The Effect Of PACAP/PAC1R On Temperature-regulated Reproductive Activities Of Black Threaded Rat

The Phodopus sungorus belongs to Cricetinae,Phodopus.It has typical seasonal reproduction characteristics,and the breeding time is generally from March to September every year.It is one of the main grassland rodents in Inner Mongolia.The mouse is slow to move,easy to catch,easy to breed and breed,and 4 to 8 per litter.It can be used as an ideal material for studying the seasonal reproduction mechanism of animals.In this experiment,the Phodopus sungorus was used as the research object.Besides,the ambient temperature is an important environmental factor affecting the seasonal reproduction activities of animals.The seasonal reproductive activity of mammals is regulated by positive feedback from the complex nervous system.Pituitary adenylate cyclase activating polypeptide(PACAP)and pituitary adenylate cyclase activating peptide receptor(PAC1R)play an important role in this regulatory pathway.PACAP and PAC1 R can regulate the activity of hypothalamic gonadotropin releasing hormone(GnRH)neurons,affecting the synthesis and secretion of GnRH,and GnRH through the pituitary portal system,further affecting the secretion of sex hormones,then participates in the regulation of animal gonadal development and animal reproduction.However,there is no report on how the temperature regulates the molecular mechanism of the seasonal reproduction of the Phodopus sungorus.In order to investigate the molecular mechanism of PACAP/PAC1 R in temperature-regulating seasonal breeding activities of the Phodopus sungorus,taken the Phodopus sungorus as study object,used the method of molecular biology and bioinformatics,the nucleotide sequence of PACAP/PAC1 R was cloned and the amino acid sequence was analyzed.In addition,the feed intake of the Phodopus sungorus,the differential expression of PACAP/PAC1 R in the hypothalamus and the promotion of serum were also studied under different temperature conditions.The concentration of follicle stimulating hormone(FSH)and gonadotropin releasing hormone(GnRH),the correlation between serum hormone concentration and PACAP/PAC1 R gene expression level were analyzed.The main results were as follows.1.By gene cloning,the nucleotide sequence of PACAP/PAC1 R was obtained for the first time.The length of PACAP cDNA was 574 bp,including the complete reading frame 534 bp and part of the 5’ non-coding area,encoding 176 amino acid.PAC1 R cDNA sequence is 1467 bp in length,including the complete reading frame 1407 bp and part of the 3’ non-coding area,encoding a total of 467 amino acids.2.Through amino acid sequence structure analysis,the results showed that PACAP contains vesoactive intestinal polypeptide(VIP)/glucagons/secretin/and gastric inhibitory polypeptide(GIP).PAC1 R is a seven-transmembrane protein belonging to the G protein coupled receptor family.3.Multiple-sequence comparison analysis showed that the amino acid sequence encoded by the PACAP/PAC1 R gene of the Phodopus sungorus was compared with many species such as rat(Rattus norvegicus)and mouse(Mus musculus).The evolution of species is relatively conservative.4.By analyzing the feed intake of the black-footed gray-footed rats,the results showed that there was no significant difference between the low temperature,high temperature and suitable temperature groups 24 days ago(P>0.05),but on the 24 th day,the low temperature was significant difference to the suitable temperature group(P<0.05).And the food intake in the low temperature group was significantly higher than that in the high temperature group.Therefore,as an important regulatory factor,temperature will have a great impact on the physiological activities of the Phodopus sungorus.5.By analyzing the differential expression of PACAP/PAC1 R in the hypothalamus,PACAP/PAC1 R was expressed in the hypothalamus of females at the optimum temperature(21℃P<0.05).The concentration of GnRH and FSH was affected by temperature.The concentration of FSH hormone was higher in the optimum temperature(21°C)and high temperature(29°C)groups.The difference reached a significant level(P < 0.05)between the optimum temperature(21°C)group and the low temperature(8°C)group.The concentration of GnRH hormone was opposite,the optimum temperature(21°C)is higher than the low temperature(8°C)group and the high temperature(29°C)group.6.Correlation analysis showed that there was a significant positive correlation between PACAP expression and PAC1R(r=0.6644,P=0.0133)and a significant negative correlation with FSH concentration(r=-0.6273,P=0.0290).However,there was no correlation with GnRH(r=-0.3738,P=0.2575).Therefore,temperature is the main environmental factor affecting the expression of PACAP/PAC1 R in the hypothalamus of the Phodopus sungorus.PACAP/PAC1 R regulates the FSH,and then regulates the reproductive activity of the Phodopus sungorus,but the GnRH was regulated by a number of factors.The regulation effect of PACAP/PAC1 R on the activity of GnRH neurons did not reach a significant level in this study,so the specific mechanism needs further study.In summary,we studied the differential expression of PACAP/PAC1 R gene in the hypothalamus of black-footed gray-footed rats,and investigated the effects of temperature on the expression of PACAP/PAC1 R.The mechanism reveals its complex seasonal reproductive regulation pathways,enriching the theoretical basis for research on rodent control,population dynamics,and seasonal fluctuations of populations.