- Email: [email protected]
Feeding prepubescent gilts a high-fat diet induces molecular changes in the hypothalamus-pituitary-gonadal axis and predicts early timing of puberty
Accepted Manuscript Feeding prepubescent gilts a high fat diet induces molecular changes in the hypothalamus - pituitary - gonadal axis and predicts the early timing of puberty Yong zhuo, Ph.D. Dongsheng Zhou, Ph.D. Lianqiang Che, Ph.D. Zhengfeng Fang, Ph.D Yan Lin, Ph.D. De Wu, Ph.D. PII:
S0899-9007(14)00037-9
DOI:
10.1016/j.nut.2013.12.019
Reference:
NUT 9196
To appear in:
Nutrition
Received Date: 29 July 2013 Revised Date:
5 December 2013
Accepted Date: 25 December 2013
Please cite this article as: zhuo Y, Zhou D, Che L, Fang Z, Lin Y, De Wu , Feeding prepubescent gilts a high fat diet induces molecular changes in the hypothalamus - pituitary - gonadal axis and predicts the early timing of puberty, Nutrition (2014), doi: 10.1016/j.nut.2013.12.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Feeding prepubescent gilts a high fat diet induces molecular changes in the hypothalamus - pituitary -
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gonadal axis and predicts the early timing of puberty
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Yong zhuo Ph.D. 1, Dongsheng Zhou Ph.D. 1,2, Lianqiang Che Ph.D. 1, Zhengfeng Fang Ph.D1., Yan Lin
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Ph.D.1,De Wu Ph.D. 1*
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Education of China, Sichuan Agricultural University, Ya’an, P.R. China. 625014, and 2Shenzhen Premix Inve
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Nutrition CO. LTD, Shenzhen, P.R. China, 518103
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Institute of Animal Nutrition, and Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of
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Running head: High fat feeding predicts early attainment of puberty
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* Corresponding author. Tel: 86 835 2885107. Fax:86 835 2885065
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Email: [email protected]
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ACCEPTED MANUSCRIPT Objective
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The onset of puberty in females has occurred earlier over the past decades, presumably due to improved
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nutrition in developed countries. However, the underlying molecular mechanisms responsible for the early
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attainment of puberty by enforced nutrition remain largely unknown. In the present study, we evaluated the
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hormonal and gene expression changes in prepubescent gilts fed a high fat diet and investigated whether these
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changes could predict the early timing of puberty.
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Methods
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Forty gilts were daily fed a basal diet (LE) or basal diet with an additional 270 g/d or 340 g/d of fat (HE) during
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the prepubescent phase. Blood samples were collected during the prepubescent phase to detect hormonal
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secretion changes in insulin-like growth factor-I (IGF-I), kisspeptin, estradiol, progesterone and leptin. The gene
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expressions at in the hypothalamus - pituitary - gonadal axis were examined on d 73 of the experiment (average
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age on d 177) during the prepubescent phase.
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Results
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An HE diet resulted in accelerated bodyweight gain and back-fat thickness at the P2 point compared with LE
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gilts during the prepubescent phase. Gilts that were fed the HE diets attained puberty 12 days earlier than LE
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gilts, and a larger proportion of HE gilts reached puberty at d 180 or d 190 of age. A post-mortem analysis
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revealed a promoted development of the uterus and ovary tissue that was characterised by a 53.7% and 29.5%
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increase in the uterine and ovary weight, respectively, and an increased length of the uterine horn and oviduct
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tissue in HE gilts. Real-time quantitative PCR revealed that HE gilts had higher Kiss-1, G-protein coupled
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receptor 54 (GPR54), gonadotropin-releasing hormone (GnRH) and oestrogen receptor α (ERα) mRNA
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expression levels in the hypothalamic anteroventral periventricular nucleus (AVPV); the leptin receptor (ObR)
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mRNA expression level was higher in the hypothalamic arcuate nucleus (ARC) and ovary tissue; the insulin-like
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growth factor
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follicle-stimulating hormone (FSH) and luteinising hormone (LH) mRNA expression levels were higher in the
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pituitary gland.
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Conclusion
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These data showed that the consumption of additional fat can facilitate early attainment of puberty, which can be
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predicted by the changes in secreted hormones and gene expression in the hypothalamus - pituitary - gonadal
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axis.
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Keywords
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High fat diet; Hormone; Gene expressions; Puberty; Gilts
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receptor (IGF- R) expression was higher in the pituitary and ovary tissues, and the
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Introduction The onset of puberty in females, as measured by the age at menarche, is estimated to have advanced by
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6-12 months per 100 years between the 18th century and the 21th century in several northern European countries
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[1]. This declining age of puberty has been attributed to accelerated growth due to improved nutrition, e.g.,
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obese girls tend to mature earlier than normal or thin girls [2-4]. Thus, the accelerated growth rate due to
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nutrition fortification seems to be a more important index in predicting the early onset of puberty [5]. Although
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body condition and the timing of puberty show a strong link in developing girls, the underlying mechanism
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remains largely unknown.
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Puberty is a complex biological process that involves the secretion of gonadotropin-releasing hormone
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(GnRH) by the hypothalamus, sexual development, adrenal maturation, pubertal development and
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gametogenesis. Notably, the secretion of GnRH by the hypothalamus represents the first known step in the
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reproductive cascade to initiate the activation of pituitary and gonadal function. Therefore, understanding the
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neuroendocrine control of GnRH secretion may provide insight into the normal reproduction or disorder of the
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pubertal process. Recently, pharmacological and genetic studies revealed that GPR54, a G protein–coupled
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receptor gene, may act as a gatekeeper for normal GnRH physiology and puberty [6]. Kisspeptins, which are
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encoded by the metastasis suppressor gene Kiss-1, are a natural ligand for GPR54 to elicit a GnRH surge and
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puberty onset [7-8]. Kiss-1 and GPR54 may be involved in the early timing of puberty due to accelerated growth.
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Thus, their gene expression levels represent an interesting research topic.
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In the present study, developing gilts were fed an oil-rich diet in order to induce hormonal and molecular
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changes in the hypothalamus, pituitary tissue and gonadal tissues. These changes were used to assess whether
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hormonal and molecular changes could predict the early onset of puberty.
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Materials and methods
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All experimental procedures were approved by the Animal Care and Use Committee of Sichuan
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Agricultural University.
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Animals and diets Forty Landrace × Yorkshire crossbred gilts of initially similar body weight (55 ± 1.5 kg) and age (104 ± 2 d)
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were paired and fed one of two nutritional regimens. Twenty gilts were fed basal diets (LE) formulated to
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provide 3.22 Mcal/kg digestible energy, 19.1% crude protein and 3.25% ether extract, as recommended by NRC
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(1998). LE gilts in the 55 to 100 kg phase and 100 kg to puberty phase were fed a 1.8 kg and 2.1 kg basal diet,
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respectively. The other twenty gilts (HE) were fed basal diets containing an additional 270 g/d or 340 g/d fat
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during these two phases, respectively. Finally, due to additional fat intake, HE gilts consumed 40.7% and 44.1%
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more digestible energy than LE gilts during the 55 to 100 kg phase and 100 kg to puberty phase, respectively.
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The intakes of protein, minerals and vitamins were similar for both LE and HE gilts (Table 1). Gilts were housed
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individually (2 m × 0.8 m) in a breeding facility and fed twice daily at 0830 and 1430 h, and water was
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provided ad libitum. The environment temperature was controlled at 20 - 24
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simultaneously reared and fed LE or HE diets and used to substitute gilts that were culled due to lameness or
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illness. If gilts in the LE or HE were culled, the corresponding paired gilts were discarded as well.
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Determination of body weight and back-fat thickness
. Another subgroup of gilts was
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The fasted bodyweights were determined in the morning prior to feeding at the beginning of the experiment
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and at d 52, d 59, d 66 and d 73 of the experiment. The back-fat thickness was measured 6 cm from the midline
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of the last rib (P2 thickness) using a LEAN MEATER (RENCO-LEAN MEATER 23249, USA) at beginning of
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experiment and at d 52, d 59, d 66 and d 73 of experiment.
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Oestrus detection
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Oestrus was not induced by exogenous hormone administration for these gilts. Oestrus was carefully
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encourage pubertal oestrus. Oestrus was detected by only one experienced stockperson based on behavioural
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and vulvar characteristics. The appearance of a pink vulva and vaginal orifice mucous were important signs of
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oestrus initiation, while standing still under applied back pressure (standing reflection) was used as an important
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behavioural criterion to establish onset of oestrous. The age at puberty was recorded on the day of oestrus.
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Tissue sample collection
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In present study, gilts began to express oestrus at d 72 of experiment. To examine nutrition-induced
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molecular changes in the hypothalamus-pituitary- gonadal axis, 10 pair-fed prepubescent gilts from
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corresponding LE and HE groups were slaughtered to collect tissue samples from the hypothalamus, pituitary
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tissue and gonadal tissue.
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The brains were removed from the skull immediately after slaughter, and the excess tissues were removed.
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The hypothalamus samples were collected as previously described [9]. Briefly, the hypothalamus was dissected
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along the following boundaries: laterally 8 mm from either side of the third ventricle, longitudinally 8 mm from
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the optic chiasm to the posterior border of the mammillary bodies and 20 mm above the top surface of the
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thalamus. The anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of the hypothalamus
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were dissected out as previously described [9].
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The uterus, oviduct and ovary tissues were removed from the abdominal cavity immediately after slaughter
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and pruned for excess intestinal and other viscera tissues. The uterus was weighed, and the length of the uterus
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and oviduct were measured. These tissues were washed three times with PBS followed by drying with paper
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towels prior to weighing.
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All hypothalamic, pituitary and ovary samples were washed with PBS, dried with paper, frozen in liquid nitrogen and stored at -70
for the future analysis of gene expressions.
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Determination of hormone concentrations To explore the hormonal secretion changes of gilts fed a diet containing added oil, 10 mL blood samples
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were collected from both LE and HE gilts by jugular puncture at the beginning of the experiment and at d 52, d
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59, d 66 and d 73 of the experiment during the prepubescent phase. The blood samples were centrifuged at
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2400×g for 15 min at 4 ºC to collect serum and stored at -20 ºC for the future analysis of hormone
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concentrations.
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An enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of IGF-I
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(commercial kit from Alpco diagnostics, USA), kisspeptin (commercial kit from Phoenix pharmaceuticals, Inc.
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USA), leptin (commercial kit from R&D, USA), oestradiol (commercial kit from R&D, USA) and progesterone
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(commercial kit from R&D, USA). The detection limits of IGF-I, kisspeptin, oestradiol, progesterone and leptin
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were 0.09 ng/mL, 0.01 ng/mL, 1.1 pg/mL, 0.01 ng/mL and 0.01 ng/mL, respectively. The intra- and inter-assay
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coefficients of variation were 5.3% and 5.9%, 5.0% and 4.7%, 5.6% and 6.9%, 5.7% and 6.8%, 5.4% and 6.1%,
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for IGF-I, kisspeptin, estradiol, P4 and leptin, respectively.
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Gene expressions
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The gene expression levels were detected using a real-time quantitative polymerase chain reaction (PCR).
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The primers for the target genes are presented in table 2. The mRNA expression levels of Kiss-1, GPR54, GnRH,
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follicle-stimulating hormone (FSH), luteinising hormone (LH), insulin-like growth factor
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insulin receptor (Ins-R), leptin receptor (Ob-R), oestrogen receptor α (ERα) and progesterone (P4R) were
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determined with real-time quantitative PCR. Total RNA was isolated from tissue samples using Trizol Reagent
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(Invitrogen, USA) according to the manufacturer’s instructions. The quality and purity of RNA samples was
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determined by electrophoresis through a 1% denaturing agarose gel and separately assessed using a nucleic
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acid/protein analyser (Beckman DU-800, USA) from the OD260:OD280. The ribose nucleic acid samples were
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receptor (IGF-
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1µg of total RNA was taken for reverse transcription using the AMV First Strand cDNA Synthesis Kit (TaKaRa,
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Dalian, China). cDNA synthesis was performed using the PrimeScriptTM reagent kit (TaKaRa, Dalian, China)
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according to the manufacturer's instructions. The concentration of RNA in the final preparations was calculated
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from the OD260, and the samples were diluted with Rnase-free dH2O (TaKaRa, Dalian, China) for reverse
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transcription. The cDNA was easily dissolved in H2O (TaKaRa, Dalian, China) for real-time quantitative PCR.
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Real-time quantitative PCR was performed in a CFX-96 Real-Time PCR detection System (Bio-Rad, USA).
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Briefly, 12.5 µL SYBR Premix Ex TaqTM (contains Taq HS, dNTP, MgCl2, SYBR Green), 0.5 µL of each
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primer and 2.0 µL cDNA were included in a 25 µL PCR. The RT-PCR cycling conditions were defined as
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follows: an initial pre-denaturing cycle at 95
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denaturation at 95
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collect fluorescence. The melting curve conditions were defined as follows: 1 cycle of denaturation at 95℃ for
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10 s, followed by a gradual ramp from 65℃ to 95℃ at 0.5℃/s. The relative expression of the target gene was
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calculated as described previously [10]. The relative gene expression levels were normalised to those of the
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house-keeping gene, β-actin. The outcomes were expressed as fold changes relative to average mRNA levels of
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genes in LE groups.
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Statistical analysis
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for 1 min, followed by 40 cycles of amplification, defined by for 30 s, followed by plate reading to
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for 5 s, annealing for 30 s and extension at 72
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Gilts were fed individually in the present study, and mRNA expression levels, hormone concentrations,
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tissue weight, bodyweight and P2 back-fat thickness from corresponding LE and HE gilts were analysed using
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paired T-tests in the SPSS (10.5) software package. The results were presented as the means ± SEM. A
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chi-square test was used to analyse the differences in the fractions of gilts that reached puberty before age 180
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d, 190 d and 210 d. Statistical significance was declared when P < 0.05.
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Results
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Age at puberty in gilts As shown in table 3, gilts fed HE diets reached puberty twelve days earlier than gilts fed LE diets (P =
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0.08). The rate of gilts that reached puberty by age d 180, d 190 and d 210 in LE and HE gilts were 13% versus
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47%, 47% versus 73% and 93% versus 87%, respectively.
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Bodyweight and back-fat thickness in response to LE and HE diets
As shown in Fig. 1 and Fig. 2, the body weight and back-fat thickness at P2 point of gilts fed HE diets were
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significantly higher than those of LE gilts at d 52, d 59, d 66 and d 73 of the experiment (P < 0.01).
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Uterus and ovary tissue development in response to LE and HE diets
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As shown in table 4, the development of the uterus and ovarian tissue were significantly accelerated in gilts
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that were fed the HE diet. The uterus weight, body of the uterus, length of the left horn of the uterus, length of
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the right horn of the uterus, length of the left oviduct, length of the right oviduct and ovary weight were
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significantly decreased in gilts fed LE diets as compared to gilts fed HE diets (P < 0.05).
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Hormonal changes in response to LE and HE diets
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As shown in Fig. 3, the HE diet did not affect the circulating oestradiol concentrations at d 0, d 52, d 59
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and d 66 of the experiment, but gilts fed HE diets had significantly higher oestradiol concentrations than LE
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gilts at d 73 of the experiment (P < 0.05). The circulating insulin-like growth factor
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were higher in gilts fed HE diets than in gilts fed LE diets at d 52, d 59, d 66 and 73 of the experiment (P <
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0.05). The HE diet did not affect the circulating leptin concentrations at d 52 and d 59 of the experiment, but this
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concentration was higher in gilts fed HE diets than gilts fed LE diets at d 66 and 73 of the experiment (P < 0.05).
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The HE diet did not affect the circulating kisspeptin and progesterone concentrations during the entire
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experiment period.
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(IGF-
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Gene expressions The gene expression levels of gilts fed LE or HE diets are presented in figure 4 (A-D). As shown in figure 4
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(A), the expression of Kiss-1, GPR54, GnRH and ERα mRNA in the hypothalamic AVPV from HE gilts were
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increased compared with LE gilts (P < 0.05), but this difference did not persist in the hypothalamic ARC (figure
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4B). The expression levels of ObR mRNA in the hypothalamic ARC (figure 4B) and ovary tissues (figure 4D)
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were higher in HE gilts compared with LE gilts (P < 0.05), but this difference did not persist in the hypothalamic
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AVPV (figure 4A) or in pituitary tissue (figure 4C). The expression of IGF-
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4C) and ovary tissues (figure 4D) in HE gilts compared with LE gilts (P < 0.05). FSH and LH mRNA
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expressions in pituitary gland (figure 4C) were increased in gilts fed HE diets compared with gilts fed LE diets
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(P < 0.05). Ins-R (figure 4C and figure 4D) and P4R expressions (figure 4A and figure 4B) in their respective
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tissues were not affected by diets.
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Discussion
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Puberty is the process of physical changes by which a child’s body matures into an adult body that is
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capable of fertilisation to reproduce offspring. A recent study indicated that girls have entered puberty at
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increasingly younger ages of the past several decades, and prepubescent obesity is a predictor of early onset
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puberty. Therefore, underlying mechanisms that are involved in this process are an interesting subject of study.
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In the present study, gilts that consumed extra fat reached puberty twelve days earlier than gilts that were a
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low fat diet, and a larger proportion of gilts that were fed a high fat diet reached puberty at d 190 of age.
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Undoubtedly, the higher energy consumption as a result of the extra fat intake was the leading cause of early
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onset puberty. Several earlier studies conducted in humans also indicated that obese girls experienced menstrual
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cycles earlier than girls that were not overweight [3-4]. These results fit the hypothesis that girls must achieve a
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minimum fatness and bodyweight in order to trigger the onset of menstrual cycles [11]. Generally, bodyweight
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and the initiation of the hormonal events of puberty are strongly associated, even though bodyweight or body fat
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might not be an absolute determinant of puberty onset [12]. Little evidence is currently available to elucidate the complex biologic process by which excess nutrition
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induces early puberty onset. The hypothalamic activation and secretion of GnRH is currently known as the
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crucial up-stream regulator that initiates the reproductive cascade to stimulate the pulsatile release of FSH and
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LH [13]. In the present study, gilts that were fed a HE diet showed higher expression levels of the GnRH gene in
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the hypothalamus AVPV. Coincidently, the FSH and LH expression in the pituitary gland increased in response
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to this GnRH expression change. Therefore, the higher GnRH, FSH and LH expression was expected to induce
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early puberty onset in the present study. Given the master position of hypothalamic GnRH in controlling the
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gonadotropic axis, GnRH was noted as the target of multiple regulators of central and peripheral origin, and a
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wide array of excitatory and inhibitory circuits that govern GnRH secretion have been identified [14-15]. Based
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on genetic and pharmacological studies, an unsuspected key role for the Kiss-1/GPR54 system in the
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hypothalamic control of the GnRH-gonadotropin axis has recently been accepted as a gatekeeper of puberty [16].
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Kiss-1 was originally identified as a metastasis suppressor gene that encodes a number of structurally related
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peptides, which are termed kisspeptins [17]. The direct central administration of kisspeptins to hypothalamic
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GnRH neurons could stimulate GnRH release and puberty onset [18]. In the present study, the gene expression
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of Kiss-1/GPR54 was evaluated in the hypothalamic AVPV and ARC. The Kiss-1 and GPR54 mRNA expression
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levels in the AVPV increased coincidently with the increased hypothalamic AVPV GnRH mRNA expression.
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Interestingly, we did not find a difference in the Kiss-1/GPR54/GnRH gene expression level in the hypothalamic
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ARC in the present study. Kiss-1 is expressed most abundantly in the ARC and AVPV [9], and the kisspeptin
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neurons in these two areas appear to be functionally different [19-21]. The results of the present study also
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indicated that Kiss-1/GPR54 might function differently in the AVPV and ARC. However, the distinct role of
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AVPV and ARC in linking nutrition and GnRH secretion requires further examination. Nevertheless, given the
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“gatekeeper” role of the kisspeptin/GPR54 system in initiating puberty onset, the increased hypothalamic kiss-1
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and GPR54 mRNA expression level is likely to be an important mediator of early onset puberty. The hormonal changes appeared to be a crucial event that linked the nutritional effects on reproductive
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maturity and puberty initiation [22-23]. In the present study, the oestradiol concentration was not affected during
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the prepubescent phase but was higher when approaching the age of puberty. Oestradiol was necessary in order
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to initiate oestrus and ovulation, and it has recently been observed to play important positive role in the
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epigenetic regulation of Kiss-1 expression [24]. The increased mRNA expression of ERα in the hypothalamic
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AVPV confirmed the previous hypothesis that oestradiol signalling may participate in the positive feedback
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regulation of GnRH secretion by up-regulating Kiss-1 expression [20, 25]. The hormone leptin is produced by
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adipose tissue and was initially thought to primarily regulate energy balance by decreasing food intake and
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increasing energy expenditure [26]. Additionally, leptin treatment prevents the effects of fasting on reproductive
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processes in a variety of species [27], which implies that it has a role in regulating the reproductive process.
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Because obese girls reach puberty earlier than normal or thin girls, leptin is proposed as an important signal that
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triggers puberty. When normal prepubescent female mice were injected with leptin, they consumed less food
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and grew at a slower rate than the controls but reproduced up to 9 days earlier than the controls [28]. In the
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present study, the circulating leptin concentration increased in gilts fed extra fat. To explore the changes in the
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leptin signalling pathway in the hypothalamus - pituitary - gonadal axis, the leptin receptor mRNA expression,
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ObR, was measured using RT-PCR; the expression of ObR was higher in the hypothalamic ARC and ovarian
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tissue, which suggests that leptin plays an important role in the regulation of reproductive maturation. Contrary
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to ObR expression changes in the ARC, the ObR expression in the hypothalamic AVPV remained unchanged
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[29]. ARC was recently considered as a hypothalamic centre that couples energy metabolism and reproduction.
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reproduction [30]. However, the mechanism by which metabolic signalling changes in the hypothalamic ARC to
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potentially be transmitted to the hypothalamic AVPV and induce the activation of the Kiss-1/GPR54 system
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remains unclear. In addition to the neuroendocrine control of FSH and LH, metabolic signalling could act
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directly on the pituitary - gonadal axis to promote reproductive maturation. IGF-
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and gonadal tissues, and IGF-
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culture of pituitary cell [31]. In the present study, the peripheral IGF-
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were fed a fat-rich diet, and the expression of IGF-
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gilts. These findings indicate that the early maturation of reproduction could be attributed, at least in part, to the
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increased IGF-
signalling. However, we could attribute these phenomena to either the direct effects of
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peripheral IGF-
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regulator of reproductive neuroendocrine function [32].
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Conclusion
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R was expressed in pituitary
administration could stimulate FSH and LH secretions during the in vitro
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concentration increased in gilts that
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in the brain has also been proposed as a
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Collectively, the results presented herein suggest that accelerated growth and body fat accumulation induce
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molecular changes in the Kiss-1/GPR54 system and the hormonal secretion, which predict an early onset of
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puberty.
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Acknowledgements
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This work was supported by the National Natural Science Foundation (30871804) of P.R China. The authors
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also wish to thank the staff in the laboratory for their on-going assistance.
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[27] Schneider JE, Zhou D, Blum RM: Leptin and metabolic control of reproduction. Horm Behav 2000; 37(4):306-326. [28] Chehab FF, Mounzih K, Lu R, Lim ME. Early onset of reproductive function in normal female mice treated
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with leptin. Science 1997; 275(5296):88–90. [29] Bouret SG, Draper SJ, Simerly RB. Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 2004; 304:108 –110. [30] Sawchenko PE. Toward a new neurobiology of energy balance, appetite, and obesity: the anatomists weigh
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in. J Comp Neurol 1998; 402:435–441.
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Biol Reprod 2007; 76(6):1016-1024.
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[32] Daftary SS, Gore AC. IGF-1 in the brain as a regulator of reproductive neuroendocrine function. Exp Biol Med (Maywood) 2005; 230(5):292-306.
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Figure 1. Changes in bodyweight of gilts fed LE or HE diets at different days of the experiment. LE denotes
365
gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning
366
day of the experiment. ** denotes P < 0.01. The results are expressed as the mean values ± SEM, n = 20 gilts
367
per group.
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Figure 2. P2 thickness of gilts fed LE or HE diets at different days of the experiment. LE denotes gilts fed basal
369
diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the
370
experiment ** denotes P < 0.01. The results are expressed as the mean values ± SEM, n = 20 gilts per group.
371
Figure 3. Changes in hormone concentrations of prepubescent gilts fed LE or HE diets. LE denotes gilts fed
372
basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil supplements. D 0 was
373
the beginning day of the experiment. * denotes P < 0.05. The blood samples were collected from HE gilts and
374
corresponding paired LE gilts to determine the concentrations of hormones. The results are expressed as the
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mean values ± SEM, n = 20 gilts per group.
Figure 4. Relative mRNA expression levels in the respective tissue samples of prepubescent gilts fed LE or
377
HE diets. Figure 4 (A, B, C and D) denotes the relative mRNA expressions in AVPV (anteroventral
378
periventricular nucleus), ARC (arcuate nucleus), pituitary and ovarian tissues, respectively. LE denotes
379
gilts fed basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil
380
supplements. * denotes P < 0.05 versus LE. The tissue samples were collected from HE gilts and
381
corresponding paired LE gilts to determine the gene expression levels. The results are expressed as the
382
mean values ± SEM, n = 5 gilts per group.
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ACCEPTED MANUSCRIPT 397 Table 1 Daily nutrient intake in gilts fed LE or HE diets1
398
55 - 100 kg
100 kg - puberty HE
LE
HE
Intake of basal diet (g/d)
1800
1800
2100
2100
Oil intake (g/d)
0
270
0
340
Total feed intake (g/d)
1800
2070
2100
2440
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LE
5.80
8.16
6.76
9.74
Crude protein
344
344
401
401
Starch
842
842
983
983
Lipid
59
329
68
408
51
51
59
59
16
16
19
19
Calcium
17
17
20
20
Phosphorus
13
13
15
15
9
9
10
10
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Lysine
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Digestible energy (Mcal/d)
Crude fibre
Available Phosphorus
399
1
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Calculated daily nutrient intake (g/d)
LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements.
400 401 402 403
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Table 2. Primer sequences of genes Genebank
Amplification
Gene
Annealing
Primer sequence length (bp) 5’-GGGGGACCTCATCGTGCCAG-3’
Kiss-1
AB466320
179
GPR54
DQ459345
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5’-CGGAAACACAGTCACATA-3’
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5’-CGCCTGCCGCTTTCCGTA-3’ 5’-GCTCTACTCTACCCCCTA-3’
temperature (
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accession No.
65
115
60
119
62
117
61
142
60.5
129
59
102
59.5
101
60
110
60
5’- CTGCTGACTCTGTGTGTAGTG -3’ GnRH
NM_214274.1
5’-ACCTCTTTGGCCATCTCTTG-3’
5’-GTGGTGTGCTGGCTATTGCTAC-3’ FSH
NM_213875
TE D
5’-CCAGGTACTTTCACGGTCTCGTA-3’ 5’-AGAGACTGCTGTTGTGGCTGCT-3’ LH
NM_214080
5’-CTGGTGGTAAAGGTGATGCAGAC-3’
IGF-
R
NM_214172
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5’-GGAGATTTTGGGATGACGAGAG-3’
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5’-ATCAGAGTGCGTGGTGAAGACT-3’
5’-GTGTTGTGATTGGAAGCATTTAT -3’
Ins-R
AF102858
5’-GGGGCTCATGGAGTCACGGT-3’
5’-ATTTCTGCTATCTGGCTATA-3’ Ob-R
NM_001024587 5’-GTTTCACCACGGAATC-3’ 5’-CTGGACAAGATCACAGACACCT-3’
ERα
NM_214220 5’-CTGAAGTGAGACAGGATGAGGAG-3’
20
)
ACCEPTED MANUSCRIPT 5’-CCCTAGCTCACAGCGTTTCT-3’ P 4R
GQ903679.1
184
61
104
60
5’- CACCATCCCTGCCAATATCT -3’ 5’-GGCCGCACCACTGGCATTGTCAT-3’ β-actin
DQ845171.1
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5’-AGGTCCAGACGCAGGATGGCG-3’
406
GPR54, G-protein coupled receptor 54; GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating
407
hormone; LH, luteinizing hormone; IGF- R, insulin-like growth factor
408
Ob-R, leptin receptor; ERα, oestrogen receptor α; P4R, progesterone receptor;
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receptor; Ins-R, insulin receptor;
420 421 422 423
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ACCEPTED MANUSCRIPT 424 Table 3 Age at puberty and rate of puberty until d 180, d 190 and d 210 of age in gilts fed LE or HE diets1,2,3 LE
HE
Significance
Number of gilts, n
15
15
--
Age at puberty (d)
191.3 ± 5.6
179.0 ± 5.5
P = 0.08
Rate of gilts attaining puberty until age 180 d (%)
13 (2)
47 (7)
P = 0.11
Rate of gilts attaining puberty until age 190 d (%)
47 (7)
73 (11)
P = 0.26
Rate of gilts attaining puberty until age 210 d (%)
93 (14)
87 (13)
P = 1.0
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Items
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425
1
LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements.
427
2
The data on the rate of gilts that reached puberty at 180 d, 190 d and 210 d were analysed Chi-square test.
428
3
Number in the bracket indicates number of gilts that reached oestrus.
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ACCEPTED MANUSCRIPT 442 Table 4 Uterus and ovary tissue development in gilts fed LE or HE diets1,2,3 HE
Significance
Number of gilts, n
5
5
--
Uterus weight (g)
276.6 ± 44.1
425.2 ± 52.5
Body of uterus (cm)
21.6 ± 2.9
29.5 ± 2.0
Length of left horn of uterus (cm)
51.3 ± 7.5
69.7 ± 7.2
Length of right horn of uterus (cm)
44.3 ± 5.1
62.5 ± 5.2
Length of left oviduct (cm) Length of right oviduct (cm) Ovary weight (g)
**
18.1 ± 3.9
27.3 ± 2.7
*
6.5 ± 0.5
8.4 ± 0.3
*
2
446
prepubescent gilts were slaughtered at d 73 of the experiment and d 177 of age.
447
3
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LE denotes gilts fed basal diet, HE denotes gilts fed basal diet with additional oil supplements. These
* denotes P < 0.05, ** denotes P < 0.01.
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450
*
*
445
449
*
27.5 ± 2.7
1
448
*
18.4 ± 2.4
444
Data were presented as the means ± SEM
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LE
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443
451 452 453 454
23
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Figure 1. Changes in bodyweight of gilts fed LE or HE diets at different days of the experiment. LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the experiment. ** denotes P < 0.01. The results are expressed as the mean
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values ± SEM, n = 20 gilts per group.
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Figure 2. P2 thickness of gilts fed LE or HE diets at different days of the experiment. LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the experiment ** denotes P < 0.01. The results are expressed as the mean values ±
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SEM, n = 20 gilts per group.
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Figure 3. Changes in hormone concentrations of prepubescent gilts fed LE or HE diets. LE denotes
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gilts fed basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the experiment. * denotes P < 0.05. The blood samples were collected from HE gilts and corresponding paired LE gilts to determine the concentrations of hormones. The results are expressed as the mean values ± SEM, n = 20 gilts per group.
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ACCEPTED MANUSCRIPT Figure 4. Relative mRNA expression levels in the respective tissue samples of prepubescent gilts fed LE or HE diets. Figure 4 (A, B, C and D) denotes the relative mRNA expressions in AVPV (anteroventral periventricular nucleus), ARC (arcuate nucleus), pituitary and ovarian tissues, respectively. LE denotes gilts
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fed basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil supplements. * denotes P < 0.05 versus LE. The tissue samples were collected from HE gilts and corresponding paired LE gilts to determine the gene expression levels. The results are expressed as the mean values ± SEM, n = 5 gilts
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per group.
S0899-9007(14)00037-9
DOI:
10.1016/j.nut.2013.12.019
Reference:
NUT 9196
To appear in:
Nutrition
Received Date: 29 July 2013 Revised Date:
5 December 2013
Accepted Date: 25 December 2013
Please cite this article as: zhuo Y, Zhou D, Che L, Fang Z, Lin Y, De Wu , Feeding prepubescent gilts a high fat diet induces molecular changes in the hypothalamus - pituitary - gonadal axis and predicts the early timing of puberty, Nutrition (2014), doi: 10.1016/j.nut.2013.12.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Feeding prepubescent gilts a high fat diet induces molecular changes in the hypothalamus - pituitary -
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gonadal axis and predicts the early timing of puberty
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Yong zhuo Ph.D. 1, Dongsheng Zhou Ph.D. 1,2, Lianqiang Che Ph.D. 1, Zhengfeng Fang Ph.D1., Yan Lin
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Ph.D.1,De Wu Ph.D. 1*
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1
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Education of China, Sichuan Agricultural University, Ya’an, P.R. China. 625014, and 2Shenzhen Premix Inve
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Nutrition CO. LTD, Shenzhen, P.R. China, 518103
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Institute of Animal Nutrition, and Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of
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Running head: High fat feeding predicts early attainment of puberty
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* Corresponding author. Tel: 86 835 2885107. Fax:86 835 2885065
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Email: [email protected]
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ACCEPTED MANUSCRIPT Objective
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The onset of puberty in females has occurred earlier over the past decades, presumably due to improved
25
nutrition in developed countries. However, the underlying molecular mechanisms responsible for the early
26
attainment of puberty by enforced nutrition remain largely unknown. In the present study, we evaluated the
27
hormonal and gene expression changes in prepubescent gilts fed a high fat diet and investigated whether these
28
changes could predict the early timing of puberty.
29
Methods
30
Forty gilts were daily fed a basal diet (LE) or basal diet with an additional 270 g/d or 340 g/d of fat (HE) during
31
the prepubescent phase. Blood samples were collected during the prepubescent phase to detect hormonal
32
secretion changes in insulin-like growth factor-I (IGF-I), kisspeptin, estradiol, progesterone and leptin. The gene
33
expressions at in the hypothalamus - pituitary - gonadal axis were examined on d 73 of the experiment (average
34
age on d 177) during the prepubescent phase.
35
Results
36
An HE diet resulted in accelerated bodyweight gain and back-fat thickness at the P2 point compared with LE
37
gilts during the prepubescent phase. Gilts that were fed the HE diets attained puberty 12 days earlier than LE
38
gilts, and a larger proportion of HE gilts reached puberty at d 180 or d 190 of age. A post-mortem analysis
39
revealed a promoted development of the uterus and ovary tissue that was characterised by a 53.7% and 29.5%
40
increase in the uterine and ovary weight, respectively, and an increased length of the uterine horn and oviduct
41
tissue in HE gilts. Real-time quantitative PCR revealed that HE gilts had higher Kiss-1, G-protein coupled
42
receptor 54 (GPR54), gonadotropin-releasing hormone (GnRH) and oestrogen receptor α (ERα) mRNA
43
expression levels in the hypothalamic anteroventral periventricular nucleus (AVPV); the leptin receptor (ObR)
44
mRNA expression level was higher in the hypothalamic arcuate nucleus (ARC) and ovary tissue; the insulin-like
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growth factor
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follicle-stimulating hormone (FSH) and luteinising hormone (LH) mRNA expression levels were higher in the
47
pituitary gland.
48
Conclusion
49
These data showed that the consumption of additional fat can facilitate early attainment of puberty, which can be
50
predicted by the changes in secreted hormones and gene expression in the hypothalamus - pituitary - gonadal
51
axis.
52
Keywords
53
High fat diet; Hormone; Gene expressions; Puberty; Gilts
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receptor (IGF- R) expression was higher in the pituitary and ovary tissues, and the
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Introduction The onset of puberty in females, as measured by the age at menarche, is estimated to have advanced by
69
6-12 months per 100 years between the 18th century and the 21th century in several northern European countries
70
[1]. This declining age of puberty has been attributed to accelerated growth due to improved nutrition, e.g.,
71
obese girls tend to mature earlier than normal or thin girls [2-4]. Thus, the accelerated growth rate due to
72
nutrition fortification seems to be a more important index in predicting the early onset of puberty [5]. Although
73
body condition and the timing of puberty show a strong link in developing girls, the underlying mechanism
74
remains largely unknown.
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Puberty is a complex biological process that involves the secretion of gonadotropin-releasing hormone
76
(GnRH) by the hypothalamus, sexual development, adrenal maturation, pubertal development and
77
gametogenesis. Notably, the secretion of GnRH by the hypothalamus represents the first known step in the
78
reproductive cascade to initiate the activation of pituitary and gonadal function. Therefore, understanding the
79
neuroendocrine control of GnRH secretion may provide insight into the normal reproduction or disorder of the
80
pubertal process. Recently, pharmacological and genetic studies revealed that GPR54, a G protein–coupled
81
receptor gene, may act as a gatekeeper for normal GnRH physiology and puberty [6]. Kisspeptins, which are
82
encoded by the metastasis suppressor gene Kiss-1, are a natural ligand for GPR54 to elicit a GnRH surge and
83
puberty onset [7-8]. Kiss-1 and GPR54 may be involved in the early timing of puberty due to accelerated growth.
84
Thus, their gene expression levels represent an interesting research topic.
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In the present study, developing gilts were fed an oil-rich diet in order to induce hormonal and molecular
86
changes in the hypothalamus, pituitary tissue and gonadal tissues. These changes were used to assess whether
87
hormonal and molecular changes could predict the early onset of puberty.
88
Materials and methods
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All experimental procedures were approved by the Animal Care and Use Committee of Sichuan
90
Agricultural University.
91
Animals and diets Forty Landrace × Yorkshire crossbred gilts of initially similar body weight (55 ± 1.5 kg) and age (104 ± 2 d)
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were paired and fed one of two nutritional regimens. Twenty gilts were fed basal diets (LE) formulated to
94
provide 3.22 Mcal/kg digestible energy, 19.1% crude protein and 3.25% ether extract, as recommended by NRC
95
(1998). LE gilts in the 55 to 100 kg phase and 100 kg to puberty phase were fed a 1.8 kg and 2.1 kg basal diet,
96
respectively. The other twenty gilts (HE) were fed basal diets containing an additional 270 g/d or 340 g/d fat
97
during these two phases, respectively. Finally, due to additional fat intake, HE gilts consumed 40.7% and 44.1%
98
more digestible energy than LE gilts during the 55 to 100 kg phase and 100 kg to puberty phase, respectively.
99
The intakes of protein, minerals and vitamins were similar for both LE and HE gilts (Table 1). Gilts were housed
100
individually (2 m × 0.8 m) in a breeding facility and fed twice daily at 0830 and 1430 h, and water was
101
provided ad libitum. The environment temperature was controlled at 20 - 24
102
simultaneously reared and fed LE or HE diets and used to substitute gilts that were culled due to lameness or
103
illness. If gilts in the LE or HE were culled, the corresponding paired gilts were discarded as well.
104
Determination of body weight and back-fat thickness
. Another subgroup of gilts was
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The fasted bodyweights were determined in the morning prior to feeding at the beginning of the experiment
106
and at d 52, d 59, d 66 and d 73 of the experiment. The back-fat thickness was measured 6 cm from the midline
107
of the last rib (P2 thickness) using a LEAN MEATER (RENCO-LEAN MEATER 23249, USA) at beginning of
108
experiment and at d 52, d 59, d 66 and d 73 of experiment.
109
Oestrus detection
110
Oestrus was not induced by exogenous hormone administration for these gilts. Oestrus was carefully
5
ACCEPTED MANUSCRIPT checked in order to ensure that puberty was detected. All gilts were exposed (with fence) to mature boars to
112
encourage pubertal oestrus. Oestrus was detected by only one experienced stockperson based on behavioural
113
and vulvar characteristics. The appearance of a pink vulva and vaginal orifice mucous were important signs of
114
oestrus initiation, while standing still under applied back pressure (standing reflection) was used as an important
115
behavioural criterion to establish onset of oestrous. The age at puberty was recorded on the day of oestrus.
116
Tissue sample collection
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In present study, gilts began to express oestrus at d 72 of experiment. To examine nutrition-induced
118
molecular changes in the hypothalamus-pituitary- gonadal axis, 10 pair-fed prepubescent gilts from
119
corresponding LE and HE groups were slaughtered to collect tissue samples from the hypothalamus, pituitary
120
tissue and gonadal tissue.
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The brains were removed from the skull immediately after slaughter, and the excess tissues were removed.
122
The hypothalamus samples were collected as previously described [9]. Briefly, the hypothalamus was dissected
123
along the following boundaries: laterally 8 mm from either side of the third ventricle, longitudinally 8 mm from
124
the optic chiasm to the posterior border of the mammillary bodies and 20 mm above the top surface of the
125
thalamus. The anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of the hypothalamus
126
were dissected out as previously described [9].
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The uterus, oviduct and ovary tissues were removed from the abdominal cavity immediately after slaughter
128
and pruned for excess intestinal and other viscera tissues. The uterus was weighed, and the length of the uterus
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and oviduct were measured. These tissues were washed three times with PBS followed by drying with paper
130
towels prior to weighing.
131 132
All hypothalamic, pituitary and ovary samples were washed with PBS, dried with paper, frozen in liquid nitrogen and stored at -70
for the future analysis of gene expressions.
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Determination of hormone concentrations To explore the hormonal secretion changes of gilts fed a diet containing added oil, 10 mL blood samples
135
were collected from both LE and HE gilts by jugular puncture at the beginning of the experiment and at d 52, d
136
59, d 66 and d 73 of the experiment during the prepubescent phase. The blood samples were centrifuged at
137
2400×g for 15 min at 4 ºC to collect serum and stored at -20 ºC for the future analysis of hormone
138
concentrations.
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An enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of IGF-I
140
(commercial kit from Alpco diagnostics, USA), kisspeptin (commercial kit from Phoenix pharmaceuticals, Inc.
141
USA), leptin (commercial kit from R&D, USA), oestradiol (commercial kit from R&D, USA) and progesterone
142
(commercial kit from R&D, USA). The detection limits of IGF-I, kisspeptin, oestradiol, progesterone and leptin
143
were 0.09 ng/mL, 0.01 ng/mL, 1.1 pg/mL, 0.01 ng/mL and 0.01 ng/mL, respectively. The intra- and inter-assay
144
coefficients of variation were 5.3% and 5.9%, 5.0% and 4.7%, 5.6% and 6.9%, 5.7% and 6.8%, 5.4% and 6.1%,
145
for IGF-I, kisspeptin, estradiol, P4 and leptin, respectively.
146
Gene expressions
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The gene expression levels were detected using a real-time quantitative polymerase chain reaction (PCR).
148
The primers for the target genes are presented in table 2. The mRNA expression levels of Kiss-1, GPR54, GnRH,
149
follicle-stimulating hormone (FSH), luteinising hormone (LH), insulin-like growth factor
150
insulin receptor (Ins-R), leptin receptor (Ob-R), oestrogen receptor α (ERα) and progesterone (P4R) were
151
determined with real-time quantitative PCR. Total RNA was isolated from tissue samples using Trizol Reagent
152
(Invitrogen, USA) according to the manufacturer’s instructions. The quality and purity of RNA samples was
153
determined by electrophoresis through a 1% denaturing agarose gel and separately assessed using a nucleic
154
acid/protein analyser (Beckman DU-800, USA) from the OD260:OD280. The ribose nucleic acid samples were
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7
receptor (IGF-
R),
ACCEPTED MANUSCRIPT treated with RNase free DNase-I (TaKaRa, Dalian, China) to eliminate trace genome DNA, and approximately
156
1µg of total RNA was taken for reverse transcription using the AMV First Strand cDNA Synthesis Kit (TaKaRa,
157
Dalian, China). cDNA synthesis was performed using the PrimeScriptTM reagent kit (TaKaRa, Dalian, China)
158
according to the manufacturer's instructions. The concentration of RNA in the final preparations was calculated
159
from the OD260, and the samples were diluted with Rnase-free dH2O (TaKaRa, Dalian, China) for reverse
160
transcription. The cDNA was easily dissolved in H2O (TaKaRa, Dalian, China) for real-time quantitative PCR.
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Real-time quantitative PCR was performed in a CFX-96 Real-Time PCR detection System (Bio-Rad, USA).
162
Briefly, 12.5 µL SYBR Premix Ex TaqTM (contains Taq HS, dNTP, MgCl2, SYBR Green), 0.5 µL of each
163
primer and 2.0 µL cDNA were included in a 25 µL PCR. The RT-PCR cycling conditions were defined as
164
follows: an initial pre-denaturing cycle at 95
165
denaturation at 95
166
collect fluorescence. The melting curve conditions were defined as follows: 1 cycle of denaturation at 95℃ for
167
10 s, followed by a gradual ramp from 65℃ to 95℃ at 0.5℃/s. The relative expression of the target gene was
168
calculated as described previously [10]. The relative gene expression levels were normalised to those of the
169
house-keeping gene, β-actin. The outcomes were expressed as fold changes relative to average mRNA levels of
170
genes in LE groups.
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Statistical analysis
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for 1 min, followed by 40 cycles of amplification, defined by for 30 s, followed by plate reading to
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for 5 s, annealing for 30 s and extension at 72
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Gilts were fed individually in the present study, and mRNA expression levels, hormone concentrations,
173
tissue weight, bodyweight and P2 back-fat thickness from corresponding LE and HE gilts were analysed using
174
paired T-tests in the SPSS (10.5) software package. The results were presented as the means ± SEM. A
175
chi-square test was used to analyse the differences in the fractions of gilts that reached puberty before age 180
176
d, 190 d and 210 d. Statistical significance was declared when P < 0.05.
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Results
178
Age at puberty in gilts As shown in table 3, gilts fed HE diets reached puberty twelve days earlier than gilts fed LE diets (P =
180
0.08). The rate of gilts that reached puberty by age d 180, d 190 and d 210 in LE and HE gilts were 13% versus
181
47%, 47% versus 73% and 93% versus 87%, respectively.
182
Bodyweight and back-fat thickness in response to LE and HE diets
As shown in Fig. 1 and Fig. 2, the body weight and back-fat thickness at P2 point of gilts fed HE diets were
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significantly higher than those of LE gilts at d 52, d 59, d 66 and d 73 of the experiment (P < 0.01).
185
Uterus and ovary tissue development in response to LE and HE diets
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As shown in table 4, the development of the uterus and ovarian tissue were significantly accelerated in gilts
187
that were fed the HE diet. The uterus weight, body of the uterus, length of the left horn of the uterus, length of
188
the right horn of the uterus, length of the left oviduct, length of the right oviduct and ovary weight were
189
significantly decreased in gilts fed LE diets as compared to gilts fed HE diets (P < 0.05).
190
Hormonal changes in response to LE and HE diets
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As shown in Fig. 3, the HE diet did not affect the circulating oestradiol concentrations at d 0, d 52, d 59
192
and d 66 of the experiment, but gilts fed HE diets had significantly higher oestradiol concentrations than LE
193
gilts at d 73 of the experiment (P < 0.05). The circulating insulin-like growth factor
194
were higher in gilts fed HE diets than in gilts fed LE diets at d 52, d 59, d 66 and 73 of the experiment (P <
195
0.05). The HE diet did not affect the circulating leptin concentrations at d 52 and d 59 of the experiment, but this
196
concentration was higher in gilts fed HE diets than gilts fed LE diets at d 66 and 73 of the experiment (P < 0.05).
197
The HE diet did not affect the circulating kisspeptin and progesterone concentrations during the entire
198
experiment period.
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(IGF-
) concentrations
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Gene expressions The gene expression levels of gilts fed LE or HE diets are presented in figure 4 (A-D). As shown in figure 4
201
(A), the expression of Kiss-1, GPR54, GnRH and ERα mRNA in the hypothalamic AVPV from HE gilts were
202
increased compared with LE gilts (P < 0.05), but this difference did not persist in the hypothalamic ARC (figure
203
4B). The expression levels of ObR mRNA in the hypothalamic ARC (figure 4B) and ovary tissues (figure 4D)
204
were higher in HE gilts compared with LE gilts (P < 0.05), but this difference did not persist in the hypothalamic
205
AVPV (figure 4A) or in pituitary tissue (figure 4C). The expression of IGF-
206
4C) and ovary tissues (figure 4D) in HE gilts compared with LE gilts (P < 0.05). FSH and LH mRNA
207
expressions in pituitary gland (figure 4C) were increased in gilts fed HE diets compared with gilts fed LE diets
208
(P < 0.05). Ins-R (figure 4C and figure 4D) and P4R expressions (figure 4A and figure 4B) in their respective
209
tissues were not affected by diets.
210
Discussion
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Puberty is the process of physical changes by which a child’s body matures into an adult body that is
212
capable of fertilisation to reproduce offspring. A recent study indicated that girls have entered puberty at
213
increasingly younger ages of the past several decades, and prepubescent obesity is a predictor of early onset
214
puberty. Therefore, underlying mechanisms that are involved in this process are an interesting subject of study.
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In the present study, gilts that consumed extra fat reached puberty twelve days earlier than gilts that were a
216
low fat diet, and a larger proportion of gilts that were fed a high fat diet reached puberty at d 190 of age.
217
Undoubtedly, the higher energy consumption as a result of the extra fat intake was the leading cause of early
218
onset puberty. Several earlier studies conducted in humans also indicated that obese girls experienced menstrual
219
cycles earlier than girls that were not overweight [3-4]. These results fit the hypothesis that girls must achieve a
220
minimum fatness and bodyweight in order to trigger the onset of menstrual cycles [11]. Generally, bodyweight
10
ACCEPTED MANUSCRIPT 221
and the initiation of the hormonal events of puberty are strongly associated, even though bodyweight or body fat
222
might not be an absolute determinant of puberty onset [12]. Little evidence is currently available to elucidate the complex biologic process by which excess nutrition
224
induces early puberty onset. The hypothalamic activation and secretion of GnRH is currently known as the
225
crucial up-stream regulator that initiates the reproductive cascade to stimulate the pulsatile release of FSH and
226
LH [13]. In the present study, gilts that were fed a HE diet showed higher expression levels of the GnRH gene in
227
the hypothalamus AVPV. Coincidently, the FSH and LH expression in the pituitary gland increased in response
228
to this GnRH expression change. Therefore, the higher GnRH, FSH and LH expression was expected to induce
229
early puberty onset in the present study. Given the master position of hypothalamic GnRH in controlling the
230
gonadotropic axis, GnRH was noted as the target of multiple regulators of central and peripheral origin, and a
231
wide array of excitatory and inhibitory circuits that govern GnRH secretion have been identified [14-15]. Based
232
on genetic and pharmacological studies, an unsuspected key role for the Kiss-1/GPR54 system in the
233
hypothalamic control of the GnRH-gonadotropin axis has recently been accepted as a gatekeeper of puberty [16].
234
Kiss-1 was originally identified as a metastasis suppressor gene that encodes a number of structurally related
235
peptides, which are termed kisspeptins [17]. The direct central administration of kisspeptins to hypothalamic
236
GnRH neurons could stimulate GnRH release and puberty onset [18]. In the present study, the gene expression
237
of Kiss-1/GPR54 was evaluated in the hypothalamic AVPV and ARC. The Kiss-1 and GPR54 mRNA expression
238
levels in the AVPV increased coincidently with the increased hypothalamic AVPV GnRH mRNA expression.
239
Interestingly, we did not find a difference in the Kiss-1/GPR54/GnRH gene expression level in the hypothalamic
240
ARC in the present study. Kiss-1 is expressed most abundantly in the ARC and AVPV [9], and the kisspeptin
241
neurons in these two areas appear to be functionally different [19-21]. The results of the present study also
242
indicated that Kiss-1/GPR54 might function differently in the AVPV and ARC. However, the distinct role of
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AVPV and ARC in linking nutrition and GnRH secretion requires further examination. Nevertheless, given the
244
“gatekeeper” role of the kisspeptin/GPR54 system in initiating puberty onset, the increased hypothalamic kiss-1
245
and GPR54 mRNA expression level is likely to be an important mediator of early onset puberty. The hormonal changes appeared to be a crucial event that linked the nutritional effects on reproductive
247
maturity and puberty initiation [22-23]. In the present study, the oestradiol concentration was not affected during
248
the prepubescent phase but was higher when approaching the age of puberty. Oestradiol was necessary in order
249
to initiate oestrus and ovulation, and it has recently been observed to play important positive role in the
250
epigenetic regulation of Kiss-1 expression [24]. The increased mRNA expression of ERα in the hypothalamic
251
AVPV confirmed the previous hypothesis that oestradiol signalling may participate in the positive feedback
252
regulation of GnRH secretion by up-regulating Kiss-1 expression [20, 25]. The hormone leptin is produced by
253
adipose tissue and was initially thought to primarily regulate energy balance by decreasing food intake and
254
increasing energy expenditure [26]. Additionally, leptin treatment prevents the effects of fasting on reproductive
255
processes in a variety of species [27], which implies that it has a role in regulating the reproductive process.
256
Because obese girls reach puberty earlier than normal or thin girls, leptin is proposed as an important signal that
257
triggers puberty. When normal prepubescent female mice were injected with leptin, they consumed less food
258
and grew at a slower rate than the controls but reproduced up to 9 days earlier than the controls [28]. In the
259
present study, the circulating leptin concentration increased in gilts fed extra fat. To explore the changes in the
260
leptin signalling pathway in the hypothalamus - pituitary - gonadal axis, the leptin receptor mRNA expression,
261
ObR, was measured using RT-PCR; the expression of ObR was higher in the hypothalamic ARC and ovarian
262
tissue, which suggests that leptin plays an important role in the regulation of reproductive maturation. Contrary
263
to ObR expression changes in the ARC, the ObR expression in the hypothalamic AVPV remained unchanged
264
[29]. ARC was recently considered as a hypothalamic centre that couples energy metabolism and reproduction.
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266
reproduction [30]. However, the mechanism by which metabolic signalling changes in the hypothalamic ARC to
267
potentially be transmitted to the hypothalamic AVPV and induce the activation of the Kiss-1/GPR54 system
268
remains unclear. In addition to the neuroendocrine control of FSH and LH, metabolic signalling could act
269
directly on the pituitary - gonadal axis to promote reproductive maturation. IGF-
270
and gonadal tissues, and IGF-
271
culture of pituitary cell [31]. In the present study, the peripheral IGF-
272
were fed a fat-rich diet, and the expression of IGF-
273
gilts. These findings indicate that the early maturation of reproduction could be attributed, at least in part, to the
274
increased IGF-
signalling. However, we could attribute these phenomena to either the direct effects of
275
peripheral IGF-
action or central IGF-
276
regulator of reproductive neuroendocrine function [32].
277
Conclusion
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R was expressed in pituitary
administration could stimulate FSH and LH secretions during the in vitro
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concentration increased in gilts that
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in the brain has also been proposed as a
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Collectively, the results presented herein suggest that accelerated growth and body fat accumulation induce
279
molecular changes in the Kiss-1/GPR54 system and the hormonal secretion, which predict an early onset of
280
puberty.
281
Acknowledgements
282
This work was supported by the National Natural Science Foundation (30871804) of P.R China. The authors
283
also wish to thank the staff in the laboratory for their on-going assistance.
284
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with leptin. Science 1997; 275(5296):88–90. [29] Bouret SG, Draper SJ, Simerly RB. Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 2004; 304:108 –110. [30] Sawchenko PE. Toward a new neurobiology of energy balance, appetite, and obesity: the anatomists weigh
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Biol Reprod 2007; 76(6):1016-1024.
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[32] Daftary SS, Gore AC. IGF-1 in the brain as a regulator of reproductive neuroendocrine function. Exp Biol Med (Maywood) 2005; 230(5):292-306.
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Figure 1. Changes in bodyweight of gilts fed LE or HE diets at different days of the experiment. LE denotes
365
gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning
366
day of the experiment. ** denotes P < 0.01. The results are expressed as the mean values ± SEM, n = 20 gilts
367
per group.
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Figure 2. P2 thickness of gilts fed LE or HE diets at different days of the experiment. LE denotes gilts fed basal
369
diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the
370
experiment ** denotes P < 0.01. The results are expressed as the mean values ± SEM, n = 20 gilts per group.
371
Figure 3. Changes in hormone concentrations of prepubescent gilts fed LE or HE diets. LE denotes gilts fed
372
basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil supplements. D 0 was
373
the beginning day of the experiment. * denotes P < 0.05. The blood samples were collected from HE gilts and
374
corresponding paired LE gilts to determine the concentrations of hormones. The results are expressed as the
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mean values ± SEM, n = 20 gilts per group.
Figure 4. Relative mRNA expression levels in the respective tissue samples of prepubescent gilts fed LE or
377
HE diets. Figure 4 (A, B, C and D) denotes the relative mRNA expressions in AVPV (anteroventral
378
periventricular nucleus), ARC (arcuate nucleus), pituitary and ovarian tissues, respectively. LE denotes
379
gilts fed basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil
380
supplements. * denotes P < 0.05 versus LE. The tissue samples were collected from HE gilts and
381
corresponding paired LE gilts to determine the gene expression levels. The results are expressed as the
382
mean values ± SEM, n = 5 gilts per group.
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391 392 393
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ACCEPTED MANUSCRIPT 397 Table 1 Daily nutrient intake in gilts fed LE or HE diets1
398
55 - 100 kg
100 kg - puberty HE
LE
HE
Intake of basal diet (g/d)
1800
1800
2100
2100
Oil intake (g/d)
0
270
0
340
Total feed intake (g/d)
1800
2070
2100
2440
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LE
5.80
8.16
6.76
9.74
Crude protein
344
344
401
401
Starch
842
842
983
983
Lipid
59
329
68
408
51
51
59
59
16
16
19
19
Calcium
17
17
20
20
Phosphorus
13
13
15
15
9
9
10
10
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Digestible energy (Mcal/d)
Crude fibre
Available Phosphorus
399
1
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LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements.
400 401 402 403
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Table 2. Primer sequences of genes Genebank
Amplification
Gene
Annealing
Primer sequence length (bp) 5’-GGGGGACCTCATCGTGCCAG-3’
Kiss-1
AB466320
179
GPR54
DQ459345
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5’-CGGAAACACAGTCACATA-3’
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5’-CGCCTGCCGCTTTCCGTA-3’ 5’-GCTCTACTCTACCCCCTA-3’
temperature (
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accession No.
65
115
60
119
62
117
61
142
60.5
129
59
102
59.5
101
60
110
60
5’- CTGCTGACTCTGTGTGTAGTG -3’ GnRH
NM_214274.1
5’-ACCTCTTTGGCCATCTCTTG-3’
5’-GTGGTGTGCTGGCTATTGCTAC-3’ FSH
NM_213875
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5’-CCAGGTACTTTCACGGTCTCGTA-3’ 5’-AGAGACTGCTGTTGTGGCTGCT-3’ LH
NM_214080
5’-CTGGTGGTAAAGGTGATGCAGAC-3’
IGF-
R
NM_214172
EP
5’-GGAGATTTTGGGATGACGAGAG-3’
AC C
5’-ATCAGAGTGCGTGGTGAAGACT-3’
5’-GTGTTGTGATTGGAAGCATTTAT -3’
Ins-R
AF102858
5’-GGGGCTCATGGAGTCACGGT-3’
5’-ATTTCTGCTATCTGGCTATA-3’ Ob-R
NM_001024587 5’-GTTTCACCACGGAATC-3’ 5’-CTGGACAAGATCACAGACACCT-3’
ERα
NM_214220 5’-CTGAAGTGAGACAGGATGAGGAG-3’
20
)
ACCEPTED MANUSCRIPT 5’-CCCTAGCTCACAGCGTTTCT-3’ P 4R
GQ903679.1
184
61
104
60
5’- CACCATCCCTGCCAATATCT -3’ 5’-GGCCGCACCACTGGCATTGTCAT-3’ β-actin
DQ845171.1
RI PT
5’-AGGTCCAGACGCAGGATGGCG-3’
406
GPR54, G-protein coupled receptor 54; GnRH, gonadotropin-releasing hormone; FSH, follicle-stimulating
407
hormone; LH, luteinizing hormone; IGF- R, insulin-like growth factor
408
Ob-R, leptin receptor; ERα, oestrogen receptor α; P4R, progesterone receptor;
SC
M AN U
409 410 411 412
TE D
413 414
418 419
AC C
417
EP
415 416
receptor; Ins-R, insulin receptor;
420 421 422 423
21
ACCEPTED MANUSCRIPT 424 Table 3 Age at puberty and rate of puberty until d 180, d 190 and d 210 of age in gilts fed LE or HE diets1,2,3 LE
HE
Significance
Number of gilts, n
15
15
--
Age at puberty (d)
191.3 ± 5.6
179.0 ± 5.5
P = 0.08
Rate of gilts attaining puberty until age 180 d (%)
13 (2)
47 (7)
P = 0.11
Rate of gilts attaining puberty until age 190 d (%)
47 (7)
73 (11)
P = 0.26
Rate of gilts attaining puberty until age 210 d (%)
93 (14)
87 (13)
P = 1.0
SC
Items
RI PT
425
1
LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements.
427
2
The data on the rate of gilts that reached puberty at 180 d, 190 d and 210 d were analysed Chi-square test.
428
3
Number in the bracket indicates number of gilts that reached oestrus.
M AN U
426
429 430
TE D
431 432
435 436 437
AC C
434
EP
433
438 439 440 441 22
ACCEPTED MANUSCRIPT 442 Table 4 Uterus and ovary tissue development in gilts fed LE or HE diets1,2,3 HE
Significance
Number of gilts, n
5
5
--
Uterus weight (g)
276.6 ± 44.1
425.2 ± 52.5
Body of uterus (cm)
21.6 ± 2.9
29.5 ± 2.0
Length of left horn of uterus (cm)
51.3 ± 7.5
69.7 ± 7.2
Length of right horn of uterus (cm)
44.3 ± 5.1
62.5 ± 5.2
Length of left oviduct (cm) Length of right oviduct (cm) Ovary weight (g)
**
18.1 ± 3.9
27.3 ± 2.7
*
6.5 ± 0.5
8.4 ± 0.3
*
2
446
prepubescent gilts were slaughtered at d 73 of the experiment and d 177 of age.
447
3
TE D
EP
LE denotes gilts fed basal diet, HE denotes gilts fed basal diet with additional oil supplements. These
* denotes P < 0.05, ** denotes P < 0.01.
AC C
450
*
*
445
449
*
27.5 ± 2.7
1
448
*
18.4 ± 2.4
444
Data were presented as the means ± SEM
RI PT
LE
M AN U
Items
SC
443
451 452 453 454
23
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
Figure 1. Changes in bodyweight of gilts fed LE or HE diets at different days of the experiment. LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the experiment. ** denotes P < 0.01. The results are expressed as the mean
AC C
EP
TE D
values ± SEM, n = 20 gilts per group.
SC
RI PT
ACCEPTED MANUSCRIPT
M AN U
Figure 2. P2 thickness of gilts fed LE or HE diets at different days of the experiment. LE denotes gilts fed basal diets, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the experiment ** denotes P < 0.01. The results are expressed as the mean values ±
AC C
EP
TE D
SEM, n = 20 gilts per group.
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
EP
Figure 3. Changes in hormone concentrations of prepubescent gilts fed LE or HE diets. LE denotes
AC C
gilts fed basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil supplements. D 0 was the beginning day of the experiment. * denotes P < 0.05. The blood samples were collected from HE gilts and corresponding paired LE gilts to determine the concentrations of hormones. The results are expressed as the mean values ± SEM, n = 20 gilts per group.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Figure 4. Relative mRNA expression levels in the respective tissue samples of prepubescent gilts fed LE or HE diets. Figure 4 (A, B, C and D) denotes the relative mRNA expressions in AVPV (anteroventral periventricular nucleus), ARC (arcuate nucleus), pituitary and ovarian tissues, respectively. LE denotes gilts
RI PT
fed basal diets with a low energy intake, HE denotes gilts fed basal diets with additional oil supplements. * denotes P < 0.05 versus LE. The tissue samples were collected from HE gilts and corresponding paired LE gilts to determine the gene expression levels. The results are expressed as the mean values ± SEM, n = 5 gilts
AC C
EP
TE D
M AN U
SC
per group.