly regulation of PG activity, we measured the concentration of MEL secreted into the culture medium using radioimmunoassay (RIA).Within the control group, the mean MEL concentration was greater (P < 0.05) for PG explants collected during the SD season than for those collected during the LD season (Figure 5).GHRL treatment decreased (P < 0.001) MEL concentrations in the cultures of PG explants obtained from ewes in the SD season compared with concentrations in the controls.
A decline (P < 0.05) in MEL secretion in PG explants collected from sheep during the SD season was observed within the GHRL treatment group (Figure 5).

Discussion
Our results confirm the direct effect of GHRL on the secretory activity of the PG and the process of MEL biosynthesis in sheep.Ghrelin inhibited PG hormone secretion, melatonin, and its effect was observed during the SD photoperiod.Differences in MEL secretion have also been reported during in vitro study of ovine pineal explants treated with GHRL, regardless of the applied dose [6] and in in vivo study after infusions of GHRL into the third ventricle of sheep brain at or after the sunset [8].However, in rats no changes were observed in either MEL concentration or AA-NAT and HIOMT activity levels in the PG after the subcutaneous injection of GHRL before the middle of the dark phase [25].The aforementioned studies suggest that the species, route of administration of GHRL and the pathway through which GHRL acts may be important in the regulation of PG activity.
The further factor modulating action of GHRL on PG activity has been shown to be a photoperiod.During an SD, GHRL inhibited MEL secretion and had no significant effect on MEL concentration during an LD.In GHRL-treated explants the level of TPH1 transcripts increased during an SD, whereas it did not change during an LD.These differences were not reflected in the protein concentration of TPH1.An increase in TPH1 concentration was only observed within the group of GHRL-treated PG explants during the SD season compared with concentrations during the LD season.The results suggest that TPH1 may not play a key role in the GHRL-dependent regulation of MEL secretion in sheep.Because neither the TPH1 mRNA expression profile nor the TPH1 protein concentration profile correspond to the previously described changes in MEL secretion from the PG, it cannot be determined which mechanism of TPH1 regulation (transcriptional or posttranscriptional) is predominant in MEL biosynthesis in sheep.The results of Privat et al. [17] and Namboodiri et al. [26] partially confirmed those findings.They showed that the required TPH1 mRNAs and proteins are available at the beginning of the dark period in both the PG in sheep and in cultured pinealocytes.The fast availability of TPH1 decides about the rapid (~30 min) increase in MEL secretion after light removal in sheep [26], while several hours are required in the case of rats, possibly reflecting the time necessary for de novo synthesis and accumulation of TPH1 and AA-NAT messengers [27].Huang et al. [28] also suggest that, in rats, pineal TPH1 protein expression displays marked diurnal rhythms, and cAMP-dependent TPH1 phosphorylation is responsible for daily changes in 5-HT availability, on which further stages of MEL biosynthesis are dependent.
The GHRL-induced increase in TPH1 mRNA expression during the SD season is surprising in the context of the concurrent inhibitory effect of GHRL on MEL secretion.After the theory of a constitutive level of 5-HT existing in the PG was undermined, TPH1 has been considered to be the main enzyme determining the availability of the substrate (5-HT) for MEL production [29].Data of in vivo experiments in sheep indicated that administration of TPH1 caused an increase in the levels of 5-HT, NAS and MEL in the PG, as well as MEL in the blood.
However, the activity of AA-NAT and HIOMT did not change, indicating that the augmentation in MEL was due to the increase 5-HT acetylation resulting from the rise in 5-HT concentration [26].Privat et al. [17] showed, that while working on in vitro culture of ovine pinealocytes an inhibitor of TPH1 activity (parachlorophenylalanine, pCPA) almost totally suppressed the induction of MEL synthesis.Similarly, Miguez et al. [18] observed the inhibition of MEL secretion from rat pinealocytes in in vitro conditions after supplementation with culture medium with pCPA added.
Ghrelin-induced changes in the level of TPH1 transcripts may indicate that there is a relationship between GHRL and the serotoninergic system at the level of the PG that, however, it does not directly affect MEL secretion.So far, such interactions have been demonstrated in the hypothalamus [30], hippocampus [31], dorsal raphe and amygdala [32].They primarily involve the regulation of energy homeostasis, emotional behavior and the ability to remember and learn.
In these nerve centers, GHRL inhibits [30,31] or boosts [32]  involving GHSR-1a and the 5-HT receptor 5-HT2C was identified in the hypothalamus and hippocampus.Moreover, experiments on rodents [35] and ruminants [36] have shown that 5-HT2C and/or 5-HT2B receptors are directly involved in regulating MEL biosynthesis and secretion.In light of those results a further analysis is needed to determine the effect of GHRL on the level of expression of 5-HT, its metabolites and its receptors in the PGs of sheep.
Regulation of the biosynthesis and secretion of MEL from the PG is a complex process, primarily due to species differences.Between species such as rodents and sheep or cattle and primates, there are differences in the specific way norepinephrine affects α-and β-adrenergic receptors, as well as in the activation of an intracellular regulatory pathway dependent on cAMP, cGMP and Ca 2+ [37].However, the most attention has been paid to the species-specific regulation of AA-NAT synthesis.The nocturnal elevation of AA-NAT activity is dependent on AA-NAT gene transcription [38] and the role of the RNA-binding protein heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AA-NAT in rats [39].However, in sheep [14,19,20], cattle [21] and humans [14], it mainly depends on changes in AA-NAT phosphorylation and post-translational modifications of the AA-NAT protein, such as proteasomal degradation.In present study, GHRL decreases p31T-AA-NAT expression and AA-NAT protein concentration in the ovine PG, and the effect of its activity was the strongest during the SD season.A decrease in AA-NAT protein phosphorylation at threonine-31 of the Nterminus of the polypeptide chain is important for the biosynthesis and secretion of MEL in sheep [19] and primates [23].Phosphorylated AA-NAT together with 14-3-3 proteins forms a complex in which AA-NAT retains its catalytic activity and is protected against dephosphorylation and enzymatic degradation.Light causes the immediate destruction of this complex and proteolytic degradation of AA-NAT, resulting in a immediate suppression of MEL secretion in sheep [40].Present studies indicate that the decrease in AA-NAT protein concentration and p31T-AA-NAT expression after GHRH treatment corresponded to a simultaneous reduction in MEL secretion from the PG.This result reveals that GHRL regulates the secretory activity of the PG of sheep by influencing AA-NAT protein synthesis.The next step will be to examine the impact of GHRL on the c-AMP-dependent, protein kinase Amediated phosphorylation mechanism involving 14-3-3 proteins.
Ghrelin may affect the PG through several pathways, including via the SCN, which is reached by efferent nerve fibers from the arcuate, paraventricular and dorsomedial hypothalamic nuclei, where GHRL and its receptor have been identified [1, 41,42].Information from the abovementioned centers is amplified and synchronized in the SCN with light, which enters through the retinohypothalamic tract, and then, "ready nerve instructions" are sent via multineuronal tracks from the SCN to the PG [1].In the second case, the GHRL produced in the central nervous system (CNS) or in the periphery reaches the PG through the cerebrospinal fluid (CSF).The PG is in direct contact with the CSF due to the incomplete ependymal lining in the pineal recess, an extension of the third ventricle [43].Studies carried out on sheep [29], humans [44] and rodents [45] have confirmed the presence of GHRL in the CSF, through which, it is able to reach most of the forebrain, midbrain and hindbrain areas where GHSR-1a gene expression has been reported.Intracerebroventricular infusions of GHRL into the CSF allows GHRL to influence the PG directly or indirectly through the nerve centers of the brain, such as the mediobasal hypothalamus, that are functionally and anatomically connected with the PG, in which GHSR-1a has been identified [1].Cabral et al. [45] also detected GHRL uptake in the ependymal cells of the ventricular system of the brain, where GHSR-1a is presumably not present.The PG is rich in fenestrated capillaries and canaliculi, through which, even large molecules such as peptides and proteins, including GHRL, can probably penetrate from the CSF into the gland [43].To reach the majority of neural structures connected to the PG, peripherally administered GHRL must first cross the blood-brain barrier (BBB).This is crucial, especially in sheep, in which GHRL administered intravenously crosses the BBB to a very small (< 1:1000) extent, and the concentration of this hormone in the CSF is about one thousand times lower than it is in the blood plasma [46].
In addition to the sympathetic pathways that initiate MEL synthesis, there are nerve projections of the central areas of the brain that modulate the activity of the PG.There are several neurotransmitters and neuropeptides that are involved in this process, including glutamate, γ-aminobutyric acid, acetylcholine, neuropeptide Y, somatostatin, vasopressin, substance P, calcitonin gene-related peptide, oxytocin, histamine and the neuronal isoform of nitric oxide synthase [47,48].In sheep, the above list is complemented by GHRL, leptin (a hormonal indicator of energy metabolism) and orexin B (which controls the rhythm of sleep and wakefulness, as well as energy homeostasis) [6,8].Interestingly, well-described interactions between GHRL and leptin [49] also occur in sheep at the level of the PG [6].Among these ruminants, photoperiod is an additional factor that interferes with these interactions, as demonstrated by the fact that GHRL has been shown to inhibit the leptin-stimulated increase in MEL secretion during an SD and strengthen the impact of leptin on PG during an LD [6].
To sum up, the results presented here have substantially contributed in the latest research regarding the role of GHRL as an important neurobiological factor that regulates numerous processes in animals.Our results indicate that GHRL, so far known mainly as a hormonal indicator of energy metabolism, influences the secretory activity of the PG and MEL biosynthesis, which demonstrates that the mechanism underlying MEL release is not completely understood.These results may also bring us closer to understanding the function of the PG in humans.Between human and sheep PGs, there is anatomical homology of gland vascularization and its distribution in relation to brain ventricles, particularly the position of the PG relative to the third ventricle [50] is comparable, which makes study using ovine PG valuable to carry on.were performed under sterile conditions and a dim red light.This light was insufficiently bright (< 3 lux) and at an improper wavelength (622 -780 nm) to influence MEL production [51].
Briefly, PGs collected from each ewe (n = 8/season) were dissected and sliced sagittally into 0.5to 2-mm thick strips (n = 36/season).Each PG explant was selected randomly, placed on a stainless steel grid covered with lens paper and incubated in a gas-liquid interface in 1 ml of Dulbecco's modified Eagle's medium (Biomed, Lublin, Poland).The explants were incubated in a 12-well tissue culture dish (Corning Netwell Insert, 15-mm membrane diameter, New York, NY) with 95% humidified air and 5% CO2 at 37°C for a total of 5.0 hours.After a 60 min period of equilibration, PG explants were challenged with either 0 (control) or 10 ng/ml of ovine GHRL (PolyPeptide Labs, Strasburg, France), and incubated for another 4 hrs.The dose of GHRL was based on previous studies [4].Sixty min after GHRL treatment 50 μl of medium was harvested from each well.Samples were stored at −20°C until RIA for melatonin.
Not treated explants (n = 4), collected from PGs immediately after euthanization and explants from the controls and the GHRL-treated groups, harvested from culture every hour of 4 hrs incubation (4 explants per group per hour), were frozen in liquid nitrogen and then stored at -80°C for subsequent analysis.Analysis include the determination of TPH1 mRNA expression by Real-time PCR, measurement of p31T-AA-NAT protein expression via western blotting, and determination of TPH1 and AA-NAT protein concentrations via ELISA.Pineal RNA and protein extracts from sheep PGs were obtained using a commercial NucleoSpin ® RNA/Protein kit (Marchery-Nagel, Oensingen, Switzerland), which allows for the isolation of total RNA (approx.70 μg) and protein (approx.1200 μg) from the same sample.

Isolation of total RNA and protein from pineal samples
According to the instructions provided by the manufacturer of the NucleoSpin® RNA/Protein kit, the frozen tissue was first mechanically fragmented using a rotor stator homogenizer (Omni TH™; Omni International Inc., Kennesaw, GA, USA) equipped with an Omni Soft Tissue Tip™.After isolating total RNA, its quality was verified by determining the ratio of 28S subunits to 18S subunits after separating the RNA samples on an agarose gel with ethidium bromide.The purity of the total RNA was measured spectrophotometrically using a NanoDrop 2000/2000c device (Thermo Scientific, Waltham, MA, USA).The concentration of isolated protein was measured using bicinchonic acid (BCA) (Sigma Aldrich, Poznan, Poland).

4.3.Real-time PCR analysis of pineal samples
Real-time PCR was used to measure TPH1 mRNA levels.The RNA (0.5 µg) was reverse transcribed to cDNA using Quantiscript reverse transcriptase and RT primer mix (QuantiTect® Reverse Transcription Kit; Qiagen, Hilden, Germany) by incubating the samples at 42°C for 15 min.The reaction was terminated by heating samples to 94°C for 3 min.Genomic DNA was removed by incubating the samples at 42°C for 2 min in the presence of gDNA Wipeout Buffer (QuantiTect® Reverse Transcription Kit).Amplification of cDNA was performed using TaqMan® Gene Expression Master Mix (Life Technologies, Foster City, CA, USA) and an Applied Biosystems 7300 Real-Time PCR System.Primers and probes were designed using Primer Express software v.2.0 (Applied Biosystems, Foster City, CA, USA).The products were amplified using the following primers, each at a final concentration of 900 nM: Using diluted samples, the amplification efficacies for the TPH1 (target gene) and β-actin (reference gene) amplimers were found to be identical.There was no significant variation in the Ct values for β-actin among the treatment groups.

4.4.Enzyme-linked immunosorbent assay of TPH1 and AA-NAT protein concentrations
The concentrations of the TPH1, AA-NAT proteins in the ovine PG explants and the concentrations of estradiol in plasma were determined via enzyme immunoassay using commercially available kits (rat TPH1 ELISA kit, Wuhan Fine Biological Technology Co., Ltd., Wuhan, China; sheep AA-NAT ELISA kit, Cusabio, Wuhan, China; Sheep E2 Estradiol Elisa kit, MyBioSource, San Diego, USA) according to the manufacturer's specifications.All samples MEL concentrations were assayed in 10 µl of medium according to the method of Fraser et al. [53] as modified by Misztal et al. [54] and measured per 1 g of tissue.Ovine anti-melatonin serum (AB/S/01) was obtained from Stockgrand Ltd. (Guildford, Surrey, UK).Synthetic MEL (Sigma Aldrich, Poznan, Poland) was used for standards, and [O-methyl-3H] melatonin (Amersham Biosciences, Buckinghamshire, UK) was used as the radiolabeled tracer.The range of the calibration curve was 0.4 to 50 ng/mL, and the working dilution of the antiserum was 1:4000.Bound and free fractions were separated using dextran-coated charcoal after an overnight incubation at 4°C.The sensitivity of the assay was 14.6 ± 2.0 pg/ml, and the intra-and inter-assay CVs were 7.8% and 9.1%, respectively.

Figure 1A .
Figure 1A.Mean (± SEM) TPH1 mRNA expression in ovine PG explants incubated in the presence of GHRL (10 ng/ml) for experiments carried out during the SD and LD seasons.TPH1mRNA expression is reported in arbitrary units relative to β-actin mRNA expression.The mean ΔCt value calculated for the explants not exposed to exogenous hormone (control) during the appropriate season was used as a calibrator.** denote significant differences from the control (P < 0.01).

Figure 1B .
Figure 1B.Mean (± SEM) TPH1 mRNA expression in ovine PG explants incubated for the indicated time in the presence of GHRL (10 ng/ml) for experiments carried out during the SD season.TPH1 mRNA expression is reported in arbitrary units relative to β-actin mRNA expression.The mean ΔCt value calculated for the explants not exposed to exogenous hormone

Figure 1C .
Figure 1C.Mean (± SEM) TPH1 mRNA expression in ovine PG explants incubated for the indicated time in the presence of GHRL (10 ng/ml) for experiments carried out during the LD season.TPH1 mRNA expression is reported in arbitrary units relative to β-actin mRNA expression.The mean ΔCt value calculated for the explants not exposed to exogenous hormone (control) at the appropriate time point was used as a calibrator.** denote significant differences from the control (P < 0.001).

Figure 1D .
Figure 1D.Mean (± SEM) basal endogenous TPH1 mRNA expression in ovine PGs collected during the SD and LD seasons and frozen immediately after tissue isolation.TPH1 mRNA expression is reported in arbitrary units relative to β-actin mRNA expression.The mean ΔCt value calculated for PGs isolated in the LD season was used as a calibrator.

Figure 2A .
Figure 2A.Mean (± SEM) TPH1 protein concentrations in ovine PG explants incubated in the presence of GHRL (10 ng/ml) for experiments carried out during the SD and LD seasons.Values with asterisks are significantly different: * (P < 0.05).

Figure 2C .
Figure 2C.Mean (± SEM) TPH1 protein concentrations in ovine PG explants obtained during the LD season.After 60 min of equilibration, PG explants were incubated with medium alone

Figure 3A .
Figure 3A.Representative western blots showing pT31-AA-NAT protein levels in ovine PG explants collected during the SD season and treated with 10 ng/ml of GHRL for 1, 2, 3 and 4 hrs.Protein bands were quantified via densitometry.The results are shown as the percentage of pT31-AA-NAT protein relative to the control protein levels.The blots were stripped and reprobed with an anti-actin antibody to control for the amounts of protein loaded onto the gel.Each column represents the mean (± SEM).* P < 0.05 and ** P < 0.01 indicate significant differences relative to the control.

Figure 3B .
Figure 3B.Representative western blots showing pT31-AA-NAT protein levels in ovine PG explants collected during the LD season and treated with 10 ng/ml of GHRL for 1, 2, 3 and 4 hrs.Protein bands were quantified via densitometry.The results are shown as the percentage of pT31-AA-NAT protein relative to the control protein levels.The blots were stripped and reprobed with an anti-actin antibody to control for the amounts of protein loaded onto the gel.Each column represents the mean (± SEM).* P < 0.05 indicates significant differences relative to the control.

Figure 4A .
Figure 4A.Mean (± SEM) AA-NAT protein concentrations in ovine PG explants incubated in the presence of GHRL (10 ng/ml) for experiments carried out during the SD and LD seasons.Values with asterisks are significantly different: ** (P < 0.01).

Figure 4B .
Figure 4B.Mean (± SEM) AA-NAT protein concentrations in the ovine PG explants obtained during the SD season.After 60 min of equilibration, PG explants were incubated with medium

Figure 4C .
Figure 4C.Mean (± SEM) AA-NAT protein concentrations in ovine PG explants obtained during the LD season.After 60 min of equilibration, PG explants were incubated with medium alone (control) or with medium containing GHRL (10 ng/ml) for 1, 2, 3 and 4 hrs.

Figure 5 .
Figure 5. Mean (± SEM) MEL concentrations in media collected from cultures of PG explants obtained during the SD and LD seasons.After 60 min of equilibration, PG explants were incubated with medium alone (control) or with medium containing GHRL (10 ng/ml) for 1 hr.Values with asterisks are significantly different: * (P < 0.05), ** (P < 0.01).

Posted: 28 February 2017 doi:10.20944/preprints201702.0101.v1
animal-related procedures used in these studies were approved by the Local Agricultural Animal Care and Use Committee of Krakow (protocol #80/2012).The study was carried out at the Experiment Station in the Department of Animal Biotechnology, Agricultural University of [8][9][10]lish Longwool ovariectomized ewes (a breed with pronounced reproductive seasonality; 4-5 y of age; mean body weight of 70 ± 5 kg), each bearing a subcutaneous estradiol implant, were used.In the present study, mean concentrations of estradiol in implanted ewes were 2-4 pg/ml as previously reported[8].Hormonal implants provide a constant level of estradiol negative feedback without the complications associated with ovarian cyclicity.Ovariectomized, steroid-treated females and castrated, steroid treated males have proven to be good models for studying the effects of nutrition on the neuroendocrine axis[8][9][10].Preprints (www.preprints.org)| NOT PEER-REVIEWED |