论文部分内容阅读
AbstractThis study was conducted to analyze the regulation mechanisms of TAC1, TACR1, TACR2 and TACR3 genes on reproduction of goat under different photoperiods. The expression conditions of TAC1, TACR1, TACR2 and TACR3 genes in 12 tissues (oviduct, ovary, uterus, gluteus, mesenteric fat, brain, cerebellum, medulla oblongata, hart, lung, liver, kidney) of adult Henan Huai goat under different photoperiods (short day light, Light 8 h∶dark 16 h, and long day light, light 16 h∶dark 8 h) were analyzed by qPCR method. TAC1, TACR1, TACR2 and TACR3 genes were expressed in all the 12 tissues of goats, with different expression characteristics; and the expression levels of all the genes were affected by photoperiod and changing of light signal between light and dark under short photoperiod. Shifting of light signal from dark to light was more conductive to the expression of these genes in all tissues than that of light signal from light to dark. There were significant differences in the expression levels of genes between light shifting from light to dark and from dark to light when the genes were expressed at a higher level in some tissues. TAC1 and TACR2 genes were expressed at a higher level than TACR1 and TACR3 genes in the various tissues, which implied that TACR2 is a receptor given priority to combine with TAC1 when TAC1 is functional.
Key wordsGoat; TAC1; TACR1; TACR2; TACR3; Tissue Expression; Photoperiod
Received: May 23, 2018Accepted: August 27, 2018
Supported by National Natural Science Foundation of China (31472095); Earmarked Fund for China Agriculture Research System (CARS38).
Yuqin WANG (1972-), female, P. R. China, Professor, PhD, devoted to research about genetic breeding and production.
*Corresponding author. Email: wangyq6836@163.com.
Tachykinins (TACs) constitute a peptide family, and the bestknown peptides include substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and hemokinin1 (HK1)［1-2］. Researches have demonstrated that TAC1 could produce four kinds of different mRNA subtypes (a, β, γ and δ), which could code SP (a, β, γ and δ) and NKA (β and γ)［3-4］. TACs are a type of polypeptides having biological activity, which are widely distributed in central nervous system and peripheral nervous system［5-6］. In recent years, researches have shown that TACs play an important role in regulating the reproductive function of mammal animals including human and mice through hypothalamuspituitarygonad axis, and especially, researches on hypothalamuspituitarygonad axis have shown that TACs influence the release of GnRH through hypothalamic neuron, and play an important role in regulating reproductive processes including animal oestrus, ovulation and gestation. The reproductive phenomenon and reproductive law of mammals have shown that melatonin secreted by pituitary and pineal directly acts on pars tuberalis of hypophysis to regulate the expression of thyroid stimulating hormone (TSH) which then feeds back the information to hypothalamus and regulates the reproduction pathway［7］, but there is also another pathway in hypophysis which regulates seasonal release of prolactin (PRL)［8-9］. These researches are of great significance to the revealing of the reproductive mechanism of mammals and the improvement of animal husbandry productivity. However, few studies have been conducted on goats. In this study, directing at the action mechanism of TAC1, the expression of TAC1 and its receptors genes in goat tissues including hypothalamus and hypophysis were studied under different illumination conditions, and the effects of light signal shifting on the expression of TAC1 and its receptors genes in various tissues were analyzed under different photoperiods, so as to further lay a foundation for revealing the regulation mechanism of TAC1 and its receptors genes on animal reproduction. Materials and Methods
Animal materials
18 healthy multiparous Henan goats over 1 year old were selected and fed under the same feeding and management and environment conditions. The goats ate food and drank water freely. Among the goats raised under artificial short photoperiod (SP= light 8 h∶dark 16 h) for 42 d, three goats were slaughtered after 4 h of illumination, and another three goats were slaughtered after 4 h of dark treatment following the illumination. The remaining goats were then raised under long photoperiod (LP= light 16 h∶dark 8 h). On the 7th day of raising the goats under the long photoperiod condition, three goats were slaughtered at 4 h of light exposure, and another three goats were slaughtered at 4 h of dark following the light exposure. On the 28th day of raising the goats under the long photoperiod condition, three goats were slaughtered at 4 h of light exposure, and another three goats were slaughtered at 4 h of dark following the light exposure. After slaughter, 12 tissues including oviduct, ovary, uterus, gluteus, mesenteric fat, brain, cerebellum, medulla oblongata, heart, lung, liver and kidney were collected and preserved in liquid nitrogen.
RNA extraction
RNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; and reverse transcription kit (PrimeScriptTM RT Reagent Kit) and fluorescent dye (SYBR Premix Ex TaqTM II) were purchased from TaKaRa (Dalian). Total RNA (Tiangen, Beijing) of various tissues were extracted with animal tissue total RNA extraction kit, and its purity and concentration were detected with Nanodrop2000. The integrity of RNA was detected by 1.5% agarose gel electrophoresis.
Primer design
According to the mRNA sequences of TAC1, TACR1, TACR2 and TACR3 genes provided by GenBank (accession number: XM_005678953.2, XM_005686308, XM_005699135 and XM_018049248), cross exon primers were designed using software Primer Premier 6.0 with GAPDH (XM_005680968.3) as reference gene. The primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. Primer names and sequences, annealing temperatures and amplified fragments are shown in Table 1.
Table 1Primers of TAC1, TACR1, TACR2 and TACR3 genes in goat
PrimernamePrimer sequenceAnnealingtemperature∥℃Productsize∥bp
TAC1AGAAGAAATCGGAGCCAAC
GAATCAGCATCCCGTTTG5594
TACR1CCAGAGCATCCCAACAAG
TCGTGGTAGCGGTCAGAG56100 TACR2CTCTCGTGGTGATGTTCG
CACGAACTTCTTCTTGGC5378
TACR3CCATCATTGACCCCTTG
AGCCTTTAGCTGCTCATG5481
GAPDHGTGATGGGCGTGAACC
ATGGCGTGGACAGTGG5592
cDNA synthesis
cDNA was synthesized with a first strand cDNA systhesis kit through reverse transcription. The reverse transcription had a total volume of 20 μl, as shown in Table 2. The reverse transcription was started at 37 ℃ for 15 min and completed at 85 ℃ for 5 s, obtaining the first chain of cDNA. The product was diluted by 5 times and subjected to PCR detection using housekeeping gene GAPDH, and the qualified cDNA was preserved at -20 ℃ for later detection of target genes.
Realtime fluorescent quantitative PCR
The reaction system of the realtime fluorescent quantitative PCR had a total volume of 20 μl, as shown in Table 3. The PCR was started with predenaturation at 95 ℃ for 5 min, followed by 40 cycles of denaturation at 95 ℃ for 10 s and annealing at 60 ℃ for 30 s. After the reaction, the melting curve was analyzed.
Table 2System of reverse transcription
ReagentVolume of each reagent
PrimeScript RT Enzyme Mix I1.0 μl
Oligo dT Primer1.0 μl
Random 6 mers1.0 μl
5×PrimeScript Buffer (for Real Time)4.0 μl
RNA1.0 μg
RNaseFree ddH2OAdded to a total volume of 20 μl
Table 3System of realtime fluorescent quantitative PCR
ReagentVolume of each reagent∥μl
SYBR Premix Ex Taq II10
Forward primer 0.8
Reverse primer 0.8
cDNA template 2
RNaseFree ddH2O6.4
Data analysis
Fluorescence quantitative detection was performed on a Roche Light Cycler480 Fluorescence quantitative PCR meter. Each sample was detected for three times with GAPDH as reference gene. The relative expression levels of target genes were calculated by 2 -△△CT method. Data were subjected to single factor ANOVA variance analysis with software SPSS19.0, and all data were represented as "mean ± standard deviation".
Results and Analysis
Tissue expression of TAC1, TACR1, TACR2 and TACR3 under short photoperiod
TAC1 gene
The experimental animals were raised under short photoperiod with 8 h of illumination daily. The experimental data showed that TAC1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in the 12 tissues except brain and kidney. Among the various tissues, TAC1 was expressed at the highest level in lung at the two
SP1 represents the expression level of TAC1 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TAC1 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 1Tissue expression of TAC1 in goat under short photoperiod relative expression of TAC1
time points, followed by ovary, liver, kidney, mesenteric fat, cerebellum, heart, brain and uterus, and the lowest in oviduct. The changing order of light and dark caused a significant difference in expression level of the gene in lung, ovary and liver.
TACR1 gene
TACR1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark in all tissues. At both the two time points, it was expressed at the highest level in liver, followed by lung, uterus, kidney, medulla oblongata, mesenteric fat, oviduct, heart, cerebellum and brain, and the lowest in muscle. The changing order of light and dark caused a significant difference in expression level of the gene in lung, ovary and liver.
SP1 represents the expression level of TACR1 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TACR1 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 2Tissue expression of TACR1 in goats under short photoperiod
Yuqin WANG et al. Expression of TAC1 and Its Receptors TACR1, TACR2, TACR3 Genes in Different Goat Tissues Under Different Photoperiods
SP1 represents the expression level of TACR2 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TACR2 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 3Tissue expression of TACR2 in goats under short photoperiod
TACR2 gene
TACR2 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in all the tissues except lung. It was expressed at the highest level in mesenteric fat, followed by brain, medulla oblongata, liver, uterus, ovary, muscle, heart, oviduct and brain. In the tissues with a higher expression level, the changing order made a significant difference in the expression level in these tissues. TACR3 gene
Also, TACR3 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in all the tissues except kidney. The expression level was the highest in liver, followed by oviduct, ovary, kidney, mesenteric fat, uterus, cerebellum, heart, medulla oblongata, muscle and brain. A significant difference was observed in the expression level of the gene between the two time points in liver, oviduct and ovary.
SP1 represents the expression level of TACR3 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TACR3 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 4Tissue expression of TACR3 in goats under short photoperiod
SP1 represents the expression level of TAC1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TAC1 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 5Tissue expression of TAC1 in goats on the 7th day under long photoperiod
It could be seen that under short photoperiod, the expression levels of tachykinin TAC1 and its receptors TACR1, TACR2 and TACR3 genes were affected by illumination, and shifting of signal from dark to light was more conductive to the expression of these genes in all tissues than that of signal from light to dark. In tissues with a higher expression level, the changing order of light and dark caused significant differences in the expression of these genes.
Tissue expression of TAC1, TACR1, TACR2 and TACR3 under long photoperiod
On the 7th day of raising under long photoperiod
After raising the goats under short photoperiod, they were then raised under long photoperiod. The data showed that TAC1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in all the tissues except brain and heart. However, except for the significant differences in the expression level in ovary and liver, there were no significant differences in other tissues.
SP1 represents the expression level of TACR1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR1 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 6Tissue expression of TACR1 in goats on the 7th day under long photoperiod
SP1 represents the expression level of TACR2 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR2 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 7Tissue expression of TACR2 in goats on the 7th day under long photoperiod
After entering the long photoperiod, TACR1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark in all tissues. The expression level was the highest in liver, with significant differences from other tissues.
Under the long photoperiod, TACR2 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in various tissues except lung and liver. The expression level was the highest in mesenteric fat, and the lowest in brain.
SP1 represents the expression level of TACR3 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR3 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 8Tissue expression of TACR3 in goats on the 7th day under long photoperiod
Under long photoperiod, TACR3 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark in various tissues. It was expressed at the highest level in liver, followed by oviduct, ovary, kidney, mesenteric fat, lung, uterus, cerebellum and heart, and the lowest in brain.
SP1 represents the expression level of TAC1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TAC1 in tissues at 4 h of dark following the light exposure under long photoperiod. "*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 9Tissue expression of TAC1 in goats on the 28th day under long photoperiod
On the 28th day of raising under long photoperiod
On the 28th day after entering long photoperiod, TAC1 was expressed at a lower level in uterus, brain, cerebellum, ovary and kidney at 4 h after turning from dark to light than at 4 h after turning from light to dark following 16 h of light exposure. The expression level of TAC1 gene was the highest in lung.
SP1 represents the expression level of TACR1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR1 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 10Tissue expression of TACR1 in goats on the 28th day under long photoperiod
On the 28th day after entering long photoperiod, TACR1 gene was expressed at the highest level in liver at 4 h after turning from dark to light, followed by lung, oviduct and kidney.
SP1 represents the expression level of TACR2 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR2 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 11Tissue expression of TACR2 in goats on the 28th day under long photoperiod
On the 28th day after entering long photoperiod, TACR2 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark following 16 h of light exposure in various tissues. The expression level of TACR2 was the highest in lung, followed by oviduct, liver and ovary.
SP1 represents the expression level of TACR3 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR3 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark. Fig. 12Tissue expression of TACR3 in goats on the 28th day under long photoperiod
On the 28th day after entering long photoperiod, TACR3 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark following 16 h of light exposure, in various tissues except cerebellum. The expression level of TACR3 was the highest in mesenteric fat, followed by oviduct, liver, lung and ovary.
Discussion
TACs play an important role in regulating reproductive function through thalamus, hypophysis and gonad. Among all tachykinins, P substance (SP), neurokinin A (NKA) and neurokinin B (NKB) have been deeply studied. They function by acting on different types of G proteincoupled receptors NK1, NK2 and NK3［10］. Researches have shown that in the reproductive system of human, tachykinins exist in ovary, uterus［11-12］, testis and prostate［13-14］. Sastry et al.［15］ once reported that SP could inhibit or stimulate the movement of human sperm. A lot of experimental data also show that TACs could play a role in the reproductive function of chordate and protochordata［16-17］. Ravina et al.［18］ demonstrated that tachykinin receptors appeared in human sperm, and acted vigorously in regulating sperm function, and SP and NKA coded by TAC1 were also expressed in sperm. Researches also showed that SP and NKA also could improve the activity of sperm, and the specificity of such effect has been demonstrated by culture experiment using NK1 and NK2 receptor inhibitors. Furthermore, NK1 and NK2 receptor genes and TAC1 genes are expressed in human sperm, so it is reckoned that TACs might play a role in sperm movement, thereby causing fertilization［19］. Besides, studies on TACs and their receptors also have been reported in medical fields including human reproduction, such as the diagnosis of the pathological change in body tissues using the function of tachykinin genes［20-21］, study on the effects of hypobaric hypoxia on the gene expression profile and TAC1 variation of rat cerebral cortex［22］, study on burn wound healing［23］ and study on colon cancer diagnosis and treatment［24-26］.
In this study, it was found that under short photoperiod, TAC1 and its receptors TACR1, TACR2 and TACR3 genes were all expressed in ovary, uterus and oviduct, and the expression levels were higher at 4 h of light exposure than at 4 h after light removal. Specifically, TAC1 gene was expressed at the highest level in ovary; TACR1 and TACR2 genes were expressed at the highest levels in uterus; and TACR3 gene was expressed at the highest level in oviduct. After entering long photoperiod, the expression pattern of TAC1 did not changed on the 7th day; and similar to short photoperiod, TACR1 was expressed at the highest level in uterus, TACR2 showed the highest expression level in oviduct, and TACR3 was expressed at the highest level in oviduct, followed by ovary. At this time, TAC1 and its receptors TACR1, TACR2 and TACR3 genes were expressed at a higher level at 4 h of light exposure than at 4 h after light removal. On the 28th day after entering long photoperiod, TAC1 and its receptors TACR1, TACR2 and TACR3 genes were all expressed in ovary, uterus and oviduct. Specifically, TAC1 gene was expressed in ovary, oviduct and uterus, and expressed at the highest level in ovary, followed by oviduct; and TACR1 gene was expressed at the highest level in oviduct, followed by uterus and ovary. However, the expression levels in these tissues decreased significantly compared with short photoperiod. The expression level of TACR2 was the highest in oviduct, followed by ovary and uterus. TACR3 was expressed at the highest level in oviduct, followed by ovary and uterus. TACR1 and TACR2 were expressed at a higher level at 4 h after light removal than at 4 h of light exposure, which was different from the expression patterns of TAC1 and TACR1 at this time. In this study, the expression of tachykinin TAC1 and its receptor genes in various tissues was not only related to daily illumination time, but also affected by the changing order of light and dark. Such phenomenon is also related to the seasonality of the reproduction of many animals in nature. Animals including horses and donkeys rut and mate in spring when the sunshine duration is prolonging, while sheep and goats rut and mate in autumn when animals sunshine duration is shortening. Animals, which rut and mate when the sunshine duration is prolonging, are known as longday animals, such as horses, donkeys and ferrets, and those, which rut and mate when the sunshine duration is shortening, are called shortday animals, such as goats and sheep. Shortening illumination time could improve shortday male animals reproductive capacity, which does not mean that long illumination would adversely influence the development of these animals reproductive organs. Treating shortday animals under long photoperiod for a certain period before mating could make animals rut more remarkably, thereby achieving a higher rate of pregnancy［27］. Researches have shown that among the 19 milk goats subjected to long photoperiod treatment (19 h/d) for 70 d, 16 female goats showed the symptom of oestrus 62 d after the treatment when animals were in anestrus, 15 only rutted and ovulated, and 11 were fertilized after mating, while no one in the natural control group rutted and mated. Therefore, the promotion of long photoperiod treatment on the reproductive activity of shortday animals has a posteffect, which might be because long photoperiod treatment before mating season stimulates adenohypophysis to secrete a large quantity of prolactin (PRL), the feedback of which inhibits the secretion of reproductionrelated hormones by hypothalamus and adenohypophysis, resulting in reductions of luteinizing hormone (LH), folliclestimulating hormone (FSH) and estrogen levels in animal blood. The termination of long illumination leads to a rapid reduction in blood PRL, the inhibition of which on hypothalamus and adenohypophysis was thus rapidly weakened, and low LH, FSH and estrogen levels are fed back and cause the improvement of the secretion of gonadotropin releasing hormone, LH, FSH and estrogen in large quantities, which then induces animal rutting and ovulation［28］. These studies show that the seasonality of animal reproduction indicates that light has a profound effect on the reproduction of livestocks, while there have been reports on the effects of different illumination time and the presence and absence of light signal on the expression of TAC1 and its receptors TACR1, TACR2 and TACR3 genes. In order to study the action mechanisms of TACs and their receptor genes in the reproductive regulation of mammals, a number of studies have been conducted on sheep and seasonal rodent animals (Syrian hamster)［29-30］, and it was found that some important pathway factors in hypothalamushypophysisgonad axis signal pathway play a role of regulating animal reproduction, which further reveals that mechanism of TAC1 as a long illumination signal to regulate reproductive hormone secretion［31-32］. However, few studies have been conducted on goats. In this study, the expression patterns of TAC1 and its receptors TACR1, TACR2 and TACR3 genes were preliminarily analyzed under different photoperiods and different signal changing orders. Due to their complicated action mechanisms and limited length of the study, the expression of these genes was not analyzed in all tissues separately, and an supplementary experiment will be further conducted to perform further analysis.
Conclusions
In this study, the expression conditions of TAC1 and its receptors TACR1, TACR2 and TACR3 genes were investigated in various tissues of goats under short photoperiod and on the 7th and 28th day after entering long photoperiod at 4 h after turning from dark to light and at 4 h after turning from light to dark. The results showed that TAC1, TACR1, TACR2 and TACR3 genes were expressed in various tissues under different illumination conditions, and in various tissues, TAC1 and TACR2 genes showed the highest expression levels, which were higher than TACR1 and TACR3 genes, suggesting that when TAC1 plays its role, TACR1 is a receptor given priority to combine with TAC1. At 4 h of light exposure on animals raised under short photoperiod and at 4 h of light exposure on animals raised under long photoperiod on the 7th and 28th day, TAC1 gene was expressed at a higher level under short photoperiod than under long photoperiod in various tissues except heart, with a significant difference in ovary. TACR2 gene was expressed at a higher level in cerebellum, medulla oblongata and uterus than at 4 h of light exposure on the 7th and 28th day after entering long photoperiod, while the expression level was higher in mesenteric fat, liver, ovary, lung, gluteus and oviduct at 4 h of light exposure on the 7th day after entering long photoperiod than at 4 h of light exposure to the animals under short photoperiod. And the expression level was the highest in mesenteric fat. The expression level of TACR2 gene was higher in oviduct, lung and liver at 4 h of light exposure on animals after entering long photoperiod than at 4 h of light exposure on animals under short photoperiod. And the expression level was the highest in lung, followed by oviduct. In the case of darkness, i.e., at 4 h of dark after 8 h of illumination under short photoperiod and after 16 h of illumination on the 7th and 28th day after entering long photoperiod, TAC1 gene was expressed at a higher level in lung and heart under long photoperiod than under short photoperiod, and at a higher level in other tissues under short photoperiod than under long photoperiod. And the expression level was the highest in lung on the 7th day after entering long photoperiod. TACR1 gene showed an expression trend on the 7th day similar to the 28th day. It could thus be seen that TAC1 and its receptor genes were extensively expressed in various goat tissues under different photoperiods, and their expression levels were affected by the changing manner of light signal, while the influence mechanisms needs further study. References
［1］ TAFELMEYER P, JOHNSSON N, JOHNSSON K. Transforming a (beta/alpha)8barrel enzyme into a splitprotein sensor through directed evolution［J］. Chem. Biol, 2004, 11(5): 681-689
［2］ HAZBUN TR, MALMSTROM L, ANDERSON S, et al. Assigning function to yeast proteins by integration of technologies［J］. Mol Cell, 2003, 12(6): 1353-1365
［3］ WESTWICK JK, MICHNICK SW. Proteinfragment complementation assays (PCA) in small GTPase research and drug discovery［J］. Methods Enzymol, 2005, 407: 388-401
［4］ ZHU H, BILGIN M, BANGHAM R, et al. Global analysis of protein activities using proteome chips［J］. Science, 2001, 293(5537): 2101-2105.
［5］ GIBSON SJ, BLOOM SR, POLAK JM. A novel substance P pathway linking the dorsal and ventral horn in the upper lumbar segments of the rat spinal cord［J］. Brain Res, 1984, 301: 243-51.
［6］ MAGGIO JE. Tachykinins［J］. Annu Rev Neurosci, 1988, 11: 13-28.
［7］ HANON EA, LINCOLN GA, FUSTIN JM, et al. Ancestral TSH mechanism signals summer in a photoperiodic mammal［J］. Curr. Biol., 2008, (18): 1147-1152.
［8］ STIRLAND JA, JOHNSTON JD, CAGAMPANG FR, et al. Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalisderived factor［J］. Neuroendocrinol, 2001, (13): 147-157.
［9］ GRAHAM ES, WEBSTER CA, HAZLERIGG DG, et al. Evidence for the biosynthesis of a prolactinreleasing factor from the ovine pars tuberalis, which is distinct from thyrotropinreleasing hormone［J］. Neuroendocrinol, 2002, (14): 945-954.
［10］ PENNEFATHER JN,LECCI A, CANDENAS ML, et al. Tachykinins and tachykinin receptors a growing family［J］. Life Sci, 2004, (74): 1445-1463
［11］ TRAURIG H, SARIA A, LEMBECK F. Substance P in primary afferent neurons of the female rat reproductive system［J］. NaunynSchmiedebergs archives of pharmacology, 1984, 326(4): 343-346.
［12］ PENNEFATHER JN, PATAK E, PINTO FM, et al. Mammalian tachykinins and uterine smooth muscle: the challenge escalates［J］. European journal of pharmacology, 2004, 500(1-3): 15-26.
［13］ LOWRY PJ. Has the mechanism by which the endocrine placenta scavenges the mother whilst sparing the foetus been unmasked［J］. Journal of molecular endocrinology, 2003, 31(3): 341-347.
［14］ CHIWAKATA C, BRACKMANN B, HUNT N, et al. Tachykinin (substanceP) gene expression in Leydig cells of the human and mouse testis［J］. Endocrinology, 1991, 128(5): 2441-8. ［15］ DEBELJUK L, RAO JN, BARTKE A. Tachykinins and their possible modulatory role on testicular function: a review ［J］. International journal of andrology, 2003, 26(4): 202-210.
［16］ PINTO FM, ALMEIDA TA, HERNANDEZ M, et al. mRNA expression of tachykinins and tachykinin receptors in different human tissues［J］. European journal of pharmacology, 2004, 494(2-3): 233-239.
［17］ SASTRY BV, JANSON VE, OWENS LK. Significance of substance P and enkephalinpeptide systems in the male genital tract［J］. Annals of the New York Academy of Sciences, 1991, 632: 339-353.
［18］ DEBELJUK L, LASAGA M. Modulation of the hypothalamopituitarygonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide gamma［J］. Peptides, 1999, 20(2): 285-299.
［19］ SATAKE H, OGASAWARA M, KAWADA T, et al. Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family［J］.The Journal of biological chemistry, 2004, 279(51): 53798-805.
［20］ CANDENAS L, LECCI A, PINTO FM, et al. Tachykinins and tachykinin receptors: effects in the genitourinary tract［J］. Life sciences, 2005, 76(8): 835-62.
［21］ RAVINA CG, SEDA M, PINTO FM, et al. A role for tachykinins in the regulation of human sperm motility［J］. Hum Reprod, 2007, 22(6): 1617-1625.
［33］ PINTO FM, RAVINA CG, SUBIRAN N, et al. Autocrine regulation of human sperm motility by tachykinins［J］. Reproductive Biology and Endocrinology, 2010, 8: 104.
［22］ GAO HS, HUANG F, WANG YZ, et al. the effects of hypobaric hypoxia on the changes of gene expression profile and TAC1 and MT1A in rats cerebral cortex［J］. Chinese Journal of Rehabilitation Medicine, 2013, 28(8): 699-707. (in Chinese)
［23］ MIN J, LI MY, LIU YH, et al. The explore of interaction between SPAG6 and TAC1［J］. National Journal of Andrology, 2016, 22(12): 1059－1064. (in Chinese)
［24］ ZHANG Y. The protective role of transient receptor potential vanilloid 1 via substance P in apoptosis post acute myocardial infarction in mice［D］. Chongqing: Chongqing Medical University, 2015. (in Chinese)
［25］ WANG XH. Effect of neurokinin A on wound healing of deep second degree burn in rats［D］. Shanghai: Shanghai Jiao Tong University, 2014. (in Chinese)
［38］ LU Y. Study on two agonists of neurokinin receptor on colon cancer cell proliferation［D］. Hanzhou: Zhejiang SciTech University, 2015. (in Chinese) ［26］ WRANGLE J, MACHIDA EO, DANILOVA L, et al. Functional identification of cancerspecific methylation of CDOl, HOXA9, and TACl for the diagnosis of lung cancer ［J］. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research.2014, 20(7):1856-1864.
［27］ MENG F, DEMORROW S, VENTER J, et al. Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth［J］. American Journal of Physiology Gastrointestinal and Liver Physiology, 2014, 306(9):G759-68.
［28］ WANG QY. Domestic livestock ecology in China［M］. Beijing: China Agriculture Press, 2008. (in Chinese)
［29］ BONDURANT RH, DARIEN BJ, MUNRO CJ, et al. Photoperiod induction of fertile oestrus and changes in LH and progesterone concentrations in yearling dairy goats (Capra hircus)［J］. J Reprod Fertil, 1981, 63(1): 1-9.
［30］ WOOD SH, CHRISTIAN HC, MIEDZINSKA K, et al. Binary switching of calendar cells in the pituitary defines the phase of the circannual cycle in mammals［J］. Curr Biol, 2015, 25(20): 2651-2662.
［44］ SHONA H WOOD, HELEN C CHRISTIAN, KATARZYNA MIEDZINSKA, et al. Binary switching of calendar cells in the pituitary defines the phase of the circannual cycle in mammals［J］. Curr Biol, 2015, 25(20): 2651-2662.
［31］ WEEMS PW, GOODMAN RL, LEHMAN MN. Neural mechanisms controlling seasonal reproduction: principles derived from the sheep model and its comparison with hamsters［J］. Front Neuroendocrinol, 2015, 37: 43-51.
［32］ DUPR SM, MIEDZINSKA K, DUVAL CV, et al. Identification of Eya3 and TAC1 as longday signals in the sheep pituitary［J］. Curr Biol, 2010, 20(9): 829-35.
Key wordsGoat; TAC1; TACR1; TACR2; TACR3; Tissue Expression; Photoperiod
Received: May 23, 2018Accepted: August 27, 2018
Supported by National Natural Science Foundation of China (31472095); Earmarked Fund for China Agriculture Research System (CARS38).
Yuqin WANG (1972-), female, P. R. China, Professor, PhD, devoted to research about genetic breeding and production.
*Corresponding author. Email: wangyq6836@163.com.
Tachykinins (TACs) constitute a peptide family, and the bestknown peptides include substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and hemokinin1 (HK1)［1-2］. Researches have demonstrated that TAC1 could produce four kinds of different mRNA subtypes (a, β, γ and δ), which could code SP (a, β, γ and δ) and NKA (β and γ)［3-4］. TACs are a type of polypeptides having biological activity, which are widely distributed in central nervous system and peripheral nervous system［5-6］. In recent years, researches have shown that TACs play an important role in regulating the reproductive function of mammal animals including human and mice through hypothalamuspituitarygonad axis, and especially, researches on hypothalamuspituitarygonad axis have shown that TACs influence the release of GnRH through hypothalamic neuron, and play an important role in regulating reproductive processes including animal oestrus, ovulation and gestation. The reproductive phenomenon and reproductive law of mammals have shown that melatonin secreted by pituitary and pineal directly acts on pars tuberalis of hypophysis to regulate the expression of thyroid stimulating hormone (TSH) which then feeds back the information to hypothalamus and regulates the reproduction pathway［7］, but there is also another pathway in hypophysis which regulates seasonal release of prolactin (PRL)［8-9］. These researches are of great significance to the revealing of the reproductive mechanism of mammals and the improvement of animal husbandry productivity. However, few studies have been conducted on goats. In this study, directing at the action mechanism of TAC1, the expression of TAC1 and its receptors genes in goat tissues including hypothalamus and hypophysis were studied under different illumination conditions, and the effects of light signal shifting on the expression of TAC1 and its receptors genes in various tissues were analyzed under different photoperiods, so as to further lay a foundation for revealing the regulation mechanism of TAC1 and its receptors genes on animal reproduction. Materials and Methods
Animal materials
18 healthy multiparous Henan goats over 1 year old were selected and fed under the same feeding and management and environment conditions. The goats ate food and drank water freely. Among the goats raised under artificial short photoperiod (SP= light 8 h∶dark 16 h) for 42 d, three goats were slaughtered after 4 h of illumination, and another three goats were slaughtered after 4 h of dark treatment following the illumination. The remaining goats were then raised under long photoperiod (LP= light 16 h∶dark 8 h). On the 7th day of raising the goats under the long photoperiod condition, three goats were slaughtered at 4 h of light exposure, and another three goats were slaughtered at 4 h of dark following the light exposure. On the 28th day of raising the goats under the long photoperiod condition, three goats were slaughtered at 4 h of light exposure, and another three goats were slaughtered at 4 h of dark following the light exposure. After slaughter, 12 tissues including oviduct, ovary, uterus, gluteus, mesenteric fat, brain, cerebellum, medulla oblongata, heart, lung, liver and kidney were collected and preserved in liquid nitrogen.
RNA extraction
RNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; and reverse transcription kit (PrimeScriptTM RT Reagent Kit) and fluorescent dye (SYBR Premix Ex TaqTM II) were purchased from TaKaRa (Dalian). Total RNA (Tiangen, Beijing) of various tissues were extracted with animal tissue total RNA extraction kit, and its purity and concentration were detected with Nanodrop2000. The integrity of RNA was detected by 1.5% agarose gel electrophoresis.
Primer design
According to the mRNA sequences of TAC1, TACR1, TACR2 and TACR3 genes provided by GenBank (accession number: XM_005678953.2, XM_005686308, XM_005699135 and XM_018049248), cross exon primers were designed using software Primer Premier 6.0 with GAPDH (XM_005680968.3) as reference gene. The primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. Primer names and sequences, annealing temperatures and amplified fragments are shown in Table 1.
Table 1Primers of TAC1, TACR1, TACR2 and TACR3 genes in goat
PrimernamePrimer sequenceAnnealingtemperature∥℃Productsize∥bp
TAC1AGAAGAAATCGGAGCCAAC
GAATCAGCATCCCGTTTG5594
TACR1CCAGAGCATCCCAACAAG
TCGTGGTAGCGGTCAGAG56100 TACR2CTCTCGTGGTGATGTTCG
CACGAACTTCTTCTTGGC5378
TACR3CCATCATTGACCCCTTG
AGCCTTTAGCTGCTCATG5481
GAPDHGTGATGGGCGTGAACC
ATGGCGTGGACAGTGG5592
cDNA synthesis
cDNA was synthesized with a first strand cDNA systhesis kit through reverse transcription. The reverse transcription had a total volume of 20 μl, as shown in Table 2. The reverse transcription was started at 37 ℃ for 15 min and completed at 85 ℃ for 5 s, obtaining the first chain of cDNA. The product was diluted by 5 times and subjected to PCR detection using housekeeping gene GAPDH, and the qualified cDNA was preserved at -20 ℃ for later detection of target genes.
Realtime fluorescent quantitative PCR
The reaction system of the realtime fluorescent quantitative PCR had a total volume of 20 μl, as shown in Table 3. The PCR was started with predenaturation at 95 ℃ for 5 min, followed by 40 cycles of denaturation at 95 ℃ for 10 s and annealing at 60 ℃ for 30 s. After the reaction, the melting curve was analyzed.
Table 2System of reverse transcription
ReagentVolume of each reagent
PrimeScript RT Enzyme Mix I1.0 μl
Oligo dT Primer1.0 μl
Random 6 mers1.0 μl
5×PrimeScript Buffer (for Real Time)4.0 μl
RNA1.0 μg
RNaseFree ddH2OAdded to a total volume of 20 μl
Table 3System of realtime fluorescent quantitative PCR
ReagentVolume of each reagent∥μl
SYBR Premix Ex Taq II10
Forward primer 0.8
Reverse primer 0.8
cDNA template 2
RNaseFree ddH2O6.4
Data analysis
Fluorescence quantitative detection was performed on a Roche Light Cycler480 Fluorescence quantitative PCR meter. Each sample was detected for three times with GAPDH as reference gene. The relative expression levels of target genes were calculated by 2 -△△CT method. Data were subjected to single factor ANOVA variance analysis with software SPSS19.0, and all data were represented as "mean ± standard deviation".
Results and Analysis
Tissue expression of TAC1, TACR1, TACR2 and TACR3 under short photoperiod
TAC1 gene
The experimental animals were raised under short photoperiod with 8 h of illumination daily. The experimental data showed that TAC1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in the 12 tissues except brain and kidney. Among the various tissues, TAC1 was expressed at the highest level in lung at the two
SP1 represents the expression level of TAC1 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TAC1 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 1Tissue expression of TAC1 in goat under short photoperiod relative expression of TAC1
time points, followed by ovary, liver, kidney, mesenteric fat, cerebellum, heart, brain and uterus, and the lowest in oviduct. The changing order of light and dark caused a significant difference in expression level of the gene in lung, ovary and liver.
TACR1 gene
TACR1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark in all tissues. At both the two time points, it was expressed at the highest level in liver, followed by lung, uterus, kidney, medulla oblongata, mesenteric fat, oviduct, heart, cerebellum and brain, and the lowest in muscle. The changing order of light and dark caused a significant difference in expression level of the gene in lung, ovary and liver.
SP1 represents the expression level of TACR1 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TACR1 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 2Tissue expression of TACR1 in goats under short photoperiod
Yuqin WANG et al. Expression of TAC1 and Its Receptors TACR1, TACR2, TACR3 Genes in Different Goat Tissues Under Different Photoperiods
SP1 represents the expression level of TACR2 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TACR2 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 3Tissue expression of TACR2 in goats under short photoperiod
TACR2 gene
TACR2 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in all the tissues except lung. It was expressed at the highest level in mesenteric fat, followed by brain, medulla oblongata, liver, uterus, ovary, muscle, heart, oviduct and brain. In the tissues with a higher expression level, the changing order made a significant difference in the expression level in these tissues. TACR3 gene
Also, TACR3 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in all the tissues except kidney. The expression level was the highest in liver, followed by oviduct, ovary, kidney, mesenteric fat, uterus, cerebellum, heart, medulla oblongata, muscle and brain. A significant difference was observed in the expression level of the gene between the two time points in liver, oviduct and ovary.
SP1 represents the expression level of TACR3 in tissues at 4 h of light exposure under short photoperiod; and SP2 represents the expression level of TACR3 in tissues at 4 h of dark following the light exposure under short photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 4Tissue expression of TACR3 in goats under short photoperiod
SP1 represents the expression level of TAC1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TAC1 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 5Tissue expression of TAC1 in goats on the 7th day under long photoperiod
It could be seen that under short photoperiod, the expression levels of tachykinin TAC1 and its receptors TACR1, TACR2 and TACR3 genes were affected by illumination, and shifting of signal from dark to light was more conductive to the expression of these genes in all tissues than that of signal from light to dark. In tissues with a higher expression level, the changing order of light and dark caused significant differences in the expression of these genes.
Tissue expression of TAC1, TACR1, TACR2 and TACR3 under long photoperiod
On the 7th day of raising under long photoperiod
After raising the goats under short photoperiod, they were then raised under long photoperiod. The data showed that TAC1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in all the tissues except brain and heart. However, except for the significant differences in the expression level in ovary and liver, there were no significant differences in other tissues.
SP1 represents the expression level of TACR1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR1 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 6Tissue expression of TACR1 in goats on the 7th day under long photoperiod
SP1 represents the expression level of TACR2 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR2 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 7Tissue expression of TACR2 in goats on the 7th day under long photoperiod
After entering the long photoperiod, TACR1 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark in all tissues. The expression level was the highest in liver, with significant differences from other tissues.
Under the long photoperiod, TACR2 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark, in various tissues except lung and liver. The expression level was the highest in mesenteric fat, and the lowest in brain.
SP1 represents the expression level of TACR3 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR3 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 8Tissue expression of TACR3 in goats on the 7th day under long photoperiod
Under long photoperiod, TACR3 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark in various tissues. It was expressed at the highest level in liver, followed by oviduct, ovary, kidney, mesenteric fat, lung, uterus, cerebellum and heart, and the lowest in brain.
SP1 represents the expression level of TAC1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TAC1 in tissues at 4 h of dark following the light exposure under long photoperiod. "*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 9Tissue expression of TAC1 in goats on the 28th day under long photoperiod
On the 28th day of raising under long photoperiod
On the 28th day after entering long photoperiod, TAC1 was expressed at a lower level in uterus, brain, cerebellum, ovary and kidney at 4 h after turning from dark to light than at 4 h after turning from light to dark following 16 h of light exposure. The expression level of TAC1 gene was the highest in lung.
SP1 represents the expression level of TACR1 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR1 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 10Tissue expression of TACR1 in goats on the 28th day under long photoperiod
On the 28th day after entering long photoperiod, TACR1 gene was expressed at the highest level in liver at 4 h after turning from dark to light, followed by lung, oviduct and kidney.
SP1 represents the expression level of TACR2 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR2 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark.
Fig. 11Tissue expression of TACR2 in goats on the 28th day under long photoperiod
On the 28th day after entering long photoperiod, TACR2 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark following 16 h of light exposure in various tissues. The expression level of TACR2 was the highest in lung, followed by oviduct, liver and ovary.
SP1 represents the expression level of TACR3 in tissues at 4 h of light exposure under long photoperiod; and SP2 represents the expression level of TACR3 in tissues at 4 h of dark following the light exposure under long photoperiod.
"*" on the columns indicates a significant difference (P<0.05) in the expression level of the gene between 4 h of light and 4 h of dark. Fig. 12Tissue expression of TACR3 in goats on the 28th day under long photoperiod
On the 28th day after entering long photoperiod, TACR3 was expressed at a higher level at 4 h after turning from dark to light than at 4 h after turning from light to dark following 16 h of light exposure, in various tissues except cerebellum. The expression level of TACR3 was the highest in mesenteric fat, followed by oviduct, liver, lung and ovary.
Discussion
TACs play an important role in regulating reproductive function through thalamus, hypophysis and gonad. Among all tachykinins, P substance (SP), neurokinin A (NKA) and neurokinin B (NKB) have been deeply studied. They function by acting on different types of G proteincoupled receptors NK1, NK2 and NK3［10］. Researches have shown that in the reproductive system of human, tachykinins exist in ovary, uterus［11-12］, testis and prostate［13-14］. Sastry et al.［15］ once reported that SP could inhibit or stimulate the movement of human sperm. A lot of experimental data also show that TACs could play a role in the reproductive function of chordate and protochordata［16-17］. Ravina et al.［18］ demonstrated that tachykinin receptors appeared in human sperm, and acted vigorously in regulating sperm function, and SP and NKA coded by TAC1 were also expressed in sperm. Researches also showed that SP and NKA also could improve the activity of sperm, and the specificity of such effect has been demonstrated by culture experiment using NK1 and NK2 receptor inhibitors. Furthermore, NK1 and NK2 receptor genes and TAC1 genes are expressed in human sperm, so it is reckoned that TACs might play a role in sperm movement, thereby causing fertilization［19］. Besides, studies on TACs and their receptors also have been reported in medical fields including human reproduction, such as the diagnosis of the pathological change in body tissues using the function of tachykinin genes［20-21］, study on the effects of hypobaric hypoxia on the gene expression profile and TAC1 variation of rat cerebral cortex［22］, study on burn wound healing［23］ and study on colon cancer diagnosis and treatment［24-26］.
In this study, it was found that under short photoperiod, TAC1 and its receptors TACR1, TACR2 and TACR3 genes were all expressed in ovary, uterus and oviduct, and the expression levels were higher at 4 h of light exposure than at 4 h after light removal. Specifically, TAC1 gene was expressed at the highest level in ovary; TACR1 and TACR2 genes were expressed at the highest levels in uterus; and TACR3 gene was expressed at the highest level in oviduct. After entering long photoperiod, the expression pattern of TAC1 did not changed on the 7th day; and similar to short photoperiod, TACR1 was expressed at the highest level in uterus, TACR2 showed the highest expression level in oviduct, and TACR3 was expressed at the highest level in oviduct, followed by ovary. At this time, TAC1 and its receptors TACR1, TACR2 and TACR3 genes were expressed at a higher level at 4 h of light exposure than at 4 h after light removal. On the 28th day after entering long photoperiod, TAC1 and its receptors TACR1, TACR2 and TACR3 genes were all expressed in ovary, uterus and oviduct. Specifically, TAC1 gene was expressed in ovary, oviduct and uterus, and expressed at the highest level in ovary, followed by oviduct; and TACR1 gene was expressed at the highest level in oviduct, followed by uterus and ovary. However, the expression levels in these tissues decreased significantly compared with short photoperiod. The expression level of TACR2 was the highest in oviduct, followed by ovary and uterus. TACR3 was expressed at the highest level in oviduct, followed by ovary and uterus. TACR1 and TACR2 were expressed at a higher level at 4 h after light removal than at 4 h of light exposure, which was different from the expression patterns of TAC1 and TACR1 at this time. In this study, the expression of tachykinin TAC1 and its receptor genes in various tissues was not only related to daily illumination time, but also affected by the changing order of light and dark. Such phenomenon is also related to the seasonality of the reproduction of many animals in nature. Animals including horses and donkeys rut and mate in spring when the sunshine duration is prolonging, while sheep and goats rut and mate in autumn when animals sunshine duration is shortening. Animals, which rut and mate when the sunshine duration is prolonging, are known as longday animals, such as horses, donkeys and ferrets, and those, which rut and mate when the sunshine duration is shortening, are called shortday animals, such as goats and sheep. Shortening illumination time could improve shortday male animals reproductive capacity, which does not mean that long illumination would adversely influence the development of these animals reproductive organs. Treating shortday animals under long photoperiod for a certain period before mating could make animals rut more remarkably, thereby achieving a higher rate of pregnancy［27］. Researches have shown that among the 19 milk goats subjected to long photoperiod treatment (19 h/d) for 70 d, 16 female goats showed the symptom of oestrus 62 d after the treatment when animals were in anestrus, 15 only rutted and ovulated, and 11 were fertilized after mating, while no one in the natural control group rutted and mated. Therefore, the promotion of long photoperiod treatment on the reproductive activity of shortday animals has a posteffect, which might be because long photoperiod treatment before mating season stimulates adenohypophysis to secrete a large quantity of prolactin (PRL), the feedback of which inhibits the secretion of reproductionrelated hormones by hypothalamus and adenohypophysis, resulting in reductions of luteinizing hormone (LH), folliclestimulating hormone (FSH) and estrogen levels in animal blood. The termination of long illumination leads to a rapid reduction in blood PRL, the inhibition of which on hypothalamus and adenohypophysis was thus rapidly weakened, and low LH, FSH and estrogen levels are fed back and cause the improvement of the secretion of gonadotropin releasing hormone, LH, FSH and estrogen in large quantities, which then induces animal rutting and ovulation［28］. These studies show that the seasonality of animal reproduction indicates that light has a profound effect on the reproduction of livestocks, while there have been reports on the effects of different illumination time and the presence and absence of light signal on the expression of TAC1 and its receptors TACR1, TACR2 and TACR3 genes. In order to study the action mechanisms of TACs and their receptor genes in the reproductive regulation of mammals, a number of studies have been conducted on sheep and seasonal rodent animals (Syrian hamster)［29-30］, and it was found that some important pathway factors in hypothalamushypophysisgonad axis signal pathway play a role of regulating animal reproduction, which further reveals that mechanism of TAC1 as a long illumination signal to regulate reproductive hormone secretion［31-32］. However, few studies have been conducted on goats. In this study, the expression patterns of TAC1 and its receptors TACR1, TACR2 and TACR3 genes were preliminarily analyzed under different photoperiods and different signal changing orders. Due to their complicated action mechanisms and limited length of the study, the expression of these genes was not analyzed in all tissues separately, and an supplementary experiment will be further conducted to perform further analysis.
Conclusions
In this study, the expression conditions of TAC1 and its receptors TACR1, TACR2 and TACR3 genes were investigated in various tissues of goats under short photoperiod and on the 7th and 28th day after entering long photoperiod at 4 h after turning from dark to light and at 4 h after turning from light to dark. The results showed that TAC1, TACR1, TACR2 and TACR3 genes were expressed in various tissues under different illumination conditions, and in various tissues, TAC1 and TACR2 genes showed the highest expression levels, which were higher than TACR1 and TACR3 genes, suggesting that when TAC1 plays its role, TACR1 is a receptor given priority to combine with TAC1. At 4 h of light exposure on animals raised under short photoperiod and at 4 h of light exposure on animals raised under long photoperiod on the 7th and 28th day, TAC1 gene was expressed at a higher level under short photoperiod than under long photoperiod in various tissues except heart, with a significant difference in ovary. TACR2 gene was expressed at a higher level in cerebellum, medulla oblongata and uterus than at 4 h of light exposure on the 7th and 28th day after entering long photoperiod, while the expression level was higher in mesenteric fat, liver, ovary, lung, gluteus and oviduct at 4 h of light exposure on the 7th day after entering long photoperiod than at 4 h of light exposure to the animals under short photoperiod. And the expression level was the highest in mesenteric fat. The expression level of TACR2 gene was higher in oviduct, lung and liver at 4 h of light exposure on animals after entering long photoperiod than at 4 h of light exposure on animals under short photoperiod. And the expression level was the highest in lung, followed by oviduct. In the case of darkness, i.e., at 4 h of dark after 8 h of illumination under short photoperiod and after 16 h of illumination on the 7th and 28th day after entering long photoperiod, TAC1 gene was expressed at a higher level in lung and heart under long photoperiod than under short photoperiod, and at a higher level in other tissues under short photoperiod than under long photoperiod. And the expression level was the highest in lung on the 7th day after entering long photoperiod. TACR1 gene showed an expression trend on the 7th day similar to the 28th day. It could thus be seen that TAC1 and its receptor genes were extensively expressed in various goat tissues under different photoperiods, and their expression levels were affected by the changing manner of light signal, while the influence mechanisms needs further study. References
［1］ TAFELMEYER P, JOHNSSON N, JOHNSSON K. Transforming a (beta/alpha)8barrel enzyme into a splitprotein sensor through directed evolution［J］. Chem. Biol, 2004, 11(5): 681-689
［2］ HAZBUN TR, MALMSTROM L, ANDERSON S, et al. Assigning function to yeast proteins by integration of technologies［J］. Mol Cell, 2003, 12(6): 1353-1365
［3］ WESTWICK JK, MICHNICK SW. Proteinfragment complementation assays (PCA) in small GTPase research and drug discovery［J］. Methods Enzymol, 2005, 407: 388-401
［4］ ZHU H, BILGIN M, BANGHAM R, et al. Global analysis of protein activities using proteome chips［J］. Science, 2001, 293(5537): 2101-2105.
［5］ GIBSON SJ, BLOOM SR, POLAK JM. A novel substance P pathway linking the dorsal and ventral horn in the upper lumbar segments of the rat spinal cord［J］. Brain Res, 1984, 301: 243-51.
［6］ MAGGIO JE. Tachykinins［J］. Annu Rev Neurosci, 1988, 11: 13-28.
［7］ HANON EA, LINCOLN GA, FUSTIN JM, et al. Ancestral TSH mechanism signals summer in a photoperiodic mammal［J］. Curr. Biol., 2008, (18): 1147-1152.
［8］ STIRLAND JA, JOHNSTON JD, CAGAMPANG FR, et al. Photoperiodic regulation of prolactin gene expression in the Syrian hamster by a pars tuberalisderived factor［J］. Neuroendocrinol, 2001, (13): 147-157.
［9］ GRAHAM ES, WEBSTER CA, HAZLERIGG DG, et al. Evidence for the biosynthesis of a prolactinreleasing factor from the ovine pars tuberalis, which is distinct from thyrotropinreleasing hormone［J］. Neuroendocrinol, 2002, (14): 945-954.
［10］ PENNEFATHER JN,LECCI A, CANDENAS ML, et al. Tachykinins and tachykinin receptors a growing family［J］. Life Sci, 2004, (74): 1445-1463
［11］ TRAURIG H, SARIA A, LEMBECK F. Substance P in primary afferent neurons of the female rat reproductive system［J］. NaunynSchmiedebergs archives of pharmacology, 1984, 326(4): 343-346.
［12］ PENNEFATHER JN, PATAK E, PINTO FM, et al. Mammalian tachykinins and uterine smooth muscle: the challenge escalates［J］. European journal of pharmacology, 2004, 500(1-3): 15-26.
［13］ LOWRY PJ. Has the mechanism by which the endocrine placenta scavenges the mother whilst sparing the foetus been unmasked［J］. Journal of molecular endocrinology, 2003, 31(3): 341-347.
［14］ CHIWAKATA C, BRACKMANN B, HUNT N, et al. Tachykinin (substanceP) gene expression in Leydig cells of the human and mouse testis［J］. Endocrinology, 1991, 128(5): 2441-8. ［15］ DEBELJUK L, RAO JN, BARTKE A. Tachykinins and their possible modulatory role on testicular function: a review ［J］. International journal of andrology, 2003, 26(4): 202-210.
［16］ PINTO FM, ALMEIDA TA, HERNANDEZ M, et al. mRNA expression of tachykinins and tachykinin receptors in different human tissues［J］. European journal of pharmacology, 2004, 494(2-3): 233-239.
［17］ SASTRY BV, JANSON VE, OWENS LK. Significance of substance P and enkephalinpeptide systems in the male genital tract［J］. Annals of the New York Academy of Sciences, 1991, 632: 339-353.
［18］ DEBELJUK L, LASAGA M. Modulation of the hypothalamopituitarygonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide gamma［J］. Peptides, 1999, 20(2): 285-299.
［19］ SATAKE H, OGASAWARA M, KAWADA T, et al. Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family［J］.The Journal of biological chemistry, 2004, 279(51): 53798-805.
［20］ CANDENAS L, LECCI A, PINTO FM, et al. Tachykinins and tachykinin receptors: effects in the genitourinary tract［J］. Life sciences, 2005, 76(8): 835-62.
［21］ RAVINA CG, SEDA M, PINTO FM, et al. A role for tachykinins in the regulation of human sperm motility［J］. Hum Reprod, 2007, 22(6): 1617-1625.
［33］ PINTO FM, RAVINA CG, SUBIRAN N, et al. Autocrine regulation of human sperm motility by tachykinins［J］. Reproductive Biology and Endocrinology, 2010, 8: 104.
［22］ GAO HS, HUANG F, WANG YZ, et al. the effects of hypobaric hypoxia on the changes of gene expression profile and TAC1 and MT1A in rats cerebral cortex［J］. Chinese Journal of Rehabilitation Medicine, 2013, 28(8): 699-707. (in Chinese)
［23］ MIN J, LI MY, LIU YH, et al. The explore of interaction between SPAG6 and TAC1［J］. National Journal of Andrology, 2016, 22(12): 1059－1064. (in Chinese)
［24］ ZHANG Y. The protective role of transient receptor potential vanilloid 1 via substance P in apoptosis post acute myocardial infarction in mice［D］. Chongqing: Chongqing Medical University, 2015. (in Chinese)
［25］ WANG XH. Effect of neurokinin A on wound healing of deep second degree burn in rats［D］. Shanghai: Shanghai Jiao Tong University, 2014. (in Chinese)
［38］ LU Y. Study on two agonists of neurokinin receptor on colon cancer cell proliferation［D］. Hanzhou: Zhejiang SciTech University, 2015. (in Chinese) ［26］ WRANGLE J, MACHIDA EO, DANILOVA L, et al. Functional identification of cancerspecific methylation of CDOl, HOXA9, and TACl for the diagnosis of lung cancer ［J］. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research.2014, 20(7):1856-1864.
［27］ MENG F, DEMORROW S, VENTER J, et al. Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth［J］. American Journal of Physiology Gastrointestinal and Liver Physiology, 2014, 306(9):G759-68.
［28］ WANG QY. Domestic livestock ecology in China［M］. Beijing: China Agriculture Press, 2008. (in Chinese)
［29］ BONDURANT RH, DARIEN BJ, MUNRO CJ, et al. Photoperiod induction of fertile oestrus and changes in LH and progesterone concentrations in yearling dairy goats (Capra hircus)［J］. J Reprod Fertil, 1981, 63(1): 1-9.
［30］ WOOD SH, CHRISTIAN HC, MIEDZINSKA K, et al. Binary switching of calendar cells in the pituitary defines the phase of the circannual cycle in mammals［J］. Curr Biol, 2015, 25(20): 2651-2662.
［44］ SHONA H WOOD, HELEN C CHRISTIAN, KATARZYNA MIEDZINSKA, et al. Binary switching of calendar cells in the pituitary defines the phase of the circannual cycle in mammals［J］. Curr Biol, 2015, 25(20): 2651-2662.
［31］ WEEMS PW, GOODMAN RL, LEHMAN MN. Neural mechanisms controlling seasonal reproduction: principles derived from the sheep model and its comparison with hamsters［J］. Front Neuroendocrinol, 2015, 37: 43-51.
［32］ DUPR SM, MIEDZINSKA K, DUVAL CV, et al. Identification of Eya3 and TAC1 as longday signals in the sheep pituitary［J］. Curr Biol, 2010, 20(9): 829-35.