| The initiation of puberty results from the activation of the hypothalamic-pituitary-gonadal axis（HPGA）,manifested by an increase in hypothalamic secretion of gonadotropin releasing hormone（Gn RH）,stimulation of the pituitary gland to release follicle stimulating hormone（FSH）and luteinizing hormone（LH）,thus promoting gonadal development.In recent years,the incidence of precocious puberty in children has been increasing year by year,and its main pathogenesis is the early activation of HPGA function and the early enhancement of hypothalamic Gn RH-releasing activity.Currently,the main treatment strategy for precocious puberty is the application of Gn RH analogs（Gn RHa）.But it is expensive,a long treatment and comes with a range of side effects.Melatonin is a hormone secreted mainly by the pineal gland and plays an important regulatory role in many areas,such as circadian rhythm,neuroendocrine,anti-aging,anti-cancer,etc.Studies have shown that melatonin is involved in regulating the developmental process of puberty in many animals,and the level of melatonin in blood drops significantly when normal children start puberty.Gonadotropin-inhibitory hormone（Gn IH）is a hypothalamic neuropeptide that inhibits the release of Gn RH.Previous experiments of our group have shown that Gn IH can play a regulatory role on the HPGA by inhibiting opioid Proopiomelanocortin（POMC）and Kisspeptin（Kp）neurons and stimulating Neuropeptide Y（NPY）neurons to inhibit the synthesis and release of Gn RH.The aim of this study was to explore the effect of melatonin on adolescent development of female mice and whether it regulates HPGA by regulating hypothalamic Gn IH,Gn RH,Kisspeptin,POMC and NPY neurons.Based on the above findings,we further established a female central precocious puberty mouse model to investigate the therapeutic effect of melatonin on precocious puberty and explore its potential mechanism.Part 1 Melatonin exerts a regulatory effect on puberty in female mice via the hypothalamusObjective:Investigation of the effect of subcutaneous melatonin injection on pubertal development in female mice and its mechanism of action.Methods:1.Animal Treatment and Grouping:Seventy-eight pre-pubertal 22-day-old female Kunming mice were randomly divided into a 32-day-old sampling group（n=26;10 days of injection）,a 37-day-old sampling group（n=26;15 days of injection）and a 42-day-old sampling group（n=26;20 days of injection）.Each group was further randomly divided into a melatonin experimental group（MLT;n=13）and a normal saline control group（NS;n=13）.Mice in the melatonin group were injected subcutaneously with a daily dose of 1mg/kg of melatonin on the back,and the control group was injected with the same volume of saline.After the mice in each group reached the day of sampling,they were anaesthetized with a 40 mg/kg dose of 0.5%pentobarbital sodium and the eyes were immediately removed for blood,followed by the ovaries and uterus.The hypothalamus was then removed after the mice were executed by cervical dislocation.2.Observe and record the rate of vaginal opening in each group of mice.3.Weighing and recording the weight of each group of mice.4.After anesthesia and execution,the ovaries and uterus of each group of mice were weighed and the organ index was calculated.5.Observation of ovarian and uterine volume in each group of mice.6.HE staining to observe the number of corpus luteum in the ovary and to measure the thickness of the uterine wall.7.ELISA was used to detect the levels of Gn RH,Gn IH,FSH and LH in the serum of each group of mice.8.RT-q PCR was used to detect the transcript levels of Kiss-1,Gn RH,POMC,NPY,GPR147 and Gn IH genes in the hypothalamus of each group of mice.9.Western blot was used to detect the expression levels of Kiss-1,Gn RH,POMC,NPY and GPR147 proteins in the hypothalamus of each group of mice.Results:1.Melanin reduces the rate of vaginal opening in miceThe results showed that at 32 and 37 days of age,the vaginal opening rate in the MLT group was significantly lower than that in the NS group（P<0.05）.At 42 days of age,the vaginal opening rate was 100%in both groups.The findings suggest that melatonin delays the opening of the vaginal opening in mice.2.Melatonin decreases body weight in miceThe body weight of each group of mice was measured on the day of sampling.The results showed that the body weight of the mice in each group increased gradually with the age of the day.At 37 and 42 days of age,the body weight of mice in the MLT group decreased significantly compared with the NS group（P<0.05）.3.Melatonin reduces ovarian and uterine weights and organ indices in miceThe results showed that at 32 days of age,ovarian index,uterine wet weight and uterine index were significantly reduced in the MLT group compared with the NS group（P<0.05）,while ovarian wet weight was not statistically different（P>0.05）.At 37 and 42 days of age,ovarian wet weight,ovarian index,uterine wet weight and uterine index were significantly reduced in the MLT group compared with the NS group（P<0.05）.4.Melatonin reduces the volume of ovaries and uterus in miceOvaries and uteri were taken from 32,37 and 42 day old mice respectively,fixed in 4%paraformaldehyde and photographed.The results showed that the volume of ovaries and uterus was significantly reduced in the MLT group compared with the NS group.5.Melatonin inhibits ovarian luteinization in miceTen samples from each group were selected for paraffin sectioning and HE staining,and the number of corpus luteum in each sample was observed under a 4×microscope.The results showed that at 32,37 and 42 days of age,the number of ovarian corpus luteum was significantly reduced in the MLT group compared with the NS group（P<0.05）.6.Melatonin inhibits uterine wall thickening in miceThe results showed that at 32,37 and 42 days of age,the uterine wall thickness of mice in the MLT group was significantly reduced compared to the NS group（P<0.05）.7.Melatonin decreases Gn RH,FSH and LH levels and increases Gn IH levels in mice serumThe results of ELISA experiments showed that at 32 days of age,compared with the NS group,the serum levels of Gn RH and FSH in the MLT group decreased（P<0.05）,and the levels of LH and Gn IH did not differ significantly（P>0.05）.At 37 days of age,compared with the NS group,the serum levels of Gn RH,FSH and LH in the MLT group decreased significantly（P<0.05）,and the levels of Gn IH significantly increased（P<0.05）.At 42 days of age,there were no significant differences in serum Gn RH,FSH,LH,and Gn IH in the MLT group of mice compared with the NS group（P>0.05）.8.Melatonin down-regulates the transcript levels of Kiss-1,Gn RH,POMC and NPY genes and up-regulates the transcript levels of Gn IH and GPR147genes in mouse hypothalamusThe results of RT-q PCR experiments showed that at 32 days of age,compared with the NS group,the relative levels of m RNA expression of Kiss-1,Gn RH and NPY in the hypothalamus of mice in the MLT group decreased,and the relative levels of m RNA expression of Gn IH and GPR147 increased（P<0.05）.At 37 days of age,compared with the NS group,the relative levels of m RNA expression of Kiss-1,Gn RH,POMC,NPY,and Gn IH in the hypothalamus of mice in the MLT group decreased（P<0.05）,and the relative levels of m RNA expression of GPR147 were not significantly different（P>0.05）.At 42 days of age,compared with the NS group,the relative levels of m RNA expression of POMC and NPY in the hypothalamus of mice in the MLT group decreased（P<0.05）,and the relative levels of m RNA expression of GPR147 m RNA relative expression levels increased（P<0.05）,and the relative levels of m RNA expression of Kiss-1,Gn RH and Gn IH were not significantly different（P>0.05）.9.Melatonin down-regulates the expression levels of Kiss-1,Gn RH,POMC and NPY proteins and up-regulates the expression level of GPR147protein in mouse hypothalamusThe results of protein immunoblotting experiments showed that at 32 and37 days of age,compared with the NS group,the relative expression levels of Kiss-1,Gn RH,POMC and NPY proteins decreased and the relative expression levels of GPR147 protein increased in the hypothalamus of mice in the MLT group（P<0.05）;at 42 days of age,the relative expression level of Kiss-1 protein in hypothalamus decreased and the relative expression level of Gn RH protein increased in MLT group mice compared with NS group（P<0.05）,while the relative expression levels of POMC,NPY and GPR147 proteins were not significantly different（P>0.05）.Summary:Melatonin inhibits pubertal development in female mice,probably by acting on hypothalamic Gn IH neurons in mice to increase Gn IH expression and release,which in turn acts indirectly on Kiss-1,POMC and NPY neurons,thereby inhibiting the release of Gn RH and the activation of HPGA.Part 2 Melatonin exerts therapeutic effects on female central precocious puberty mice via the hypothalamic Kiss-1/GPR54 axisObjective:Investigating the therapeutic effects and mechanisms of melatonin in female central precocious puberty mice.Methods:1.Animal Treatment and Grouping:Four groups of newborn female Kunming mice were assigned at random:normal control group,precocious puberty model group,melatonin-treated group and leuprolide treated group（n=10）.At 5 days of age,except for the normal control group,all three groups were injected with 300μg of danazol subcutaneously at once to create a precocious model.300μg of danazol was dissolved in 25μl of a 1:1 mixture of ethylene glycol and ethanol by volume.Ten days after modeling,leuprolide was injected subcutaneously at a dose of 100μg/kg per day in the leuprolide treatment group（positive control drug）,melatonin at a dose of 1mg/kg per day in the melatonin treatment group,and an equal volume of saline per day in the normal control and precocious puberty model groups.2.Animal execution time:Starting from 20 days of age,the vaginal opening of each group of mice was checked and recorded daily,and after opening,vaginal smears were performed daily for observation.Mice in the model group were executed at the onset of the first interestrus,while mice in other groups were randomly executed in the same number.The injection of drugs or normal saline was stopped in each group before execution.3.Observe and record the opening time of vaginal opening in each group of mice.4.HE stained vaginal smears of mice to observe their estrous cycle.5.Weighing the weight of each group of mice.6.After execution by anaesthesia,weigh the ovaries and uterus of each group of mice and calculate the organ index.7.Observe the size of ovaries and uterus in each group of mice.8.HE staining to observe the number of corpus luteum in the ovary and to measure the thickness of the uterine wall.9.ELISA was used to detect the levels of FSH,LH and E2 in the serum of each group of mice.10.RT-q PCR was used to detect the transcript levels of Kiss-1,GPR54and Gn RH genes in the hypothalamus of each group of mice.11.Western blot was used to detect the expression levels of Kiss-1,GPR54and Gn RH proteins in the hypothalamus of each group of mice.12.RT-q PCR was used to detect the transcript level of Gn RHR gene in pituitary gland of each group of mice.13.Western blot was used to detect the expression level of Gn RHR protein in the pituitary gland of each group of mice.Results:1.Melatonin delays the opening time of the vaginal opening in female central precocious puberty miceThe results showed that the opening time of the vaginal opening was earlier in the mice in the precocious puberty model group compared with the normal control group（P<0.05）;the opening time of the vaginal opening was delayed in the mice in the melatonin and leuprolide groups compared with the precocious puberty model group（P<0.05）;between the melatonin and leuprolide groups,there was no apparent different.（P>0.05）.2.Melatonin reduces body weight in female central precocious puberty miceThe results showed that the body weight of mice in the precocious puberty model group increased compared with the normal control group（P<0.05）;the body weight of mice in the melatonin and leuprolide groups decreased compared with the precocious puberty model group（P<0.05）;between the melatonin and leuprolide groups,there was no apparent different（P>0.05）.3.Melatonin reduces ovarian and uterine weights and organ indices in female central precocious puberty miceThe results showed that compared with the normal control group,the ovarian weight,ovarian index,uterine weight and uterine index of mice in the precocious puberty model group increased（P<0.05）,the ovarian weight,ovarian index,uterine weight and uterine index of mice in the melatonin group decreased（P<0.05）,and the uterine weight and uterine index of mice in the leuprolide group decreased（P<0.05）.Compared with the precocious puberty model group,ovarian weight,ovarian index,uterine weight and uterine index were reduced in the melatonin group（P<0.05）,and ovarian weight,ovarian index,uterine weight and uterine index were reduced in the leuprolide group of mice（P<0.05）.There were no apparent differences in ovarian weight,ovarian index,uterine weight and uterine index in mice in the leuprolide group compared with the melatonin group（P>0.05）.4.Melatonin reduces the volume of ovaries and uterus in female central precocious puberty miceThe ovaries and uterus of each group of mice were taken separately,fixed in 4%paraformaldehyde and photographed.The results showed that the volume of ovaries and uterus of mice in the precocious puberty model group was significantly larger compared with the normal control group;the volume of ovaries and uterus of mice in the melatonin and leuprolide groups was significantly reduced compared with the precocious puberty model group.There was no apparent difference in the volume of ovaries and uterus between the melatonin and leuprolide groups of mice.5.Melatonin inhibits ovarian luteinization in female central precocious puberty miceThe results showed that all levels of follicles were visible in the ovaries of the normal control group and no luteal production was observed.The rate of luteal production in the ovaries of the mice in the precocious puberty model group was 2-3 per/ovary.All levels of follicles were visible in the ovaries of the mice in the melatonin and leuprolide groups and no luteal production was observed.6.Melatonin inhibits uterine wall thickening in female central precocious puberty miceThe results showed that compared with the normal control group,the uterine wall thickness of mice in the precocious puberty model group increased significantly（P<0.05）;compared with the precocious puberty model group,the uterine wall thickness of mice in the melatonin and leuprolide groups decreased significantly（P<0.05）;between the melatonin and leuprolide groups,there was no apparent different（P>0.05）.7.Melatonin reduces serum FSH,LH and E2 levels in female central precocious puberty miceThe results of ELISA experiments showed that compared with the normal control group,the serum FSH,LH and E2 levels of mice in the precocious puberty model group increased（P<0.05）.Compared with the precocious puberty model group,the serum FSH,LH and E2 levels of mice in the melatonin group decreased（P<0.05）,and the serum LH and E2 levels of mice in the leuprolide group decreased（P<0.05）.There was no apparent difference in serum FSH,LH and E2 levels in mice in the leuprolide group compared with the melatonin group（P>0.05）.8.Melatonin down-regulates transcript levels of hypothalamic Kiss-1,GPR54,and Gn RH genes in female central precocious puberty miceThe results of RT-q PCR experiments showed that compared with the normal control group,the m RNA relative expression levels of Kiss-1,GPR54and Gn RH in the hypothalamus of mice in the precocious puberty model group increased（P<0.05）;compared with the precocious puberty model group,the m RNA relative expression levels of Kiss-1,GPR54 and Gn RH in the hypothalamus of mice in the melatonin and leuprolide groups were decreased（P<0.05）.Compared with the melatonin group,the relative m RNA expression levels of Gn RH in the hypothalamus of mice in the leuprolide group were decreased（P<0.05）,and the relative m RNA expression levels of Kiss-1 and GPR54 were not apparent different（P>0.05）.9.Melatonin down-regulates the expression levels of hypothalamic Kiss-1,GPR54 and Gn RH proteins in female central precocious puberty miceThe results of protein immunoblotting experiments showed that the relative expression levels of hypothalamic Kiss-1,GPR54 and Gn RH proteins increased in the mice of the precocious puberty model group compared with the normal control group（P<0.05）;compared with the precocious puberty model group,the relative expression levels of hypothalamic Kiss-1,GPR54 and Gn RH proteins decreased in the mice of the melatonin and leuprolide groups（P<0.05）.Compared with the melatonin group,the relative expression levels of Kiss-1and Gn RH proteins in the hypothalamus of mice in the leuprolide group were decreased（P<0.05）,and the relative expression levels of GPR54 protein were not apparent different（P>0.05）.10.Melatonin down-regulates the transcript levels of the pituitary Gn RHR gene in female central precocious puberty miceThe results of the RT-q PCR experiments showed that the relative m RNA expression level of pituitary Gn RHR in mice in the precocious puberty model group increased compared with the normal control group（P<0.05）;the relative m RNA expression level of pituitary Gn RHR in mice in the melatonin and leupeptin groups decreased compared with the precocious puberty model group（P<0.05）;compared with the leuprolide group,the m RNA relative expression level of pituitary Gn RHR in the melatonin group decreased（P<0.05）.The relative m RNA expression levels of Gn RHR in the pituitary gland of mice in the leuprolide group were increased compared with the melatonin group（P<0.05）.11.Melatonin down-regulates the expression level of Gn RHR protein in the pituitary gland of female central precocious puberty miceThe results of protein immunoblotting experiments showed that the relative protein expression levels of pituitary Gn RHR in mice in the precocious puberty model group increased compared with the normal control group（P<0.05）;the relative protein expression levels of pituitary Gn RHR in mice in the melatonin and leupeptin groups decreased compared with the precocious puberty model group（P<0.05）;there was no apparent difference between the melatonin and leuprolide groups（P>0.05）.Summary:Melatonin can treat female central precocious puberty in mice,probably by inhibiting the hypothalamic Kiss-1/GPR54 axis,inhibiting the expression and release of Gn RH,and finally inhibiting the activation of HPGA.Conclusion:Subcutaneous injection of melatonin inhibits HPGA and thus pubertal development in female mice by upregulating the expression of hypothalamic Gn IH and downregulating the expression of Gn RH,Kiss-1,POMC and NPY;In addition,melatonin exerts a therapeutic effect on female central precocious puberty mice by inhibiting the hypothalamic Kiss-1/GPR54 axis,suppressing Gn RH expression and HPGA activation. |
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