The authors have declared that no competing interests exist.
Conceived and designed the experiments: SX DZ D. Wu. Performed the experiments: SX D. Wang DZ. Analyzed the data: SX D. Wang. Contributed reagents/materials/analysis tools: SX D. Wang DZ. Wrote the paper: SX D. Wang DZ YL LC ZF D. Wu.
The onset of estrus is a critical sign of female sexual maturity. The pituitary plays a vital role in this process by the secretion of reproductive hormones. To investigate the effects of nutrient restriction on reproductive function and the underlying mechanisms involved, deep RNA sequencing of pituitary gland tissue was carried out to determine the differentially expressed genes (DEGs) between gilts in normal estrus, and gilts in which anestrus was induced by nutrient restriction. Gilts which had gone through two estrus cycles were fed a normal (CON, 2.86kg/d, n = 10) or nutrient restricted (NR, 1kg/d, n = 10) diet. The NR gilts experienced another three estrus cycles, but did not express estrus symptoms at the anticipated 6th and 7th cycles. Body weight gain in NR gilts was significantly decreased by nutrient restriction. Gilts were considered as anestrus when blood progesterone concentrations lower than 1.0 ng/mL from three consecutive blood samples were recorded. Circulating concentrations of progesterone (< 1.0 ng/mL vs. 2.1 ng/mL) and estradiol (208.6 ng/mL vs. 371.8 ng/mL) were significantly lower in the NR gilts than in the CON gilts. Between 5,360,000 and 5,370,000 sequence reads per sample from the CON and NR gilts’ pituitaries were obtained and mapped to the porcine genome. Analysis of read counts revealed 185 DEGs. Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatic analysis identified that the genes identified were enriched in the GO terms “neuroactive ligand-receptor interaction”, “GnRH signaling pathway” and “immune response system”. Our findings provide a new perspective for understanding the nutrient restriction-induced reproductive impairment at the pituitary transcriptional level, and how this is linked to hormone secretion. Moreover, the transcriptomic changes in anestrus gilts associated with nutrient restriction could be a resource for targeted studies of genes and pathways potentially involved in the regulation of reproductive function and animal health.
Throughout vertebrates the onset of estrus indicates that the female organism has reached sufficient sexual maturity to reproduce. Studies have shown that reproductive function in pigs is sensitive to the status of energy reserves suggesting that weight loss in humans by extreme dieting methods may have potential negative effects on fertility [
Nutritional status, especially overall energy level, is important for the onset of estrus [
At the present time, it has become routine to obtain large-scale genetic data at the genomic and transcriptomic levels in pigs due to the development of high-throughput deep sequencing technologies. These approaches include Illumina’s RNA sequencing (RNA-seq) system, which employs whole transcriptome shotgun sequencing wherein mRNA or cDNA is mechanically fragmented, resulting in overlapping short fragments that cover the entire transcriptome. RNA-seq is powerful for unraveling transcriptome complexity, and for the identification of genes, splice variants, non-coding RNAs, and even novel transcriptional units [
The pig anestrus model is an important tool for the investigation of the influence of nutrition on estrus onset. Identification of differences in gene expression between normal and anestrus gilts could shed light on the underlying mechanisms. Therefore, using nutritionally-induced anestrus gilts, we assessed the hormonal secretion and transcriptomic responses of the pituitary by deep RNA sequencing. The mechanisms behind these nutrition-induced responses were investigated on a molecular basis.
All experimental procedures were approved by the Animal Care and Use Committee of Sichuan Agricultural University. Twenty Landrace × Yorkshire crossbred gilts having experienced two estrus cycles were used in this study. Gilts with similar bodyweight (122 ± 4.5 kg) and age (230 ± 6 days) were allocated to two nutrition allowance levels and either fed a normal diet (control, CON, 2.86 kg/d) or a nutrient restricted diet (NR, 1 kg/d) [
Ingredients (g/kg) | Calculated nutritive composition | ||
---|---|---|---|
Corn | 684.40 | Digestible energy (Mcal/kg) | 3.40 |
Soybean meal | 159.50 | Crude protein (%) | 14.30 |
Whey bran | 60.00 | Starch (%) | 47.30 |
Fish meal | 20.00 | Either extract (%) | 7.10 |
Soybean oil | 40.00 | Soluble fiber (%) | 1.62 |
Calcium Carbonate | 10.00 | Crude fiber (%) | 2.46 |
Calcium hydrophosphate | 6.80 | Neutral Detergent Fiber (%) | 10.05 |
Salt | 4.00 | Insoluble fiber (%) | 10.50 |
Vitamin premix |
0.50 | Lysine (%) | 0.97 |
Trace mineral premix |
5.00 | Calcium (g/kg) | 9.20 |
Choline chloride | 1.60 | Phosphorous (g/kg) | 6.70 |
Total | 1000.00 | Available phosphorous (g/kg) | 4.60 |
Daily feeding intake in normal and restricted gilts were 2.86 kg/d and 1 kg/d, respectively.
a Supplied per kilogram diet: Vitamin A, 16, 000 IU; Vitamin D3 4, 000 IU; Vitamin E, 10 IU; Vitamin K, 2 mg; Vitamin B1, 0.8 mg; Vitamin B2, 6.4 mg; Vitamin B12, 0.012 mg; Niacin, 10 mg; Pantothenic, 10 mg; Folic acid, 0.2 mg.
b Supplied per kilogram diet: Cu 13.5 mg, Fe 162 mg, Mn 54 mg, Zn 162 mg, I 0.55 mg, Se 0.4 mg.
The day of the second occurrence of standing heat in gilts was designated as day 0 (d 0). At d 0, the gilts in CON and NR groups were started on their respective feeding regimens. All gilts had fence-line contact with a mature boar to encourage pubertal estrus. Onset of estrus was detected by conducted by back pressure testing and examination of vulval characteristics twice daily, in the morning and afternoon, by an experienced stockperson. Gilts failed to express estrus until the 23rd day of the 6th estrus cycle (d 88) in the NR group. Blood samples (10 mL) were collected from the both groups of gilts every 3 days to determine the concentration of progesterone. When progesterone concentrations were below 1.0 ng/mL for three consecutive days in gilts who did not enter estrus, this was defined as nutritionally induced anestrus [
At d 107 (when standing heat in the 7th estrus cycle was observed in the control group) both groups of gilts were anaesthetized with an intravenous injection of Zoletil 50 (0.1 mg/kg body weight; Virbac, Carros cedex, France), and were bled by exsanguination. Brains were immediately removed from the skull and excess tissues were removed. Within a few minutes after slaughter the pituitary gland was removed and flushed with ice-cold PBS, then rapidly frozen in liquid nitrogen. All the samples were maintained at −80°C until RNA analysis were performed.
Blood samples were collected on d 0, and every 3 days from d 88 to d 107 by jugular venipuncture. Blood samples were centrifuged at 2400 g for 15 min to collect serum and stored at -20°C for future analysis of hormone concentrations. Circulating concentrations of E2 and progesterone (P) were determined using enzyme-linked immunosorbent assay kits (R&D Systems Inc., Minneapolis, MN, USA), according to the manufacturer’s recommendations. The limits for detection of E2 and P were 0.01 ng/mL, and 1.0 ng/mL, respectively. The intra- and inter-assay coefficients of variation were 6.6% and 7.5% for E2, and 5.3% and 6.4% for P.
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from frozen tissues ground in liquid nitrogen. The quality and purity of RNA samples were checked by spectrophotometry (Beckman DU-800, Los Angeles, CA, USA) from the OD260:OD280 and denaturing agarose gel electrophoresis. All RNA samples were treated with DNase-I (TaKaRa Biotechnology Co., Ltd, Dalian, China). RNA integrity and concentration were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Folsom, CA, USA).
For RNA library construction and deep sequencing, equal quantities of RNA from four individual pig pituitaries which were fed a normal diet were pooled, and RNA samples from the four nutrient restricted pig pituitaries were pooled. Approximately 4 μg of RNA from each treatment group were submitted for sequencing. In brief, after RNA extraction, poly A-containing mRNAs were purified using oligo-dT-conjugated magnetic beads and fragmented into small pieces. Using these short fragments as templates, first- and second-strand cDNA were synthesized. These cDNA fragments were subjected to end repair, addition of a single ‘A’ base, and ligation of adapters. Products were subsequently purified and amplified by PCR to construct the final cDNA libraries. Finally, the raw image files from each of the libraries were collected by the Illumina HiSeqTM 2000 sequencing platform in BGI Shenzhen (
All data is MIAME compliant. For the raw data, we filtered adaptor tags, low quality tags and tags with a copy number of 1 to obtain clean tags. Subsequently, the clean tags were classified according their copy number in the library, and the percentage of total clean tags was calculated and saturation of the library was analyzed. The
To confirm the differential expression of genes revealed by RNA-Seq, the expression of genes in the pituitary, including Kiss-1, G protein-coupled receptor 54 (GPR54), FSH, LH, GnRHR and leptin receptor (Ob-R), were measured by qPCR. Complementary DNA (cDNA) was synthesized with random primers (Invitrogen, Carlsbad, CA, USA). Real-time PCR was used to quantify mRNA expression levels using the Sybr Green Kit (Qiagen, Valencia, CA, USA). Primers are listed in
Genes | Primer sequences | Size of amplified fragment(bp) | GenBank accession number |
---|---|---|---|
Kiss-1 | F: |
179 | AB466320 |
R: |
|||
GPR54 | F: |
115 | DQ459345 |
R: |
|||
FSH | F: |
117 | NM_213875 |
R: |
|||
LH | F: |
142 | NM_214080 |
R: |
|||
GnRHR | F: |
101 | NM_214273 |
R: |
|||
Ob-R | F: |
101 | NM_001024587 |
R: |
|||
β-actin | F: |
104 | DQ845171.1 |
R: |
GPR54: G protein-coupled receptor 54, FSH: follicle stimulating hormone, LH: luteinizing hormone, GnRHR: gonadotropin-releasing hormone receptor, Ob-R: leptin receptor.
Data from gilts in the normal and nutrient-restricted groups were compared by
In the present study, nutrient restricted gilts (n = 10) which already had experienced two estrus cycles failed to express estrus at the anticipated time of the 6th and 7th estrus (d 88 and d 107). The day of the second occurrence of standing heat was designated as d 0. As shown in
Item | CON | NR | |
---|---|---|---|
BW (kg) | d 0 | 122.40 ± 6.67 | 124.05 ± 5.87 |
d 88 | 144.70 ± 7.68 |
100.15 ± 4.38 |
|
d 107 | 154.58 ± 2.32 |
97.80 ± 6.21 |
|
P2 thickness(mm) | d 0 | 13.25 ± 0.75 | 13.75 ± 0.75 |
d 88 | 16.50 ± 0.65 |
6.25 ± 0.25 |
|
d 107 | 19.05 ± 1.92 |
5.55 ± 1.38 |
a, b in the same line denotes the effects of timing on the changes of bodyweight and P2 thickness.
CON denotes normal-fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d). The day of the second occurrence of standing heat was designated as d 0.
The circulating concentrations of progesterone were determined at d 0 and every 3 days from d 88 to d 107. In the NR gilts progesterone concentrations were lower than 1.0 ng/mL and also beyond the limit of detection from d 88 to d 107. As shown in
CON denotes normally fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d). The day of the second occurrence of standing heat was designated as d 0. P, progesterone; E2, estradiol. * denotes
Circulating concentrations of E2 were determined at d 0, d 88, d 91 and d 107. E2 concentrations at d 0, d 88 and d 91 were not affected by nutrient restriction, with the exception of decreased E2 concentration in the NR gilts at d 107 compared with the CON group (208.6 ± 32.1 ng/mL vs. 371.8 ± 38.5 ng/mL,
CON denotes normally fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d). The day of the second occurrence of standing heat was designated as d 0. P, progesterone; E2, estradiol. * denotes
To investigate the regulation of the response to nutrient restriction in gilts, we obtained the global gene expression profiles in pituitary tissues using RNA-seq. We sequenced two porcine pituitary RNA-seq libraries from CON and NR groups using parallel sequencing on the Illumina platform. Sequencing quality evaluation is shown in
RNA-Seq sample | CON | NR | |
---|---|---|---|
Total tags (raw data) | 7,187,315 | 7,221,064 | |
Clean tags | 7,160,026 | 7,193,730 | |
Total distinct clean tags | 7,147,897 | 7,180,408 | |
Mapping to gene | TDCT | 5,358,288 | 5,369,164 |
DCT | 4,224,291 | 4,138,639 | |
Unambiguous Mapping | TDCT | 3,494,005 | 3,320,515 |
DCT | 2,836,845 | 2,973,311 | |
Unknown Tag | 2,923,606 | 3,041,769 |
TDCT- Total distinct clean tag; DCT- distinct clean tag. CON denotes normal-fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d).
To compare the global transcriptional changes between CON and NR pituitary tissues, we applied the same method as described previously [
The molecular function (MF), biological process (BP) and cellular component (CC) analysis based on gene ontology (GO) in the 185 identified DEGs are shown in
The 185 DEGs could be grouped into 138 pathways based on analysis using the KEGG pathway database. These pathways and the DEGs involved are shown in
To validate DEGs identified by RNA-seq, we used qRT-PCR to measure the expression of six genes. The genes were selected based on their involvement in neuroactive ligand-receptor interaction and GnRH signaling pathway (FSH, LH and GnRHR) or because they are important components of estrus onset (Kiss-1, GPR54 and Ob-R). Our results indicated that most of the genes investigated had expression levels which were consistent between RNA-Seq and qRT-PCR assays (
Results are expressed as the target/reference ratio of each sample normalized by the target/reference ratio of the calibrator. β-actin was used as a reference gene. The vertical axis indicates the fold change of transcript abundance in CON gilts compared to the NR. For the NR sample, the fold change of transcript abundance relative to the NR sample equals one, by definition. qRT-PCR: the RNA samples from independent RNA extractions from six biological replicates; RNA-Seq: the results of deep RNA sequencing. Error bars represent standard error. CON denotes normally fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d). GPR54: G protein-coupled receptor 54, FSH: follicle stimulating hormone, LH: luteinizing hormone, GnRHR: gonadotropin-releasing hormone receptor, Ob-R: leptin receptor.
The onset of estrus is the hallmark of reproductive maturity, and a sign that the female individual is sufficiently mature to produce offspring. It has been reported that nutritional factors are the major environmental influence on the timing of puberty [
In this study, the gilts were allowed to initially experience two estrus cycles. In the NR group, the nutrition allowance level was 1 kg/d, which is lower than the maintenance nutrient requirement by NRC (1998) [
To investigate the global gene expression profiles in the NR group pituitaries, we used the Illumina RNA-Seq system, a tag-based novel high-throughput transcriptome deep sequencing method. This was done using pooled biological replicates from four pigs from each group in order to obtain representative samples for deep sequencing analysis. We were able reach a sequencing depth of 7.19–7.22 million tags per library and found tags were mapped to around 5.4 million genes in the
Based on GO analysis of the 185 DEGs identified in the NR gilts, multiple biological processes were significantly affected. As far as we know, the GO term “biological process response to hormone stimulus” is the most important in reproduction, which is the initiator of the estrus process [
Moreover, several components of the “neuroactive ligand-receptor interaction pathway”, including FSH, LH, thyroid stimulating hormone receptor (TSHR), growth hormone releasing hormone receptor (GHRHR), growth hormone (GH), prolactin (PRL) were altered by nutrient restriction. These genes not only play a vital role in reproduction and lactation [
The GO terms “GnRH signaling pathway”, “oocyte meiosis” and “progesterone-mediated oocyte maturation” have well described roles in reproduction via DEGs including inositol 1,4,5-trisphosphate receptor type 1, calbindin 2 (CALM) and progesterone receptor [
The Kiss1/GPR54 (G-protein-coupled receptor) system is considered as the gate keeper of estrus [
In conclusion, this study provides evidence at the transcriptional level to explain why postpubertal gilts terminate their reproductive functions in a nutrient restricted diet. From the GO and the KEGG analysis, it was also found that nutrient restriction had effects on immunity and growth in the gilts. Based on these results, humans should also be aware of potential risks to reproductive function and even overall health when we are on a diet.
A, pituitary sample from normally fed gilts (2.86 kg/d); B, pituitary sample from nutrient restricted gilts (1 kg/d).
(TIF)
The Genes shown in red were upregulated and those shown in green were downregulated in CON relative to NR gilts. See supplementary
(TIF)
(ZIP)
CON denotes normally fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d).
(XLSX)
CON denotes normally fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d).
(XLSX)
CON denotes normally fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d).
(XLSX)
The Genes shown in red box were upregulated and those shown in green box were downregulated in CON relative to NR gilts in the hyperlink pathway figure. CON denotes normally fed gilts (2.86 kg/d) and NR denotes nutrient restricted gilts (1 kg/d).
(DOCX)
We would like to thank the staff at our laboratory for their ongoing assistance.