The Effects Of Conditionally Deleting TR? In Kiss-1 Neurons On Reproductive And Metabolic Phenotype In Mice

Background and objective: Keeping hypothalamic-pituitary-gonadal(HPG)axis intact and functional is the key to maintain normal puberty development and reproductive function.Kisspeptin synthesized by Kiss-1 neurons in hypothalamus can act directly on Gn RH neurons by binding to its specific receptor GPR54,in this way,it can stimulate Gn RH secretion and increase serum LH and FSH levels,which means to activating and maintaining the function of HPG axis.The Kiss-1/GPR54 gene mutation can lead to hypogonadotropin sexual dysfunction and results in abnormal puberty development,hypogonadism and infertility.Therefore,Kiss-1/GPR54 is the key to regulate HPG axis and reproductive function.Besides,some new studies also suggest that kisspeptin may be directly or indirectly involved in obesity,diabetes and other metabolic disorders.In addition,there is a close relationship between thyroid hormones and reproductive function.For example,hyperthyroidism or hypothyroidism patients often show varying degrees of abnormalities in reproductive function.Most studies about the impacts of thyroid hormones on reproductive function are focused on thyroid hormones directly influencing the function of testes or ovary through the peripheral pathway,but the existing conclusions can not explain all of the clinical phenomena.Increasing evidence suggests that thyroid hormones can also affect the reproductive function of mammals through the central pathway.In the previous studies,we found that kisspeptin expression was decreased in hypothalamus of hypothyroid rats,and that the chronic injection of T3 in the lateral ventricle of mice could affect HPG axis by acting on Kiss-1neurons and could also increase the intake of mice.On the basis of these findings,this study was designed to construct the mice which were conditional TR?-deleted in Kiss-1neurons and observe the changes in reproductive and metabolic function.In this way,this study was to investigate whether thyroid hormones could affect reproductive and metabolic function by interacting with TR? in Kiss-1 neurons through the central pathway,which would be important for clarifying the relationship and interaction among HPT axis,HPG axis and metabolic function and might provide a new theoretical basis or find a new target for the treatment of some reproductive or metabolic diseases.Methods: In this study,we used the CRISPR/Cas9 principle and the Cre-lox P approach to construct the mice which were conditional TR?-deleted in Kiss-1 neurons.In order to detect the changes of conditional TR?-deleted mice in reproductive and metabolic function,we evaluated the body weight,intake,age of initial vaginal opening,rhythmicity of estrus cycles,fertility,glucose tolerance and insulin tolerance,moreover,we assayed serum hormones levels such as FSH,LH,E2,T and AMH levels,respectively.Results: 1.Results of reproductive phenotype:(1)There was no significant difference in age of initial vaginal opening in Kiss1-TR? KO female mice(33.500±0.627 days,n=8)compared with WT female mice(wide-type,32.833±0.401 days,n=6).In 2-month-old females,serum FSH levels in Kiss1-TR? KO group were significantly decreased compared with that in WT group(P <0.05).Meanwhile,the serum levels of LH and E2 in Kiss1-TR? KO group were also decreased compared with that in WT group.And three of the eight TR?-deleted females showed irregular estrus cycles,the other five showed regular and complete estrus cycles.In contrast,all of the six WT females showed regular and complete estrous cycle.There existed some difference in the rhythmicity of estrus cycles between KO and WT groups though this difference was not very significant(P>0.05).Moreover,though there was no difference in serum AMH levels between KO and WT groups in 2-month-old females,serum AMH levels in Kiss1-TR? KO group were significantly decreased compared with that in WT group(P<0.05)in 6-month-old females.No matter in KO or WT group,serum AMH levels in6-month-old females were significantly decreased compared with that in 2-month-old females(P <0.05).In addition,regardless Kiss1-TR? KO females or WT females could be pregnant(both pregnancy rates were 100%).And there was no significant difference in litter size between KO(6.250±0.590)and WT(7.833±0.477)groups.(2)No matter in KO or WT group,serum FSH levels in 6-month-old males were significantly decreased compared with that in 2-month-old males(P <0.05).Meanwhile,serum LH levels in6-month-old males were significantly decreased compared with that in 2-month-old males(P <0.05)in Kiss1-TR? KO group.And in WT group,serum LH levels in6-month-old males were also decreased compared with that in 2-month-old males.However,neither the serum FSH nor LH levels displayed difference between KO and WT groups.It was worth noting that though there was no difference in serum T levels between KO and WT groups in 2-month-old males,serum T levels in Kiss1-TR? KO group were significantly decreased compared with that in WT group(P <0.05)in6-month-old males.And no matter in KO or WT group,serum T levels in 6-month-old males were significantly decreased compared with that in 2-month-old males(P <0.05).Moreover,regardless Kiss1-TR? KO males or WT males could impregnate the normal females(both impregnation rates were 100%).And there was no significant difference in litter size between KO(7.250±0.750)and WT(7.250±0.881)groups.2.Results of metabolic phenotype: Though Kiss1-TR? KO males ate and drank more than WT males in the first two weeks after weaning(P <0.05),there was no difference between KO and WT groups in the following three weeks,neither in food nor drink intake.Meanwhile,in females,there was no difference in food or drink intake between KO and WT groups.Moreover,no difference was detected in body weight,glucose tolerance or insulin tolerance between KO and WT groups,neither in males nor females.Conclusion: 1.Our present findings indicate that the interaction of thyroid hormones with TR? in Kiss-1 neurons is involved in the regulation of hypothalamus maintaining HPG axis and the regulation of ovary maintaining reserve function.It provides either a new way or a research target for thyroid hormones to regulate reproductive function through the central pathway.2.Moreover,the results suggest that there was no significant effect of conditionally deleting TR? in Kiss-1 neurons on the metabolic phenotype of mice,which can support us in deciding the next research direction and research plan.3.In this study,we only conditionally deleted the TR? gene but not TR?gene in Kiss-1 neurons.Therefore,we can not rule out the possibility that thyroid hormones may regulate reproductive function through the central pathway by acting on TR? in Kiss-1 neurons.Further studies are necessary to clarify the relationship between thyroid hormones and TR? in Kiss-1 neurons,which is important for further exploring either the central regulation of reproductive function or the possible interactions between HPT axis and HPG axis.