Additional file 2 of Gut microbiota-testis axis: FMT improves systemic and testicular micro-environment to increase semen quality in type 1 diabetes

Additional file 2: Fig. S1. Body weight and gut microbiota changes (STZ vs. Con). a Animal bodyweight. The y-axis represents the body weight (g). The x-axis represents the age (weeks). b Blood insulin levels. The y-axis represents the concentration (mIU/L). The x-axis represents the treatment. c The alpha index of the small intestine microbiota (Chao index). The y-axis represents the relative amount. The x-axis represents the treatment. d The beta index of small intestinal microbiota. The y-axis represents the relative amount. The x-axis represents the treatment. e PLS-DA (OTU) of small intestine microbiota in STZ and Con groups. f Small intestine microbiota levels at the genus level in STZ and Con groups. The y-axis represents the relative amount (%). The x-axis represents the individual microbiota. g The alpha index of the cecum microbiota (Chao index). The y-axis represents the relative amount. The x-axis represents the treatment. h The beta index of cecum microbiota. The y-axis represents the relative amount. The x-axis represents the treatment. i PLS-DA (OTU) of cecum microbiota in STZ and Con groups. j Cecum microbiota levels at the genus level in STZ and Con groups. The y-axis represents the relative amount (%). The x-axis represents the individual microbiota. k The alpha index of the colon microbiota (Chao index). The y-axis represents the relative amount. The x-axis represents the treatment. l The beta index of colon microbiota. The y-axis represents the relative amount. The x-axis represents the treatment. m PLS-DA (OTU) of colon microbiota in STZ and Con groups. n Colon microbiota levels at the genus level in STZ and Con groups. The y-axis represents the relative amount (%). The x-axis represents the individual microbiota. Fig. S2. a PLS-DA (OTU) of cecum microbiota in STZ, A10-FMT, and Con-FMT groups. b Cecum microbiota levels at the genus level in STZ, A10-FMT, and Con-FMT groups. The y-axis represents the relative amount (%). The x-axis represents the treatments. Different colors represent different microbiota. c Cladogram of the LEfSe determining the cecum microbiota difference in abundance. d Changed microbiota in the cecum. The y-axis represents the relative amount at the genus level. The x-axis represents the treatment. *p < 0.05. e PLS-DA (OTU) of colon microbiota in STZ, A10-FMT, and Con-FMT groups. f Colon microbiota levels at the genus level in STZ, A10-FMT, and Con-FMT groups. The y-axis represents the relative amount (%). The x-axis represents the treatments. Different colors represent different microbiota. g Cladogram of the LEfSe determining the difference in abundance of colon microbiota. h Changed microbiota in the colon. The y-axis represents the relative amount at the genus level. The x-axis represents the treatment. *p < 0.05. Fig. S3. Blood metabolism changes. a Blood flavonoid Malvidin 3-O-(6-O-(4-O-feruloyl-alpha-rhamnopyranosyl)-beta-glucopyranoside)-5-beta-glucopy -ranoside levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. b Blood glycerophosphocholine levels in different treatments. The y-axis represents the fold change compared to control group (Con). The x-axis represents the treatment. c Blood LysoPC (15:0) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. d Blood LysoPC [18:1(9z)] levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. e Blood LysoPC [22:6(4Z,7Z,10Z,13Z,16Z,19Z)] levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. f Blood PE [22:2(13Z,16Z)/0:0] levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. g Blood PE (22:0/0:0) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. h Blood PE [22:1(11Z)/0:0] levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. i Blood LysoPE [0:0/24:6(6Z,9Z,12Z,15Z,18Z,21Z)] levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. Fig. S4. Testicular metabolite changes. a Testicular 4,8,12,15,18-eicosapentaenoic acid levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. b Testicular EPA in different treatments. The y-axis represents the relative amount. The x-axis represents the treatment. *p < 0.05. c Testicular retinoids levels in different treatments. The y-axis represents the fold change compared to the control group (Con). The x-axis represents the treatment. d Testicular retinol levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. e Testicular 9-cis-retinal levels in different treatments. The y-axis represents the relative amount. The x-axis represents the treatment. *p < 0.05. f Testicular retinyl ester levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. g Quantitative data for Western blotting analysis in Fig. 4d. h Testicular testosterone levels in different treatments. The y-axis represents the fold change compared to control group (Con). The x-axis represents the treatment. i Testicular testosterone propionate levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. j Testicular testosterone acetate levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. k Testicular 7alpha-hydroxytestosterone levels in different treatments. The y-axis represents the relative amount. The x-axis represents the treatment. *p < 0.05. l Testicular steroids levels in different treatments. The y-axis represents the fold change compared to control group (Con). The x-axis represents the treatment. m Testicular 3b,17b-Dihydroxyetiocholane levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. n Testicular 5-Androstene-3b,16b,17a-triol levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. o Testicular 3beta,17alpha,21-Trihydroxy-pregnenone levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. p Testicular asterogenol levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. q Testicular PCs levels in different treatments. The y-axis represents the fold change compared to the control group (Con). The x-axis represents the treatment. r Testicular) PC(4:0/18:1(9Z) level. The y-axis represents the relative amount (%). The x-axis represents the treatments. s Testicular PC (8:0/8:0) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. t Testicular 6-hydroxymelatonin levels in different treatments. The y-axis represents the relative amount. The x-axis represents the treatment. *p < 0.05. Fig. S5. Additional data for testis and spleen. a Western blotting analysis of the proteins of some important genes for spermatogenesis in each treatment. n≥3/group. b PCA for RNA-seq analysis of spleen. c The functional enrichment analysis of STZ increased genes while these were decreased by A10-FMT or Con-FMT. d IHF staining of ki67 in the spleen. e Western blotting analysis of p53, Bax and Bcl-xl in the spleen. Fig. S6. A10-FMT improved liver function and systemic anti-oxidative capability. a Blood alanine aminotransferase (ALT) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. b PCA for RNA-seq analysis of liver. c The functional enrichment analysis of STZ decreased genes while these were increased by A10-FMT or Con-FMT in the liver. d The functional enrichment analysis of STZ increased genes while these were decreased by A10-FMT or Con-FMT in the liver. e Blood total triglyceride (TG) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. f Blood total cholesterol (TC) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. g Blood total antioxidant capability (T-AOC) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. h Blood total SOD levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. i Blood catalase levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. j Blood glutathione (GSH) levels. The y-axis represents the relative amount (%). The x-axis represents the treatments. k Western blotting analysis of Bax and Bcl-xl in the liver.

补充文件2：图S1。链脲佐菌素（STZ）组与对照组（Con）的体重及肠道菌群变化。a 动物体重：纵轴代表体重（g），横轴代表周龄。b 血液胰岛素水平：纵轴代表浓度（mIU/L），横轴代表处理方式。c 小肠菌群α多样性指数（Chao指数）：纵轴代表相对含量，横轴代表处理方式。d 小肠菌群β多样性指数：纵轴代表相对含量，横轴代表处理方式。e STZ组与Con组小肠菌群的偏最小二乘判别分析（PLS-DA, Partial Least Squares Discriminant Analysis，基于操作分类单元OTU, Operational Taxonomic Unit）。f STZ组与Con组小肠菌群属水平相对丰度：纵轴代表相对丰度（%），横轴代表单个菌群。g 盲肠菌群α多样性指数（Chao指数）：纵轴代表相对含量，横轴代表处理方式。h 盲肠菌群β多样性指数：纵轴代表相对含量，横轴代表处理方式。i STZ组与Con组盲肠菌群的偏最小二乘判别分析（PLS-DA，基于OTU）。j STZ组与Con组盲肠菌群属水平相对丰度：纵轴代表相对丰度（%），横轴代表单个菌群。k 结肠菌群α多样性指数（Chao指数）：纵轴代表相对含量，横轴代表处理方式。l 结肠菌群β多样性指数：纵轴代表相对含量，横轴代表处理方式。m STZ组与Con组结肠菌群的偏最小二乘判别分析（PLS-DA，基于OTU）。n STZ组与Con组结肠菌群属水平相对丰度：纵轴代表相对丰度（%），横轴代表单个菌群。
图S2。a STZ、A10-粪便菌群移植（FMT, Fecal Microbiota Transplantation）及Con-FMT组盲肠菌群的偏最小二乘判别分析（PLS-DA，基于OTU）。b STZ、A10-FMT及Con-FMT组盲肠菌群属水平相对丰度：纵轴代表相对丰度（%），横轴代表处理方式，不同颜色代表不同菌群。c 用于确定盲肠菌群丰度差异的线性判别分析效应大小（LEfSe, Linear Discriminant Analysis Effect Size）进化分支图。d 盲肠差异菌群：纵轴代表属水平相对丰度，横轴代表处理方式。*p < 0.05。e STZ、A10-FMT及Con-FMT组结肠菌群的偏最小二乘判别分析（PLS-DA，基于OTU）。f STZ、A10-FMT及Con-FMT组结肠菌群属水平相对丰度：纵轴代表相对丰度（%），横轴代表处理方式，不同颜色代表不同菌群。g 用于确定结肠菌群丰度差异的LEfSe进化分支图。h 结肠差异菌群：纵轴代表属水平相对丰度，横轴代表处理方式。*p < 0.05。
图S3。血液代谢物变化。a 血液黄酮类化合物锦葵素-3-O-(6-O-(4-O-阿魏酰基-α-鼠李吡喃糖基)-β-葡萄吡喃糖基)-5-β-葡萄吡喃糖苷水平：纵轴代表相对丰度（%），横轴代表处理方式。b 不同处理组血液甘油磷酸胆碱水平：纵轴代表相对于对照组（Con）的倍数变化，横轴代表处理方式。c 血液溶血磷脂酰胆碱（LysoPC, lysophosphatidylcholine）(15:0)水平：纵轴代表相对丰度（%），横轴代表处理方式。d 血液LysoPC [18:1(9Z)]水平：纵轴代表相对丰度（%），横轴代表处理方式。e 血液LysoPC [22:6(4Z,7Z,10Z,13Z,16Z,19Z)]水平：纵轴代表相对丰度（%），横轴代表处理方式。f 血液磷脂酰乙醇胺（PE, phosphatidylethanolamine）[22:2(13Z,16Z)/0:0]水平：纵轴代表相对丰度（%），横轴代表处理方式。g 血液PE (22:0/0:0)水平：纵轴代表相对丰度（%），横轴代表处理方式。h 血液PE [22:1(11Z)/0:0]水平：纵轴代表相对丰度（%），横轴代表处理方式。i 血液溶血磷脂酰乙醇胺（LysoPE, lysophosphatidylethanolamine）[0:0/24:6(6Z,9Z,12Z,15Z,18Z,21Z)]水平：纵轴代表相对丰度（%），横轴代表处理方式。
图S4。睾丸代谢物变化。a 睾丸4,8,12,15,18-二十碳五烯酸水平：纵轴代表相对丰度（%），横轴代表处理方式。b 不同处理组睾丸二十碳五烯酸（EPA, Eicosapentaenoic Acid）水平：纵轴代表相对含量，横轴代表处理方式。*p < 0.05。c 不同处理组睾丸类视色素水平：纵轴代表相对于对照组（Con）的倍数变化，横轴代表处理方式。d 睾丸视黄醇水平：纵轴代表相对丰度（%），横轴代表处理方式。e 不同处理组睾丸9-顺式视黄醛水平：纵轴代表相对含量，横轴代表处理方式。*p < 0.05。f 睾丸视黄酯水平：纵轴代表相对丰度（%），横轴代表处理方式。g 图4d中蛋白质免疫印迹（Western blotting）分析的定量数据。h 不同处理组睾丸睾酮水平：纵轴代表相对于对照组（Con）的倍数变化，横轴代表处理方式。i 睾丸丙酸睾酮水平：纵轴代表相对丰度（%），横轴代表处理方式。j 睾丸乙酸睾酮水平：纵轴代表相对丰度（%），横轴代表处理方式。k 不同处理组睾丸7α-羟睾酮水平：纵轴代表相对含量，横轴代表处理方式。*p < 0.05。l 不同处理组睾丸类固醇水平：纵轴代表相对于对照组（Con）的倍数变化，横轴代表处理方式。m 睾丸3β,17β-二羟基本胆烷水平：纵轴代表相对丰度（%），横轴代表处理方式。n 睾丸5-雄烯-3β,16β,17α-三醇水平：纵轴代表相对丰度（%），横轴代表处理方式。o 睾丸3β,17α,21-三羟基孕烯酮水平：纵轴代表相对丰度（%），横轴代表处理方式。p 睾丸星形甾醇（asterogenol）水平：纵轴代表相对丰度（%），横轴代表处理方式。q 不同处理组睾丸磷脂酰胆碱（PC, phosphatidylcholine）水平：纵轴代表相对于对照组（Con）的倍数变化，横轴代表处理方式。r 睾丸PC(4:0/18:1(9Z))水平：纵轴代表相对丰度（%），横轴代表处理方式。s 睾丸PC (8:0/8:0)水平：纵轴代表相对丰度（%），横轴代表处理方式。t 不同处理组睾丸6-羟基褪黑素水平：纵轴代表相对含量，横轴代表处理方式。*p < 0.05。
图S5。睾丸与脾脏补充数据。a 各处理组精子发生相关重要基因蛋白的蛋白质免疫印迹分析，每组样本量n≥3。b 脾脏转录组测序（RNA-seq）的主成分分析（PCA, Principal Component Analysis）。c STZ上调但经A10-FMT或Con-FMT处理后下调的基因的功能富集分析。d 脾脏Ki67免疫组化染色。e 脾脏p53、Bax及Bcl-xl蛋白的蛋白质免疫印迹分析。
图S6。A10-FMT改善肝功能与机体抗氧化能力。a 血液丙氨酸氨基转移酶（ALT, alanine aminotransferase）水平：纵轴代表相对丰度（%），横轴代表处理方式。b 肝脏转录组测序的PCA分析。c 肝脏中STZ下调但经A10-FMT或Con-FMT处理后上调的基因的功能富集分析。d 肝脏中STZ上调但经A10-FMT或Con-FMT处理后下调的基因的功能富集分析。e 血液总甘油三酯（TG, triglyceride）水平：纵轴代表相对丰度（%），横轴代表处理方式。f 血液总胆固醇（TC, total cholesterol）水平：纵轴代表相对丰度（%），横轴代表处理方式。g 血液总抗氧化能力（T-AOC, total antioxidant capability）水平：纵轴代表相对丰度（%），横轴代表处理方式。h 血液总超氧化物歧化酶（SOD, superoxide dismutase）水平：纵轴代表相对丰度（%），横轴代表处理方式。i 血液过氧化氢酶（CAT, catalase）水平：纵轴代表相对丰度（%），横轴代表处理方式。j 血液谷胱甘肽（GSH, glutathione）水平：纵轴代表相对丰度（%），横轴代表处理方式。k 肝脏Bax与Bcl-xl蛋白的蛋白质免疫印迹分析。