GPR54 system and thyrotropic axis activity in ewe lambs predisposed to the delayed puberty

Small Ruminant Research 144 (2016) 6–16

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Relationships between leptin, the KiSS-1/GPR54 system and thyrotropic axis activity in ewe lambs predisposed to the delayed puberty ∗ ´ Paulina Radwanska , Urszula Kosior-Korzecka Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland

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Article history: Received 8 November 2015 Received in revised form 25 July 2016 Accepted 26 July 2016 Available online 27 July 2016 Keywords: Leptin KiSS-1/GPR54 system Thyrotropic axis Ewe lambs Puberty Laparoscopy

a b s t r a c t In the present study we compared body weight gains, some factors involved in the initiation of sexual maturation (pituitary expression of KiSS-1 and G protein-coupled receptors (GPR54), plasma concentrations of kisspeptin-10 (KiSS-10), leptin, thyroid-stimulating hormone (TSH), free thyroxin (fT4)) and the time of the first ovulation in ewe lambs predisposed to delayed puberty and control animals. The experiment was carried out on 114 ewes and 64 female lambs divided according to their birth type and body weight of their mothers. All ewe lambs were weighed at birth and every two weeks thereafter, until eight months of postnatal age. From four to eight months of postnatal age at monthly intervals, blood samples were collected from the jugular vein of all ewe lambs. Then, daily weight gains were recorded and plasma KiSS-10, leptin, TSH and fT4 concentrations were analysed by ELISA using species-specific antibodies. Starting from six months of age, the pituitary expression of KiSS-1/GPR54 mRNA was determined by Real Time-PCR. The activity of the ovaries was estimated using laparoscopy. The results obtained showed that the time of the onset of puberty in the ewe lambs depended on the mother’s body mass, the plasma leptin level, and the birth type (singleton/twin). It was observed that the elevation of leptin concentration up to 3.35 ± 0.26–3.60 ± 0.19 ng/mL was associated with the initiation of puberty. Conversely, the hyperphysiological leptin levels found in ewe lambs, which were twin offspring of fatty sheep, were correlated with puberty delayed until the age of ten months. Moreover, it was found that a significant increase in pituitary KiSS-1 mRNA expression (1.40 ± 0.12–1.63 ± 0.22) (relative KiSS-1 mRNA expression level, ratio of KiSS-1 mRNA/GAPDH mRNA) and plasma KiSS-10 concentration (31.26 ± 1.54 ng/mL–32.24 ± 2.25 ng/mL) was connected with the occurrence of the first ovulation. On the other hand, GPR54 mRNA expression in the pituitary decreased around the time of the first ovulation. Also, the increase in thyroid gland activity was dependent on the mother’s body mass as well as birth type and occurred around the time of the first ovulation. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Taking into account that the onset of sexual maturation depends on the attainment of a critical body mass and sufficient accumulation of adipose tissue, delayed puberty is found in ewe lambs with a low birth weight resulting from multiple pregnancy and/or the obesity of mothers (Kosior-Korzecka et al., 2012; Rosales Nieto et al., 2014, 2013). Also leptin, which is an adipocyte-derived natural ligand of OB-R receptors, provides a link between the metabolic status and the onset of puberty (Smith et al., 2002). It initiates the activity of the hypothalamic-pituitary-ovarian (HPO) axis, inter alia through

∗ Corresponding author at: Akademicka 12, 20-033 Lublin, Poland. ´ E-mail address: [email protected] (P. Radwanska). http://dx.doi.org/10.1016/j.smallrumres.2016.07.019 0921-4488/© 2016 Elsevier B.V. All rights reserved.

indirect stimulation of gonadotropin-releasing hormone secretion. This effect is mediated, among others, via the kisspeptin/G proteincoupled receptors (KiSS-1/GPR54) system (Hausman et al., 2012). It is known that ovine kisspeptin cells in the arcuate nucleus and the preoptic area express Ob-Rb receptors and are regulated by leptin (Backholer et al., 2010). According to our previous study, leptin influences KiSS-1/GPR54 system expression also at the level of the ´ pituitary gland in pubertal ewes (Radwanska and Kosior-Korzecka, 2016). Kisspeptins are a family of neuropeptide products of the KiSS-1 gene, which are identified as endogenous ligands of G proteincoupled receptors. It has been demonstrated that the KiSS-1/GPR54 system is a key factor in the neuroendocrine control of reproduction (Pinilla et al., 2012; Tanaka et al., 2012). Taking into account that GnRH neurons express GPR54, the KiSS-1/GPR54 system can

P. Radwa´ nska, U. Kosior-Korzecka / Small Ruminant Research 144 (2016) 6–16

be a signal playing a pivotal role in the activation of GnRH neurons and in the triggering of the onset of puberty (Chaikhun et al., 2013). It has been observed that pubertal transition in rodents and primates is accompanied by a significant increase in KiSS-1 and GPR54 mRNA levels (Navarro et al., 2004; Shahab et al., 2005). Moreover, it is known that an intracerebroventricular injection of kisspeptin elevates the secretion of LH and FSH in prepubertal and adult rodents as well as in sheep (Matsui et al., 2004; Messager et al., 2005; Thompson et al., 2004). Hormones of the thyrotropic axis also participate in the regulation of reproductive processes in ruminants. It is known that almost all neurons in the brain, including GnRH neurons, express thyroid hormone receptor ␣ (␣THR), type II deiodinase and monocarboxylase transporter MCT8 (Ceballos et al., 2009; Jansen et al., 1997; Tu et al., 1997). Moreover, it is known that an increase in the plasma concentration of thyroid hormones promotes changes in the neuroendocrine axis, which result in an intensified oestrogen negative feedback, suppression of LH pulses and transition from the breeding season to the anoestrus in cyclic, mature ewes (Lehman et al., 2010). It is known that the activity of the thyrotropic axis can be modified by leptin and kisspeptin, which initiate the onset of puberty. According to our previous studies in vitro, TSH secretion from the pituitary cells of pubertal ewes is regulated by leptin. A concentration of 10−10 to 10−6 M of leptin elevates TSH secretion, while 10−5 M of leptin decreases TSH secretion from ovine ´ pituitary cells (Radwanska and Kosior-Korzecka, 2014). Moreover, short-term exposure of ovine pituitary cells to kisspeptin changes ´ TSH secretion in vitro (Radwanska and Kosior-Korzecka, 2016). However, there are no reports about the relationships between the KiSS-1/GPR54 system, leptin and thyrotropic axis activity during the onset of puberty in ewe lambs in vivo. Therefore, the aim of the present study was to analyse the changes in body weight gains, pituitary expression of KiSS-1 and GPR54, plasma concentrations of KiSS-10, leptin, thyroidstimulating hormone, free thyroxin as well as a time of the first ovulation in ewe lambs predisposed to delayed puberty and control sheep.

2. Material and methods

7

tin level (1.56 ng/mL, n = 45); II−sheep with a high body weight (76.49 ± 5.15 kg) and a high plasma leptin level (2.88 ng/mL, n = 69). Next, on the basis of the number of developing foetuses, two subgroups were identified in each group: S−single carrying ewes: IS (n = 20) and IIS (n = 22), T−twin bearing ewes: IT (n = 25) and IIT (n = 47). Similarly, the new-born ewe lambs were divided into four groups: IS−singleton offspring of normally weighing sheep (without predisposition to delayed puberty; n = 13), IT−twin offspring of normally weighing sheep (predisposed to delayed puberty due to a low birth weight resulting from multiple pregnancy; n = 15), IIS−singleton offspring of fatty sheep (predisposed to delayed puberty due to the obesity of mothers during pregnancy; n = 14) and IIT−twin offspring of fatty sheep (predisposed to delayed puberty due to a low birth weight resulting from multiple pregnancy as well as the obesity of mothers; n = 22). All ewe lambs were weighed at birth and every two weeks thereafter, until the postnatal age of eight months. Starting from four to eight months postnatal age at monthly intervals, blood samples were collected from the jugular vein (for 3 h, every 15 min). The blood was centrifuged (20 min at 4 ◦ C and 1000 x g), and plasma was stored immediately at −20 ◦ C until testing. Daily weight gains were recorded and plasma leptin, KiSS-10, TSH and fT4 concentrations were analysed by ELISA using species-specific antibodies. From the postnatal age of six to ten months, the activity of the ovaries was monitored and assessed laparoscopically for the presence, number and diameter of follicles or the presence of corpora lutea. After they reached the postnatal age of six, seven and eight months, two ewe lambs from each group were euthanized to isolate pituitary glands. Pituitaries were dissected within 10 min of sacrifice and transported within 15 min to the laboratory in cold DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma Chemicals Co., St. Louis, MO, USA) (about 4 ◦ C) supplemented with 0.1% BSA (Bovine Serum Albumin, Sigma Chemicals Co., St. Louis, MO, USA), 0.08% glucose (d-(+)Glucose, Sigma Chemicals Co., St. Louis, MO, USA), 0.59% HEPES (Sigma Chemicals Co., St. Louis, MO, USA) and gentamicin (Sigma Chemicals Co., St. Louis, MO, USA) (20 ␮g/mL). The anterior and posterior lobes of the pituitaries were separated by blunt dissection. The pituitary glands were used to analyse kisspeptin and GPR54 mRNA expression. The protocol of the study concept and all the procedures were approved by the Second Local Ethics Committee for Animal Experimentation in Lublin (Licence No. 40/2013).

2.1. Animals and experimental design The experiment was carried out on high prolific SCP sheep (Suffolk (25%) + Charolaise (25%) + Romanov (12.5%) + Polish Lowland Sheep (37.5%)): 128 multiparous, cyclic ewes (3–5 years of age, mean body weight 71.79 ± 11.32 kg) and 64 ewe lambs (the offspring of the experimental adult sheep). The whole study was performed from August to November of the next year, under natural light and temperature. At the height of the breeding season, all adult females were synchronized using intravaginal sponges impregnated with 20 mg of fluorogestone acetate (Chrono-Gest CR, Intervet International B.V.) for 16 days and 500 IU of PMSG (Folligon, Intervet International B.V.), and 7.5 mg of prostaglandin F2␣ analogue (Prosolvin, Intervet International B.V.) administered immediately after withdrawal of the sponges. Thereafter, between 56 and 58 h after removal of the sponges, laparoscopic intrauterine artificial insemination of all synchronized ewes (n = 125) was carried out. One inseminating dose of 0.1 mL of frozen-thawed semen containing 9 million spermatozoa was deposited directly into each uterine horn by using an inseminating gun. On the 40th day following insemination, an ultrasonographic study was conducted to confirm or rule out pregnancy as well as to determine the number of foetuses. The efficiency of insemination was 91.2%. Pregnant sheep (n = 114) were divided into two groups: I−sheep of average body weight (64.55 ± 4.27 kg) and a normal plasma lep-

2.2. Isolation of total RNA from pituitary glands Total RNA was isolated from pituitary glands using the TRI Reagent procedure. Firstly, pituitaries were weighed, and homogenized in TRI Reagent (Sigma Chemicals Co., St. Louis, MO, USA; 1 mL of TRI Reagent per 50–100 mg pituitary gland tissue). Then, the homogenate was centrifuged at 12 000 x g for 10 min at 2–8 ◦ C. The supernatant obtained was transferred to a new tube and incubated for 5 min at room temperature. Next, to extract RNA, 1200 ␮L of the lysate was supplemented with 120 ␮L of 1-bromo3-chloropropane (Sigma Chemicals Co., St. Louis, MO, USA), shaken for 15 s, incubated for 15 min at room temperature, and then centrifuged at 12,000 × g for 15 min. The upper aqueous and containing RNA phase was collected and supplemented with 600 ␮L of 2propanol (Sigma Chemicals Co., St. Louis, MO, USA). The mixture was incubated for 10 min at room temperature and then, centrifuged for 10 min at 12,000 x g. After removal of the supernatant, the RNA pellet was resuspended in 75% ethanol (POCH, Gliwice, Poland) and centrifuged at 12,000 x g for 5 min to obtain purified RNA sample. The quality of the RNA was determined on 1% agarose gel. RNA samples were stored immediately at −80 ◦ C until testing.

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Table 1 Changes in the body mass (kg) of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep). The data presented are mean body mass values at birth and at 28, 56, 70, 90, 110, 140, 170, 200 and 240 days of age, respectively. Groups of ewe lambs

IS IT IIS IIT

Body mass (kg) Days of postnatal life 0

28

56

70

90

110

140

170

200

240

5.35 ± 0.21A 3.65 ± 0.30AB 4.85 ± 0.40AB 3.14 ± 0.40B

13.90 ± 0.14A 11.40 ± 1.20A 11.30 ± 0.50AB 6.70 ± 0.90B

21.72 ± 1.24A 17.70 ± 0.80A 18.70 ± 0.20A 11.80 ± 1.10B

27.12 ± 1.52A 22.80 ± 0.80A 22.60 ± 1.40AB 16.90 ± 1.60B

32.54 ± 0.23A 27.30 ± 1.00B 26.70 ± 0.10BC 22.10 ± 1.40C

36.50 ± 0.28A 32.90 ± 0.90A 33.90 ± 0.20A 27.20 ± 1.30B

39.35 ± 0.64A 36.40 ± 1.30AB 38.50 ± 0.70AB 35.10 ± 2.20B

43.65 ± 0.07A 40.40 ± 2.00B 42.30 ± 0.40A 43.60 ± 2.20A

49.60 ± 0.57A 43.80 ± 1.20B 43.80 ± 1.80B 53.50 ± 2.50A

51.00 ± 0.99AB 50.10 ± 1.30A 49.80 ± 2.70AB 58.40 ± 2.10B

A,B,C−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05).

2.3. Reverse transcription Reverse transcription was performed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Lithuania). A mixture consisting of total RNA (0.1 ng to 5 ␮g), 1 ␮L primer solution (Oligo(dT)18 Primer, 100 ␮M) and nuclease-free water (to 12 ␮L), was incubated for 5 min at 65 ◦ C. After cooling and centrifugation, the mixture was supplemented with 4 ␮L reaction buffer, 1 ␮L ribonuclease inhibitor solution (RiboLock RNase Inhibitor, 20 U/␮L) and 2 ␮L of a mixture of nucleotides (10 mM). Next, 1 ␮L of reverse transcriptase solution (RevertAid M-MuLV RT, 200 U/␮L) was added to the mixture, and the whole sample was centrifuged and incubated for 60 min at 42 ◦ C. Finally, the reaction was stopped by heating the mixture for 5 min at 70 ◦ C. The cDNA obtained was stored at −20 ◦ C until Real-Time PCR analysis.

2.4. Analysis of kisspeptin and GPR54 mRNA expression in pituitary glands by real-time PCR Samples of cDNA were amplified using AmpliTaq Gold DNA polymerase (Taq Man Universal PCR Master Mix, Applied Biosystem, USA) and the Applied Biosystems 7500 (Fast Real Time PCR system, Applied Biosystem, USA). To determine the changes in the level of KiSS-1 mRNA relative to an endogenous control (Relative Quantification), cDNA (5 ng) was subjected to amplification using the following primers: 5 -CTGGTGCAGCGGGAGAAG-3 (forward primer, 500 nM), 5 -GCGAGGCCGAAGGA-3 (reverse primer, 500 nM) and a fluorescently labelled (dT-FAM/TAMRA) probe: 5 ACGTGTCCGCCTACA-3 (200 nM); amplicon size: 57 bp (Bellingham et al., 2009). To determine changes in the level of GPR54 mRNA relative to an endogenous control (Relative Quantification), cDNA (5 ng) was subjected to amplification using primers: 5 -TACATCCAGCAGGTCTCGGTG-3 (forward primer, 500 nM), 5 ACGTACCAGCGGTCCACACT-3 (reverse primer, 500 nM); dTFAM/TAMRA probe: 5 -CACGTGTGCCACTCTGACCGCC-3 (200 nM); amplicon size: 71 bp (Seminara et al., 2003). A fragment of cDNA for ovine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. It was amplified using: a forward primer: 5 -GGCGTGAACCACGAGAAGTATAA-3 (500 nM), a reverse primer: 5 -CCCTCCACGATGCCAAAGT-3 (500 nM), dT-FAM/TAMRA probe: 5 -ACCCTCAAGATTGTCAGCAATGCCTCC-3 (200 nM); amplicon size: 199 bp (Bellingham et al., 2009). After activation of DNA polymerase, with simultaneous deactivation of UNG, and after an initial denaturation of the matrix for 10 min at 95 ◦ C, 45 cycles of a two-stage PCR reaction were performed: I−denaturation for 30 s at 95 ◦ C, II−annealing, elongation of DNA and degradation of the probe for 60 s at 60 ◦ C. The −CT relative quantification method was used to determine the relative expression.

2.5. Analysis of plasma leptin, kiSS-10, TSH and fT4 concentrations Plasma KiSS-10, leptin, TSH and fT4 concentrations were analysed by ELISA (KiSS-1 (112–121) Amide/Kisspeptin-10/Metastin (45–54) Amide EIA Kit (Phoenix Pharmaceuticals Inc., CA, USA); Multi-Species Leptin RIA Kit (Linco Research, St. Charles, Mo, USA); Sheep Thyroid Stimulating Hormone ELISA Kit (NeoBiolab, Massachusetts, USA); Sheep Free Thyroxin ELISA Kit (NeoBiolab, Massachusetts, USA)) using species-specific antibodies. 2.6. Statistical analysis The results obtained were calculated using Statistica 5.0 PL and expressed as a mean and standard deviation (x ± SD). Comparisons between the experimental groups were performed using the analysis of variance and paired t-tests. Differences with a probability P ≤ 0.05 were considered significant. Pearson linear correlation coefficients were calculated to assess the relationships between the analysed variables (i.e. age of the ewe lambs, body mass, plasma leptin concentration, plasma KiSS-10 concentration, pituitary KiSS1 mRNA expression, pituitary GPR54 mRNA expression, plasma fT4 concentration and plasma TSH concentration). Linear regression (y = bx + ␣) was performed to evaluate whether months of postnatal life were predictors of changes in concentration of leptin, KiSS-10, fT4, TSH and expression of KiSS-1 and GPR54. The level of significance accepted was 0,05. 3. Results 3.1. The activity of ovaries The ewe lambs which were singleton offspring of normally weighing sheep (IS) ovulated for the first time at seven months of age. It was four weeks earlier than IT and IIS sheep, in which the first ovulation was observed at eight months of age. However, the ewe lambs which were twin offspring of fatty sheep (IIT) did not ovulate until the postnatal age of 10 months. The first ovulation was ascertained on the basis of the formation of the first corpora lutea. 3.2. Changes in the body mass of the experimental ewe lambs The body mass at birth averaged 5.35 ± 0.21 kg in IS ewe lambs (Table 1). Lower mean body masses were recorded for IT and IIS sheep (3.65 ± 0.30 kg and 4.85 ± 0.40 kg, respectively). The lowest body mass at birth was found in IIT ewe lambs (3.14 ± 0.40 kg). The daily weight gains starting from birth to 110 days of age averaged 0.28 ± 0.001 kg, 0.27 ± 0.01 kg, 0.26 ± 0.002 kg, 0.22 ± 0.01 kg in IS, IT, IIS and IIT ewe lambs, respectively (Table 2). However, from day 110 to day 240 of postnatal life, the highest daily weight gains were observed in IIT sheep (0.24 ± 0.01 kg). At the same time, the

P. Radwa´ nska, U. Kosior-Korzecka / Small Ruminant Research 144 (2016) 6–16 Table 2 Daily weight gains (kg) of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep). The data presented are mean daily weight gains values from birth to 110 days of postnatal life and from 110 to 240 days of postnatal life, respectively.

were observed after the sheep had reached the postnatal age of five months in any of the groups, compared to the younger ewes, aged four months. Leptin concentrations significantly elevated in IS, IT, IIS and IIT sheep after six months of age. In IS ewe lambs, plasma leptin concentrations reached maximum values after the seven and eight months of age (3.60 ± 0.19 ng/mL and 3.79 ± 0.17 ng/mL, respectively). This was connected with the occurrence of the first ovulation in these ewes after they reached the age of seven months. Also in IT and IIS sheep, the highest plasma leptin concentrations (3.35 ± 0.26 ng/mL and 3.50 ± 0.27 ng/mL, respectively) were recorded during the onset of puberty (after the ewes reached the age of eight months). In IIT ewe lambs significantly higher (P ≤ 0.05) plasma leptin concentrations, compared to the other groups, were observed from four to eight months of age. The maximum value in IIT sheep (7.56 ± 0.56 ng/mL) was observed when they had reached the age of eight months. Ewe lambs from this group did not ovulate until ten months of age. Plasma leptin levels were positively correlated with the age of the ewe lambs (r = 0.97, r = 0.96, r = 0.99 and r = 0.98 in IS, IT, IIS and IIT lambs, respectively). Moreover, linear regression revealed that months of postnatal life were a significant predictor of changes in leptin concentration (b = 0,80, R2 = 0,93, P = 0,007; b = 0,61, R2 = 0,93, P = 0,008; b = 0,64, R2 = 0,97, P = 0,002; b = 1,48, R2 = 0,96, P = 0,004 for IS, IT, IIS and IIT lambs, respectively). Also, a positive relationship was found between body mass and leptin concentrations (r = 0.99, r = 0.98, r = 0.97 and r = 0.98 for IS, IT, IIS and IIT lambs, respectively).

Daily weight gains (kg)

Groups of ewe lambs

Days of postnatal life

IS IT IIS IIT

0–110

110–240

0.28 ± 0.001A 0.27 ± 0.01AB 0.26 ± 0.002AB 0.22 ± 0.01B

0.11 ± 0.01A 0.13 ± 0.01AB 0.12 ± 0.02AB 0.24 ± 0.01B

9

A,B−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05).

daily weight gains for IS, IT and IIS sheep ranged from 0.11 ± 0.01 kg to 0.13 ± 0.01 kg. The occurrence of the first ovulation in IS sheep correlated with the time when they achieved 77% of maternal body mass (49.60 ± 0.57 kg), i.e. after they had reached the age of 200 days. The onset of puberty in IT and IIS sheep also correlated with the time when they reached 77% or 65% of maternal body mass (50.10 ± 1.30 kg, 49.80 ± 2.70 kg, respectively) after 240 days of age. At the same time, the body mass averaged 51.00 ± 0.99 kg in IS ewe lambs. The highest body mass was determined in IIT sheep starting from the 200th to the 240th day of postnatal life (49.80 ± 2.70 kg, 58.40 ± 2.10 kg, respectively). The ewe lambs in this group did not ovulate until the postnatal age of ten months (Table 1).

3.4. Plasma KiSS-10 concentration

3.3. Plasma leptin concentration

Plasma KiSS-10 concentrations in four-month old IS, IT, IIS and IIT ewe lambs ranged from 0.56 ± 0.10 to 1.23 ± 0.10 ng/mL (Fig. 2). There were no significant changes in kisspeptin concentrations after the sheep had reached the age of five months, compared to the younger ewes, aged four months. KiSS-10 concentration significantly increased in all groups of ewe lambs, after they had reached the age of six months. In IS sheep, the highest plasma

Plasma leptin concentrations in four-month old IS, IT and IIS female lambs ranged from 0.97 ± 0.10 ng/mL to 1.03 ± 0.09 ng/mL (Fig. 1). The sheep, which were twin offspring of fatty sheep (IIT) had significantly (P ≤ 0.05) higher leptin concentrations (2.07 ± 0.14 ng/mL). No marked changes in leptin concentration 9

B c 8

B c

7

Leptin concentration (ng/mL)

6

5

B b

IS IT

A c

4

AAA c

IIS IIT

dd 3

B a

B a AAA bbb

CC cc

2

AAA aaa

AAA aaa

1

0 4

5

6

7

8

months of postnatal life

Fig. 1. Changes in plasma leptin (ng/mL) of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep). The data presented are mean values of leptin levels in the blood collected from 08.00 to 11.00 a.m. (every 15 min) at 4, 5, 6, 7 and 8 months of age, respectively. A,B,C−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05). a,b,c,d−mean values obtained at different postnatal ages (in months) in the same experimental group and labelled with different letters differ significantly (P ≤ 0.05).

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P. Radwa´ nska, U. Kosior-Korzecka / Small Ruminant Research 144 (2016) 6–16 40

A c

35

AA dd

A c

30

KiSS-10 concentration (ng/mL)

A b

BB cc

25

IS

20

BB bb

IT IIS IIT

15

C b

C b

10

B b

5

AAAA aaaa

AAAA aaaa

0 4

5

7

6

8

months of postnatal life

Fig. 2. Changes in plasma KiSS-10 (ng/mL) of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep). The data presented are mean values of KiSS-10 levels in the blood collected from 08.00 to 11.00 a.m. (every 15 min) at 4, 5, 6, 7 and 8 months of age, respectively. A,B,C−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05). a,b,c,d−mean values obtained at different postnatal ages (in months) in the same experimental group and labelled with different letters differ significantly (P ≤ 0.05).

kisspeptin concentrations were observed after seven months of age (32.24 ± 2.25 ng/mL) and in IT, IIS ewe lambs after eight months of age (31.36 ± 2.00 ng/mL and 31.26 ± 1.54 ng/mL, respectively). This was connected with the occurrence of the first ovulation in these groups of ewe lambs. No marked changes in plasma kisspeptin concentrations were observed in IIT sheep, starting from six to eight months of age. These ewe lambs, which were predisposed to delayed puberty and did not ovulate until the age of ten months, had significantly lower plasma kisspeptin concentrations compared to IS, IT and IIS sheep. KiSS-10 concentration was positively correlated with the age of the ewe lambs (r = 0.91, r = 0.97, r = 0.97 and r = 0.85 for IS, IT, IIS and IIT ewe lambs, respectively). Linear regression revealed that months of postnatal life were a significant predictor of changes in KiSS-10 concentration in IS, IT and IIS ewe lambs (b = 8,94, R2 = 0,83, P = 0,03; b = 8,18, R2 = 0,95, P = 0,005; b = 8,36, R2 = 0,94, P = 0,006; b = 2,15, R2 = 0,73, P = 0,07 for IS, IT, IIS and IIT ewe lambs, respectively). Also, the relationship between plasma concentration of leptin and KiSS-10 was positive (r = 0.93, r = 0.98, r = 0.98 and r = 0.83 for IS, IT, IIS and IIT ewe lambs, respectively). 3.5. Relative changes in KiSS-1 and GPR54 expression in the pituitary gland The KiSS-1 mRNA expression level in six-month old IS, IT, IIS and IIT ewe lambs ranged from 0.26 ± 0.09 to 0.35 ± 0.07 (relative KiSS-1 mRNA expression level, ratio of KiSS-1 mRNA/GAPDH mRNA) (Fig. 3). After the age of seven months, a significant elevation in KiSS-1 mRNA expression (1.40 ± 0.12) was observed in IS sheep. It was connected with the occurrence of the first ovulation in these ewe lambs. At the same time, there was no significant increase in kisspeptin expression in IT, IIS or IIT sheep. After eight months of postnatal age, a marked augmentation in KiSS-1 expression (1.63 ± 0.22 and 1.59 ± 0.16, respectively) was found in IT and IIS ewe lambs. It was correlated with the onset of puberty in these groups of sheep. Therefore, the increase in plasma kisspeptin level (32.24 ± 2.25 ng/mL, 31.36 ± 2.00 ng/mL

and 31.26 ± 1.54 ng/mL, respectively), pituitary KiSS-1 expression (1.40 ± 0.12, 1.63 ± 0.22 and 1.59 ± 0.16, respectively) as well as plasma leptin concentration (3.60 ± 0.19 ng/mL, 3.35 ± 0.26 ng/mL and 3.50 ± 0.27 ng/mL, respectively) was associated with the occurrence of the first ovulation in IS, IT and IIS ewe lambs. The lowest pituitary KiSS-1 mRNA expression and a lack of its significant increase was observed in IIT ewe lambs, which did not ovulate until the age of ten months. KiSS-1 mRNA expression was positively correlated with the age of the ewe lambs (r = 0.89, r = 0.92, r = 0.90 and r = 0.99 in IS, IT, IIS and IIT ewe lambs, respectively). Also, a positive relationship was found between KiSS-1 expression and KiSS-10 plasma concentration (r = 0.54, r = 0.99, r = 0.99, r = 0.91 in IS, IT, IIS and IIT ewe lambs, respectively). Linear regression between months of postnatal life and KiSS-1 mRNA expression was as follows: b = 0,55, R2 = 0,78, P = 0,31; b = 0,68, R2 = 0,85, P = 0,26; b = 0,65, R2 = 0,82, P = 0,28; b = 0,04, R2 = 0,99, P = 0,05 for IS, IT, IIS and IIT ewe lambs, respectively. Moreover, plasma leptin concentration correlated positively with ovine pituitary KiSS-1 expression (r = 0.73, r = 0.99, r = 0.99 and r = 0.97 in IS, IT, IIS and IIT ewe lambs, respectively). The level of GPR54 mRNA expression in six-month old IS, IT, IIS and IIT ewe lambs ranged from 1.21 ± 0.13 to 1.39 ± 0.11 (relative GPR54 mRNA expression level, ratio of GPR54 mRNA/GAPDH mRNA) (Fig. 4). After the age of seven months, there was a marked drop in GPR54 mRNA expression (0.67 ± 0.08 vs. 1.21 ± 0.13) in IS sheep. At that age, IT and IIS sheep exhibited a slight decrease in GPR54 expression (1.17 ± 0.09 and 0.99 ± 0.08, respectively). The lowest GPR54 mRNA expression was found in IT and IIS ewe lambs (0.42 ± 0.15 and 0.45 ± 0.06, respectively) after they had reached the postnatal age of eight months. Conversely, there were no significant changes in pituitary GPR54 mRNA expression in IIT sheep. Pituitary GPR54 mRNA expression was negatively correlated with age (r = −0.93, r = −0.95, r = −0.98 and r = −0.77 in IS, IT, IIS and IIT ewe lambs, respectively). Linear regression revealed that months of postnatal life were not a significant predictor of changes in GPR54 mRNA expression in ewe lambs (b = −0,33, R2 = 0,87,

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Fig. 3. Relative changes in KiSS-1 expression in the pituitary of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep) at 6, 7 and 8 months of age, respectively. The values correspond to the mean ± SD of the KiSS-1 mRNA/GAPDH mRNA ratio. A,B−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05). a,b−mean values obtained at different postnatal ages (in months) in the same experimental group and labelled with different letters differ significantly (P ≤ 0.05). 1.6

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Fig. 4. Relative changes in GPR54 expression in the pituitary of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep) at 6, 7 and 8 months of age, respectively. The values correspond to the mean ± SD of the GPR54 mRNA/GAPDH mRNA ratio. A,B,C−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05). a,b,c−mean values obtained at different postnatal ages (in months) in the same experimental group and labelled with different letters differ significantly (P ≤ 0.05).

P = 0,23; b = −0,49, R2 = 0,91, P = 0,19; b = −0,41, R2 = 0,97, P = 0,12; b = −0,10, R2 = 0,60, P = 0,44 for IS, IT, IIS and IIT ewe lambs, respectively). Also, a negative correlation was found between KiSS-1 and GPR54 mRNA expression in the ovine pituitary (r = −0.99, r = −0.99, r = −0.97 and r = −0.82 in IS, IT, IIS and IIT ewe lambs, respectively) as well as between KiSS-10 concentration and GPR54 expression (r = −0.63, r = −0.98, r = −0.94 and r = −0.99 in IS, IT, IIS and IIT ewe lambs, respectively).

3.6. Changes in the concentrations of plasma free thyroxin and thyroid−stimulating hormone Plasma fT4 concentrations ranged from 10.29 ± 0.41 pg/mL to 13.20 ± 1.32 pg/mL in four-month old IT, IIS and IIT ewe lambs (Fig. 5). At that age, IS sheep had a significantly higher fT4 concentration (34.23 ± 1.71 pg/mL) compared to the other groups (IT, IIS and IIT). There were no marked changes in fT4 concentration after

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Fig. 5. Changes in plasma free thyroxin (pg/mL) of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep). The data presented are mean values of fT4 levels in the blood collected from 08.00 to 11.00 a.m. (every 15 min) at 4, 5, 6, 7 and 8 months of age, respectively. A,B,C−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05). a,b,c−mean values obtained at different postnatal ages (in months) in the same experimental group and labelled with different letters differ significantly (P ≤ 0.05).

the sheep had reached the age of five months in any of the groups, compared to younger ewes, aged four months. Plasma fT4 concentrations reached maximum values after seven and eight months of postnatal age (46.90 ± 1.08 pg/mL and 47.70 ± 1.34 pg/mL, respectively) in IS sheep. This was correlated with the onset of puberty after the age of seven months in this group of ewe lambs. Moreover, IS ewe lambs had the highest (P ≤ 0.05) fT4 concentration compared to the other experimental groups, starting from the age of four to eight months. Plasma fT4 concentrations significantly increased in IT and IIS sheep after the age of six months (23.70 ± 1.90 pg/mL and 17.21 ± 1.20 pg/mL, respectively) compared to younger ewes, aged four and five months. However, the highest fT4 concentration was observed after IT and IIS ewe lambs had reached the postnatal age of eight months (33.35 ± 2.00 pg/mL and 31.34 ± 2.20 pg/mL, respectively). This was connected with the occurrence of the first ovulation in IT and IIS ewe lambs. The lowest plasma fT4 concentration was determined in IIT ewe lambs, which did not ovulate until the age of ten months. The plasma fT4 level was positively correlated with age (r = 0.98, r = 0.99, r = 0.98 and r = 0.91 in IS, IT, IIS and IIT ewe lambs, respectively). Linear regression revealed that months of postnatal life were a significant predictor of changes in fT4 concentration in ewe lambs (b = 3,67, R2 = 0,96, P = 0,003; b = 5,22, R2 = 0,99, P = 0,0006; b = 5,47, R2 = 0,95, P = 0,005; b = 2,70, R2 = 0,83, P = 0,03 for IS, IT, IIS and IIT ewe lambs, respectively). Positive correlations were also found between body mass and fT4 concentration (r = 0.99, r = 0.99, r = 0.96 and r = 0.88 in IS, IT, IIS and IIT ewe lambs, respectively), leptin and fT4 (r = 0.99, r = 0.97, r = 0.99 and r = 0.93 in IS, IT, IIS and IIT ewe lambs, respectively), KiSS-10 and fT4 (r = 0.94, r = 0.99, r = 0.98 and r = 0.63 in IS, IT, IIS and IIT ewe lambs, respectively) as well as KiSS-1 expression and fT4 concentration (r = 0.85, r = 0.98, r = 0.99 and r = 0.99 in IS, IT, IIS and IIT ewe lambs, respectively). The mean plasma TSH concentration was 0.35 ± 0.04 ng/mL in four-month old IS ewe lambs (Fig. 6). At that age, IT and IIS sheep had significantly higher TSH concentrations (1.17 ± 0.13 ng/mL and 1.47 ± 0.11 ng/mL, respectively) than IS ewe lambs. The highest (P ≤ 0.05) plasma TSH concentration (2.08 ± 0.09 ng/mL) was determined in IIT sheep after they had reached the age of four months. TSH significantly (P ≤ 0.05) decreased after the postnatal age of five months (0.05 ± 0.02 ng/mL, 0.29 ± 0.04 ng/mL,

0.21 ± 0.02 ng/mL and 0.29 ± 0.03 ng/mL, respectively) in IS, IT, IIS and IIT sheep compared to younger ewe lambs, aged four months. The most significant drop in TSH concentration was observed in IS sheep, starting with the age of six months up till the age of eight months (0.04 ± 0.01 ng/mL), and after the age of eight months in IT, IIS and IIT sheep (0.09 ± 0.02 ng/mL, 0.04 ± 0.01 ng/mL and 0.04 ± 0.01 ng/mL, respectively). The plasma TSH level was negatively correlated with age (r = −0.72, r = −0.82, r = −0.77, and r = −0.77 in IS, IT, IIS and IIT sheep, respectively). Linear regression revealed that months of postnatal life were not a significant predictor of changes in plasma TSH concentration in ewe lambs (b = −0,06, R2 = 0,52, P = 0,17; b = −0,22, R2 = 0,68, P = 0,09; b = −0,30, R2 = 0,60, P = 0,13; b = −0,43, R2 = 0,59, P = 0,13 for IS, IT, IIS and IIT ewe lambs, respectively). A negative correlation was found between the plasma concentration of TSH and fT4 (r = −0.70, r = −0.77, r = −0.61 and r = −0.51 in IS, IT, IIS and IIT sheep, respectively). Negative relationships were also observed between plasma leptin and TSH concentration (r = −0.60, r = −0.67, r = −0.66 and r = −0.63 in IS, IT, IIS and IIT sheep, respectively), KiSS-10 and TSH (r = −0.63, r = −0.68, r = −0.65 and r = −0.70 in IS, IT, IIS and IIT sheep, respectively), and pituitary KiSS-1 expression and TSH (r = −0.99, r = −0.55, r = −0.63 and r = −0.88 in IS, IT, IIS and IIT sheep, respectively). 4. Discussion Achievement of a critical body mass and sufficient amount of adipose tissue determines the onset of puberty in mammals (Rosales Nieto et al., 2014, 2013). According to our studies, female SCP lambs reached puberty when they attained an average of 73% of their mature weight. Other reports indicate that ewe lambs enter sexual maturation when they achieve 50–70% body mass of an adult sheep (Hafez, 1952; Rosales Nieto et al., 2013). In this study, achievement of 77% body mass of a mature sheep after the postnatal age of seven months was correlated with the first ovulation in the ewe lambs which were singleton offspring of normally weighing sheep and had no predisposition to delayed puberty (IS). Moreover, sheep in this group had the highest body mass at birth as well as the highest daily weight gains starting from birth to the postnatal age of 110 days. Ewe lambs, which were twin offspring of

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months of postnatal life Fig. 6. Changes in plasma thyroid-stimulating hormone (ng/mL) of the experimental ewe lambs: IS (singleton offspring of normally weighing sheep), IT (twin offspring of normally weighing sheep), IIS (singleton offspring of fatty sheep), IIT (twin offspring of fatty sheep). The data presented are mean values of TSH levels in the blood collected from 08.00 to 11.00 a.m. (every 15 min) at 4, 5, 6, 7 and 8 months of age, respectively. A,B,C−mean values obtained at the same time for different experimental groups and labelled with different letters differ significantly (P ≤ 0.05). a,b,c−mean values obtained at different postnatal ages (in months) in the same experimental group and labelled with different letters differ significantly (P ≤ 0.05).

normally weighing sheep and were predisposed to delayed puberty due to a low birth weight resulting from multiple pregnancy (IT) ovulated four weeks later than IS sheep. The onset of puberty in this group was connected with the attainment of 77% of adult body mass. Apart from a lower birth body mass, IT sheep also showed lower daily weight gains from birth to the age of 110 days than IS sheep. This is consistent with numerous studies, which indicate that single-born lambs are heavier than twins at birth and weaning (Gardner et al., 2007; Kenyon et al., 2008). Gardner et al. (2007) have observed that the birth weight of twins is 87%, triplets 75% and quads 62% of the average body mass of singleton Mule ewe lambs. Kenyon et al. (2008) have demonstrated that Romney twins are lighter than single-born lambs, but only during the first year of age. However, according to McMillan and McDonald (1983), singleton as well as twin offspring have similar birth weights and grow at comparable rates. Moreover, it is known that ewe lambs which grow faster and accumulate fat more rapidly ovulate earlier than slower growing lambs with a lower fat accumulation rate. Therefore, single born ewes attain puberty earlier than twins (Rosales Nieto et al., 2014, 2013). Opposite findings have been reported by Kenyon et al. (2008), according to whom differences in body weight of singleton and twin offspring do not affect the onset of puberty in ewe lambs. It is also known, that maternal obesity in sheep enhances the tendency of their offspring to a low birth body weight as well as adiposity, glucose intolerance and diabetes in adult life (Ford et al., 2009; Kosior-Korzecka et al., 2012). Our studies show that singleton offspring of fatty sheep (IIS) ovulated for the first time at eight months of age. The first ovulation was correlated with the attainment of 65% adult body mass. However, twin offspring of fatty sheep (IIT) did not ovulate until the age of ten months. Moreover, sheep in this group had the lowest birth body weight and the lowest daily weight gains starting from birth to the 110th day of postnatal life compared to IS, IT and IIS ewe lambs. Interestingly, starting from the postnatal age of 200 days, IIT ewe lambs attained the highest body mass. The plasma leptin concentration is an indicator of accumulation of fat (Delavaud et al., 2000; Rosales Nieto et al., 2013). Rosales Nieto et al. (2014, 2013) have found, that ewe lambs with a higher

accumulation of fat have higher concentrations of leptin than ewe lambs with lower amounts of adipose tissue. Moreover, it is known, that leptin is a factor which trigger the onset of puberty, by initiating the HPO axis through pathways involving the kisspeptin/G proteincoupled receptors system (Hausman et al., 2012). In our study, we observed a significant progressive elevation in leptin starting from four to eight months of age. It was also observed that the elevation in leptin concentration up to 3.35 ± 0.26–3.60 ± 0.19 ng/mL was associated with the initiation of puberty in IS, IT and IIS ewe lambs. The plasma leptin concentration reached a maximum value when the IS ewe lambs had reached the postnatal age of seven and eight months. It was correlated with the occurrence of the first ovulation at seven months of age. Also in IT and IIS sheep, the highest plasma leptin concentration was observed during the onset of puberty (at eight months of age). This finding is consistent with the report of Rosales Nieto et al. (2014), which indicates that sheep with higher values of fat accumulation and leptin concentration reach puberty at a younger age. By contrast, Recabarren et al. (2004) suggest, that plasma leptin concentrations may not be connected with the onset of puberty in prepubertal ewe lambs under regular feeding. In our study, higher concentrations of leptin were observed in IIT ewe lambs. These sheep had the highest body weight compared to other groups, after 200 days of postnatal age. This hyperphysiological leptin level found in IIT sheep was correlated with a lack of ovulation up to the age of ten months. The absence of leptin signaling to stimulate the HPO axis may be caused by leptin resistance as a consequence of hyperleptinaemia. According to Kosior-Korzecka (2008), a high concentration of leptin leads to down-regulation of leptin receptors in ovine pituitary cells. The KiSS-1/GPR54 system is a key signal triggering the onset of puberty (Pinilla et al., 2012). In ewes, kisspeptin neurons in the ovine arcuate nucleus and the preoptic area are pivotal for the preovulatory GnRH/LH surge (Smith et al., 2009). According to our study, the increase in the plasma kisspeptin level up to 31.26 ± 1.54–32.24 ± 2.25 ng/mL and the elevation in pituitary KiSS-1 expression up to 1.40 ± 0.12–1.63 ± 0.22 was connected with the occurrence of the first ovulation at seven months of age in IS ewe lambs and at eight months of age in IT, IIS sheep. Our

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findings are consistent with a report of Redmond et al. (2011), which indicates that kisspeptin initiates the HPO axis during the onset of puberty in ewe lambs. An intermittent iv infusion of kisspeptin results in hourly pulses of LH, stimulation of folliculogenesis and steroidogenesis, preovulatory surge of LH, and ovulation in prepubertal ewes (Redmond et al., 2011). Other reports also demonstrate that KiSS-1 gene expression in the arcuate nucleus elevates during initiation of frequent pulsatile LH release in ewe lambs (Amstalden et al., 2010). In the present study, a significant increase in KiSS-10 secretion and KiSS-1 expression was connected with a significant elevation in leptin concentration in IS, IT and IIS ewe lambs. Also, our previous in vitro studies have demonstrated that kisspeptin secretion and expression is regulated by leptin in ´ and Kosior-Korzecka, 2016). Moreover, pubertal ewes (Radwanska it has been found that treatment of sheep of low body weight and ob/ob mice with leptin elevates cellular KiSS-1 mRNA expression in the kisspeptin neurons (Backholer et al., 2010; Smith et al., 2006). Backholer et al. (2010) have demonstrated that an icv infusion of leptin partially restores KiSS-1 expression in lean ewes. In this study, the lowest KiSS-10 concentration as well as pituitary KiSS-1 expression and a lack of ovulation up to the age of ten months was found in IIT sheep. This group of ewe lambs also had a hyperphysiological plasma leptin level. Taking into account that leptin initiates puberty via stimulation of kisspeptin, the lack of ovulation and the low level of KiSS-10 secretion and KiSS-1 expression can be connected with the absence of leptin signaling. This, in turn, may be a consequence of the down-regulation of leptin receptors under the influence of the hyperphysiological leptin concentration (KosiorKorzecka, 2008). Moreover, our previous results demonstrate that a high concentration of leptin significantly decreases KiSS-1 expression and KiSS-10 secretion in pituitary cells of pubertal ewe lambs ´ in vitro (Radwanska and Kosior-Korzecka, 2016). According to the present results, pituitary GPR54 mRNA expression decreased at the time of the first ovulation in all groups of ewes. This can be connected with the down-regulation and desensitization of GPR54 as a result of the high concentration of kisspeptin (Ramaswamy et al., 2007; Seminara et al., 2006). Also in our previous studies, a high secretion of kisspeptin and a high KiSS-1 expression, under the influence of 10−8 M of leptin, caused a reduction in the level of ´ GPR54 mRNA expression in vitro (Radwanska and Kosior-Korzecka, 2016). Hormones of the thyrotropic axis are involved in the process of sexual maturation and regulation of the reproductive function in mammals. An elevation in the thyroid gland activity occurs at oestrus in rodents and ewes (Brown-Grant, 1966; Falconer, 1970). Peeters et al. (1989) have demonstrated that the plasma T4 concentration elevates in the oestrous period, whereas the T3 level increases during the luteal phase in ewes. According to MezaHerrera et al. (2011), thyroid hormones affect the establishment of puberty in small ruminants. In goats, for example, glutamateinduced acceleration of sexual maturation is dependent on T3 concentration. In female pubertal cattle, the T3 level significantly decreases five weeks before the onset of sexual maturation, and then elevates at the onset of puberty (Leyva-Ocariz et al., 1997). In our study, fT4 increased linearly from the postnatal age of four to eight months in pubertal ewe lambs. The elevation in the fT4 concentration occurred at the time of the first ovulation in IS, IT and IIS sheep. On the other hand, IIT ewes, which did not ovulate until ten months of age, had the lowest level of fT4. These findings confirm that thyroid hormones affect the onset of puberty in ewe lambs. However, according to Wells et al. (2003), hypothyroidism, which is induced by oral administration of the thyroid inhibitor propylthiouracil (PTU) does not influence the time of the onset of puberty in ewe lambs. In this present study, we also found that the TSH concentration decreased from the age of four to eight months. The TSH concentration reached the lowest value around the onset of

puberty. This was connected with the fact that TSH is inhibited by the negative feedback of thyroid hormones (Dahl et al., 1994). Also according to Penny et al. (1983), TSH decreases with age, which can be connected with an elevated response of the thyroid gland to TSH or a decline in the metabolic clearance rate of T4. Michaud et al. (1991) have found that an elevation in TSH precedes sexual maturation, whereas an increase in thyroid hormones coincides with the onset of puberty. However, there are no other reports about changes in TSH concentration in pubertal ewes. In this present study, we also found that singleton offspring of normally weighing sheep (IS) had the highest concentration of fT4 compared to other experimental groups. Moreover, among the offspring of fatty sheep, single-born ewes had higher plasma fT4 levels than twins. This is in line with other reports, which indicate that at birth and during the preweaning period, plasma concentrations of thyroid hormones are higher in single than in twin lambs (Assane and Sere, 1990; McBee et al., 2006). Moreover, taking into account that Ob-Rb mRNA expression has been found in the hypothalamus, the pituitary and thyroid glands, the activity of the thyrotropic axis can be modified by leptin (Jin et al., 2000; Nowak et al., 2002; Tartaglia et al., 1995). According to other reports, food deprivation, which is associated with low leptin levels results in a decrease in the synthesis of TSH in the pituitary and a reduction in plasma thyroid hormone concentrations (Casanueva and Dieguez, 1999). According to Klocek-Gorka et al. (2010), leptin stimulates T3 and T4 secretion from the ovine thyroid gland in vitro. By contrast, Morrison et al. (2002) have demonstrated that an iv infusion of leptin does not significantly influence serum concentrations of T4 in growing prepubertal ewe lambs. We observed a progressive elevation in leptin concentrations and plasma concentrations of fT4 starting from four to eight months of age. Achievement of a high plasma level of leptin and fT4 was correlated with the onset of puberty in IS, IT and IIS sheep. We also found that IIT sheep, which did not ovulate until the age of ten months, had supraphysiological levels of leptin and the lowest concentrations of fT4. This lack of the stimulating action of leptin on fT4 release was probably a result of the down-regulation of the Ob-Ra and Ob-Rb receptors by the high concentrations of leptin (Kosior-Korzecka, 2008). Similarly to our results, De Oliveira et al. (2007) have noted that leptin can exert a stimulating as well as an inhibiting effect on the activity of the thyrotropic axis in rats in vivo. Acute treatment with leptin increases the levels of TSH, fT3 and fT4. However, chronic administration of leptin does not change the secretion of TSH and thyroid hormones, probably as a result of partial thyroid resistance to leptin. In the experiments reported in this article, a negative relationship was found between the concentration of leptin and that of TSH, which was inhibited by the increased level of fT4. We observed a significant reduction in TSH concentration starting from four to eight months of age. This observation is consistent with data, which indicate that leptin stimulates thyroid hormone secretion, which in turn suppresses TSH release in female rats, probably by negative feedback (Nowak et al., 2002). There are few reports showing that thyrotropic axis activity can also be modified by kisspeptin. Our previous results demonstrated that short exposure of pituitary cells of pubertal ewe lambs to kisspeptin influenced TSH secretion in vitro. KiSS-10 at a concentration of 10−11 –10−8 M significantly elevated TSH secretion from ovine pituitary cells after 2 h. Conversely, TSH secretion was not changed under the influence of KiSS-10 after 6, 12, 18, 24 ´ or 30 h (Radwanska and Kosior-Korzecka, 2016). This last result is in accordance with the findings of Luque et al. (2011), who have demonstrated that there is no significant relationship between the effect of kisspeptin and TSH secretion from pituitary cells in female baboons (Papio Anubis) in vitro. Similarly, Ramaswamy et al. (2009) have reported that administration of kisspeptin does not influence TSH secretion in male rhesus monkeys in vivo. Studies on humans have shown that acute or chronic treatment with kisspeptin does

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not affect TSH release in women (Narayanaswamy et al., 2014). However, there are no data on the relationship between kisspepin and thyrotropic axis activity in pubertal ewe lambs in vivo. In this study we found a progressive increase in KiSS-1 expression and plasma concentrations of KiSS-10 and fT4 starting from the age of four to eight months, with high values around the time of the first ovulation. Taking into account that the TSH concentration is inhibited by the negative feedback of thyroid hormones (Dahl et al., 1994), the plasma thyroid-hormone concentration reached a minimum value after eight months of age in all groups of pubertal ewe lambs. 5. Conclusions This study indicates that IS ewe lambs have the highest birth body weight and ovulate for the first time at seven months of age. This is four weeks earlier than IT and IIS sheep. The onset of puberty in the ewe lambs is dependent on the mother’s body mass and the leptin level as well as the birth type (singleton/twin). An increase in the leptin concentration was associated with the onset of puberty in IS, IT and IIS ewe lambs. By contrast, IIT ewes with a hyperphysiological level of leptin did not ovulate until the age of ten months. Furthermore the highest concentrations of KiSS-10 and the highest pituitary KiSS-1 expression were connected with the occurrence of the first ovulation in IS, IT and IIS sheep, respectively. On the other hand, pituitary GPR54 mRNA expression decreased at the time of the first ovulation. An increase in thyroid gland activity was dependent on the mother’s body mass, birth type and occurred at the time of the first ovulation. Conflict of interests The authors declare no conflict of interest. Acknowledgement The study was supported by the Polish Ministry of Science and Higher Education, grant No. NN308598439.

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