Effects Of High-fat Diet On Puberty Onset And The Morphology And Function Of GnRH Neurons In Female Mice

Objective:The hypothalamus-pituitary-gonadal(HPG)axis is a neuroendocrine system that regulates reproductive function of the body,GnRH neurons are present in the hypothalamus and secretion of GnRH regulates the expression of Luteinizing hormone(LH)and follicle-stimulating hormone(FSH)by the anterior pituitary,thus maintaining normal reproductive function.The timing of the activation of the HPG axis determines the onset of puberty.With the improvement of living standards,the incidence of central precocious puberty(CPP)caused by childhood obesity is increasing year by year.However,the mechanism of obesity-induced precocious puberty is still not clear.It is of great significance to clarify the effect of high-fat diet on the morphology and function of GnRH neurons in the hypothalamus of female mice with precocious puberty model to further reveal the mechanism of CPP production and treatment strategies.Methods:The animals were C57BL/6 GnRH-GFP female mice.After parturition,mothers were fed either a high-fat diet or a standard diet,and continued to be fed the same diet at postnatal 21.The body weight,daily intake and daily calorie intake of female rats and the onset time of puberty(i.e.the time of vaginal opening)were observed.The weight of uterus,ovary and fat were recorded.The morphology and electrophysiological characteristics of GnRH neurons were compared.All mice were divided into group A(control group)and group B(precocious puberty induced by high fat diet group)with the average onset of puberty of mice in the high fat diet group.Mice were sampled at postnatal 24(PN24)and postnatal 30(PN30):1.Serum:After deep anesthesia,severed blood was collected and mixed in a 1.5 m L anticoagulant centrifuge tube.After static centrifugation,the supernatant was extracted,and then the levels of estradiol(E2),LH and FSH were measured by ELISA.2.Brain tissue:the brain was removed and fixed with 4%paraformaldehyde(PFA);3.Acute brain separation tablet:used for electrophysiological experiment;4.Gonadal tissue:used for hematoxylin-eosin(HE)staining.To investigate the function and morphology of GnRH neurons in two groups of mice,we compared by whole-cell patch-clamp recording and laser confocal microscopy:After acute brain slice preparation(300μm),use an Neurobiotin fluid inside(1%)of potassium electrode to whole cell patch clamp recording of GnRH neurons,record contents mainly for active membrane properties(action potential amplitude,threshold,after hyperpolarization potential,half width,I_h and I_A etc.)and the passive membrane properties(input resistance,resting membrane potential,membrane capacitance),to compare GnRH neurons electrophysiological properties.After whole-cell patch-clamp recording,the brain slices were fixed in 4%PFA overnight,washed with buffer solution the next day,and incubated with rhodamine for6～8 hours.Then the slices were sealed and dried.Morphological characteristics of mouse GnRH neurons were compared by laser confocal photography.Results:(1)At postnatal 27,compared with female mice on the standard diet,the calorie intake of female mice on the high-fat diet increased significantly were 8.24±0.34kcal/d(Ctrl)vs.12.28±0.42 kcal/d(HFD).And the weight of postnatal 21 showed a significant difference,in postnatal 21,the body weight was 7.05±0.26 g(Ctrl)vs.9.62±0.26 g(HFD).The time of vaginal open was significantly advanced in high-fat diet(29.43±0.26 d)mice compared to that of the standard diet(37.86±1.15 d)mice.(2)Compared with female mice on the standard diet,the weight of subcutaneous fat,peri-gonadal fat,uterine and ovary of female mice on the high-fat diet increased significantly:respectively were 190.7±22.68 mg(Ctrl)vs.299.70±25.40 mg(HFD),23.00±3.94 mg(Ctrl)vs.62.00±12.18 mg(HFD),13.80±1.21 mg(Ctrl)vs.25.20±6.22 mg(HFD).However,at postnatal 24,the weight of subcutaneous fat,peri-gonadal fat,uterine and ovary were no significantly.(3)Compared with female mice on standard diet,HE staining showed that subcutaneous fat adipocytes and peri-gonadal fat adipocytes were enlarged.(4)Compared with female mice on standard diet,at postnatal 24 and 30,the FSH,and E2 were no significantly,however,at the postnatal 30,the LH[0.51±0.20 m IU/ml(Ctrl)vs.2.07±0.68 m IU/ml(HFD)]secretion increased significantly in high-fat diet mice.(5)Compared with female mice on standard diet,GnRH neurons were increased significantly[141.10±7.96μm~2(Ctrl)vs.183.10±7.54μm~2(HFD)],and dendritic spines density increased significantly[0.10±0.02/μm(Ctrl)vs.0.20±0.02/μm(HFD)].However,no significant changes were observed in the cell size of GnRH neurons and dendritic spine density at postnatal 24.(6)Compared with female mice on standard diet,GnRH neurons showed decreased Action Potential threshold[-27.43±1.18 m V(Ctrl)vs.-31.85±1.34 m V(HFD)]and AP half-width[1.72±0.06 ms(Ctrl)vs.1.43±0.07 ms(HFD)]at postnatal30.The discharge frequency increased[12.39±2.66 Hz(Ctrl)vs.20.83±1.78 Hz(HFD)],the amplitude of AP increased[80.75±1.88 p A(Ctrl)vs.88.14±1.63 p A(HFD)],the number of I_h current increased,and the number of I_A current decreased.The AP threshold,AP half-width and firing frequency had no significant changes at postnatal 24.Conclusions:High fat diet induced precocious puberty in female mice may be related to the increase of LH secretion,enlargement of GnRH soma and the increase of dendritic spine density,the decrease of AP half-width and the increase of firing frequency of GnRH neurons.