| Background:Hypothyroidism,a highly prevalent endocrine disorder,has significant effects on both the reproductive and neurological systems,and research related to reproductive and neurological disorders has important implications for both our population strategy and demographic structure.Reproductive and neurological disorders occur in the testis and brain,respectively.Although the testis and brain are different functional organs,they have some similar features,for instance,the blood-testis barrier and blood-brain barrier,which are the two major blood barriers in the human body,sertoli cells(referred to as supporting cells)and astrocytes,which are the largest proportion of the testis and brain,respectively.Further studies revealed a high degree of similarity in gene expression and protein fraction between these two tissues and organs.Although the pathogenesis of both diseases has been studied in depth for a long time in their respective fields,the existence of a common pathogenesis between the two diseases has not been reported yet.Given the many similarities between the testis and the brain at the tissue and molecular levels,it is important to further explore the common functional characteristics of the two organs,especially the common pathogenesis of related diseases,to reconceptualize the two different organs,as well as to learn from each other and conduct in-depth studies of reproductive and neurological diseases from a new perspective.Therefore,in this study,we first established a rat model of congenital hypothyroidism with deletion of thyroid peroxidase by knocking out the Tpo gene in the genome of SD rats using CRISPR/Cas9 gene editing technology,and a phenotype of male sterility as well as behavioral abnormalities was observed in model rats.Based on this disease model,a series of molecular and functional experiments were conducted to investigate and validate the common molecular mechanisms between the testis and the brain in the development of male reproductive disorders and neurological damage caused by hypothyroidism.Methods:1)Tpo knockout stable genetically pure mutant rats were constructed by CRISPR/Cas9 technology,and the hypothyroid phenotype of knockout rats was identified using H&E staining and ELISA for the detection of thyroid histopathology and related hormone levels.2)Single-cell transcriptome sequencing using the 10× Genomics platform was used to analyze the testicular spermatogenic cells of Tpo-/-hypothyroid rats.The cell subpopulations that were significantly altered between the two groups were calculated by taxonomic annotation of different cells in the testis;and the sequential order of each cell subpopulation in the developmental and differentiation timelines was analyzed using the Monocle2 for proposed temporal trajectory to clarify the cell subpopulations that were significantly altered.Based on the single cell transcriptome sequencing analysis,we further combined primary cell isolation,cell proliferation assay,immunofluorescence staining,transmission electron microscopy analysis,ELISA assay,H&E staining,Nissl’s staining,silver plating staining,and behavioral hanging tail assay to analyze the possible co-pathological alterations of hypothyroidism caused by Tpo knockout in the male reproductive system and the nervous system.3)Gene Ontology(GO)combined with Trajectory sequencing was used to analyze the pathways enriched by differentially expressed genes(DEGs)in subpopulations of cells with key alterations in single-cell sequencing;qRT-PCR,Western Blotting,immunofluorescence staining,in vitro induction of primary cells and established vitro cell models,ELISA,and induction of stem cells in vitro were used to validate the key genes and other molecules in testis and brain simultaneously.To investigate the possible common molecular mechanisms causing similar pathological alterations in the testis and brain of Tpo-/-rats.Results:1)Sanger sequencing and Western-Blotting results showed the successfully knockout of Tpo gene in the genome of SD rats.The levels of body size,body weight,body length and growth hormone were lower in Tpo-/-rats compared with control WT rats(p<0.01);the relative weight ratio of each parenchymal organ showed that the weight ratio of testis was significantly lower in Tpo-/-rats(p<0.01),while the weight ratio of brain was significantly higher(p<0.001).The results of H&E staining of thyroid tissue showed that thyroid follicles were smaller in Tpo-/-rats compared to WT,accompanied by follicular lumen atresia and reduced gliosis;results by ELISA tests showed that thyroid stimulating hormone(TSH)levels were significantly increased in Tpo-/-rats(p<0.01),yet the free thyroid hormone T3(p<0.01)and T4(p<0.01)levels were significantly decreased.2)By UMAP clustering analysis,13 cell clusters were obtained for each of the two groups of rat testis tissues and identified by cell type;the massive proliferation proliferation of sertoli cells in Tpo-/-rat testes was clarified by assessing the proportional distribution of each cell cluster to the total samples of the two groups in combination with Trajectory analysis.In vitro primary sertoli cells proliferation experiments corroborated the results of the single cell analysis.Comparison of testis and brain synchronization showed that both sertoli cells in testis and astrocytes in brain showed massive proliferation activation in Tpo-/-rats;combined with transmission electron microscopy analysis displayed that the integrity of blood-testis barrier and blood-brain barrier were simultaneously disrupted.Further,ELISA results of male reproduction-related hormones exhibited that the levels of prolactin,folliclestimulating hormone,testosterone,and luteinizing hormone were significantly reduced in Tpo-/-rats(p<0.01);no spermatocytes at all levels and mature spermatozoa were seen in the cross-section of spermatogenic tubules of Tpo-/-rats’ testis tissue,meanwhile,male rats were completely sterile.Nissl’s staining and silver plating of brain tissues demonstrated reduced protein synthesis and neuronal damage in the brain of Tpo-/-rats,as well as intermittent immobility and "behavioral despair" in Tpo-/-rats by conducting the tail suspension experiment.3)GO functional annotation showed that the DEGs of sertoli cells subpopulation between the two groups mainly were enriched in oxidative stress,lipid metabolism and other related pathways.The determination of oxidative stress sensors SOD,CAT,and MDA in sertoli cells,together with qRT-PCR analysis of relevant genes in a vitro thyroid hormone co-culture system,revealed that hypothyroidism caused elevated levels of oxidative stress in testicular sertoli cells.Further analysis of related gene expression using qRT-PCR at the level of primary cells cultured in vitro,as well as in the thyroid hormone co-culture system and an oxidative stress cell model,displayed that decreased thyroid hormone levels caused oxidative stress within testis sertoli cells and ultimately resulted in downregulation of Apoe expression in sertoli cells(p<0.01).Comparison of the testis and brain synchronous showed that ApoE was downregulated in testis,brain,primary sertoli cells,and astrocytes in Tpo-/-rats,and ApoEassociated lipid content tested by ELISA showing that cholesterol levels were significantly elevated in Tpo-/-rats.Also in vitro cellular assays exhibitied that high cholesterol levels led to massive proliferation of primary cells(p<0.001).Trajectory analysis combined with tissue immunofluorescence staining revealed increased numbers of SSCs and NSCs in testis and brain of Tpo-/-rats.GO functional annotation analysis showed that DEGs in stem cell subpopulations were associated with processes such as lipid metabolism,and in vitro experiments further revealed that high cholesterol levels also promoted stem cell proliferation.Conclusions:1)An animal model targeting Tpo knockout was constructed using CRIP SRCas9 technology,and by evaluating the established knockout rat thyroid tissue and related hormones,it was clear that the Tpo knockout animal model possessed a hypothyroid phenotype,providing a stable experimental disease model for this study.2)Combined with scRNA-seq analysis,it was found that corresponding pathological changes occurred in the testis and brain of Tpo-/-hypothyroidism model animals,mainly including massive proliferation of sertoli cells and astrocytes,as well as integrity damage of the blood-testis barrier and blood-brain barrier constituted by both of them respectively,which eventually resulted in similar pathological outcomes of complete azoospermia in the testis and neuronal damage in the brain.3)Hypothyroidism caused elevated levels of oxidative stress in testicular sertoli cells,the latter further causing decreased Apoe expression,which futher leads to abnormal lipid metabolism.The decreased expression of ApoE in the testis and brain,sertoli cells and astrocytes in Tpo-/-rats was clarified;as well as the promotion of sertoli cells and astrocytes proliferation by elevated cholesterol levels in Tpo-/-rats.4)It is hypothesized that abnormalities in lipid metabolism such as cholesterol may cause male reproductive disorders and neuronal damage and fail to repair them by affecting the proliferation and differentiation of SSCs and NSCs.However,the exact mechanism needs to be further research.In conclusion,this study explored and validated the common pathological changes that exist between androgenic reproductive disorders and neurological damage caused by hypothyroidism and the possible common molecular mechanisms that lead to similar pathological changes between the two through a stable genetic animal model of hypothyroidism disease.It provides a novel perspective and theoretical reference for understanding the similarities between the two organs and the common mechanisms in disease development. |
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