Experimental Study On The Role Of SPEXIN And Its Receptors In The Effect Of Diet-Induced Obesity On The Reproductive Function Of Male And Female Mice

Objective: Obesity has adverse effects on reproductive function,and the relevant mechanism is unclear.Recently,scholars found a new neuropeptide——SPEXIN,which is closely related to reproductive development.SPEXIN can produce effects by combining with its receptors-Galanin receptor 2(GalR2)and Galanin receptor 3(GalR3).The study found that the regulation of reproductive development by SPEXIN is closely related to Gonadotropin-releasing hormone(GnRH),and SPEXIN may play a role in reproductive development through reverse regulation of GnRH.Does SPEXIN expression change during diet-induced obesity? How SPEXIN plays a role in obesity impacting reproductive development by affecting GnRH secretion,and the possible regulatory pathway remains to be explored.It has been stressed that the binding of SPEXIN and receptors in the hypothalamus can regulate the expression of pro-melanocortin source(POMC),and the nerve fibers derived from POMC neurons are connected with GnRH neurons.These suggest that the information transmitted to POMC after SPEXIN and receptor binding can be further transmitted to GnRH.As a precursor peptide,POMC does not function.And α-melanotropin(α-MSH),as a product of POMC,is a key factor involved in reproductive regulation in the hypothalamus and plays a role after binding with MC4 R,which is a member of the G protein-coupled receptor family,and has been found in the GnRH neurons.Relative studies have found that ERK/CREB is a classical pathway regulating the expression of the GnRH gene,which can be activated by members of the G protein-coupled receptor family.It has been evidenced that CREB can bind to the promoter of GnRH and then affect the transcription of GnRH.This suggests that MC4 R activated by α-MSH may affect GnRH through ERK/CREB pathway.Some researchers believe that obesity in childhood can have a stronger adverse effects in adulthood.Therefore,we established an obese mouse model of obesity from childhood,then detected the expression of SPEXIN and its receptors change and made further efforts to explore the role of SPEXIN in the hypothalamus in the influence of obesity on reproductive development by regulating the secretion of GnRH,providing a theoretical basis for further elucidating the mechanism of diet-induced obesity on the reproductive function of mice.Methods: C57BL/6J mice(12 male and 24 female,8 weeks old)were selected.And two virgin female mice and one non-sibling male mouse were matched.Only two offspring(1male+1 female)were selected from one litter and were divided randomly into the control group(CON)which provides a normal diet and the high-fat group(HFD)which provides a high-fat diet.Finally,there are 24 male(female)offspring into two groups(male: CON12+HFD12;female: CON 12+HFD12).In the CON group,the mice were fed a normal diet(The offspring were weaned at the age of 3 weeks before which,12 maternal mice were fed the normal diet,after which,12 offspring were fed the same diet.)Another group was given the same fed model,just the diet kind was different.Another group was given the same fed model,just the diet kind was different.After finishing 12-week feeding,body composition,and other related indicators were detected.Then the mice were anesthetized and killed,and the sperm quality(sperm quantity,sperm motility,sperm deformity)of the male mice was detected;The histological and ultrastructural changes of mouse testis and ovaries were observed under light and transmission electron microscopes;The levels of related hormones in serum of mice were detected by ELISA kit;The expression of SPEXIN and its receptors in mouse hypothalamus,testis,and ovaries and the expression of α-MSH,ERK,p-ERK,CREB,p-CREB proteins in the hypothalamus were detected by Western blot;The expression of GnRH protein in mouse hypothalamus was detected by immunohistochemistry;The expression of SPEXIN,GalR2,GalR3 and GnRH m RNAs in mouse hypothalamus were detected by RT-PCR.Results: 1.The weight of males in the HFD group was statistically higher when weaned on the 21 st day,and the significantly different persisted for the subsequent 9 weeks.And identical results were obtained in female C57BL/6J mice.2.At the termination of the12-week experiment,compared with the control group,in the HFD group,the male and female mice’s lean body mass,fat mass,and relative coefficient were obviously higher(P<0.05).3.At the termination of the 12-week experiment,In male C57BL/6J mice,the peri testicular fat weight,perirenal fat weight,peri testicular fat index,perirenal fat index of the HFD group were significantly increased than those in the CON group.In female C57BL/6J mice,the periuterine fat weight,perirenal fat weight,periuterine fat index,perirenal fat index of the HFD group were higher than those of the CON group(P<0.05).4.At our termination of the 12-week experiment,In the HFD group,the testis coefficient of the male C57BL/6J mice was significantly reduced(P<0.05);In the HFD group,the weight of ovarian,kidney,and liver of the female C57BL/6J mice were obviously higher(P<0.05).5.The sperm quality evaluation of Male C57BL/6J mice at 12 weeks of age showed that the number of malformed sperm in the HFD group was increased significantly,and the rate of sperm malformations was nearly doubled compared with the CON group(P<0.05).6.At the termination of the 12-week experiment,the testis and ovary were stained via HE,and morphologic changes were obtained by the light microscope: In the HFD group mice,the arrangement of testicular spermatogenic tubules was irregular and disturbed.In the spermatogenic tubules,sperm cells are reduced in number and arranged in disorder.And in the HFD group of female mice,ovarian development is damaged,and more lipid droplets can be seen.7.Under the transmission electron microscope,In the mice testicular cells of the HFD group,faint chromatin was observed.Meanwhile,the morphology of the mitochondria was apparently contracted that having shrinking ridges.In the female mice ovaries,more lipid droplets were observed in the nucleus.8.At the termination of the 12-week experiment,In the HFD group,the male mice’s serum testosterone level was obviously decreased(P<0.05);In the HFD group,the female mice’s Estrogen level was apparently increased(P<0.05).9.At the termination of the 12-week experiment,Compared with the CON group,the expression of SPEXIN、GalR2、GalR3 protein and RNA of male mice hypothalamus and female mice hypothalamus in the HFD group were significantly increased(P<0.05),the expression of SPEXIN、GalR2、GalR3 protein in the male mice testicle of the HFD group was increased,and the expression of SPEXIN、GalR3 protein in the female mice ovary of the HFD group was decreased(P<0.05).10.At the termination of the 12-week experiment,In male and female mice hypothalamus,in the HFD group,the expression of GnRH protein and m RNA was obviously decreased(P<0.05).11.At the termination of the 12-week experiment,compared with the CON group,In male and female mice hypothalamus,in the HFD group,the expression of α-MSH、MC4R、p-ERK、p-CREB protein were significantly decreased(P<0.05),and the expression of CREB in the female mice hypothalamus was increased(P<0.05).Conclusion:1.Obesity can have adverse effects on the reproductive function of male and female mice;The expression of SPEXIN and its receptors in the hypothalamus of male and female obese mice increased;The expression of SPEXIN in the testis of obese male mice increased while that in the ovary of female mice decreased.2.The expression of SPEXIN and its receptors in the hypothalamus increased,while the expression ofα-MSH,MC4 R,p-ERK and p-CREB decreased,resulting in the decrease of GnRH expression,and thus affecting the level of sex hormones,which may play a role in the effect of diet-induced obesity on the reproductive function of male and female mice.