Tmc7 expression is confined to the testis, and its deletion leads to male infertility.

(a) Heatmap analysis of the expression of Tmc family proteins at embryonic day (E)10–E18 and different postnatal days (P0, P3, PD14, PD28, and PD63) in mouse testis using the Evo-devo mammalian organ database (http://apps.Kaessmannlab.org/evodevoapp/). (b) The analysis of Tmc7 expression in different tissues using the Mouse Cell Atlas database (https://bis.zju.edu.cn/MCA/). (c, d) T-Distributed Stochastic Neighbor Embedding (t-SNE) plot and violin plot showing the expression of Tmc7 in different mouse germ cells using the Male Health Atlas (http://malehealthatlas.cn/). SSC1-3: Spermatogonial stem cells I-III; Progenitor: spermatogonia progenitor cell; A1–4: A1–4 spermatogonia; In: Intermediate spermatogonia; B: Type B spermatogonia; PI: Preleptotene; L: Leptotene; Z: Zygotene; P: pachytene; D: Diplotene; M: Metaphase; RS: Round spermatid. E: Elongating spermatid. (e) The expression of Tmc7 in different adult mouse tissues was measured by RT-PCR, including the heart, liver, spleen, lung, kidney, brain, ovary, and testis. Gapdh was used as the control. (f) The expression of Tmc7 at different postnatal times (PD1, PD7, PD9, PD12, PD24, PD33, and 2 months) was measured by RT-PCR. Gapdh was used as the control. (g) Schematic illustrating the generation of Tmc7−/− mice using CRISPR/Cas9. (h) Testes from 9-week-old WT and Tmc7−/− mice. (i) Testicular weights of 9-week-old WT and Tmc7−/− mice. Four adult mice of each genotype were used. (j) Testis/body weight ratio of 9-week-old WT and Tmc7−/− mice. Four mice of each genotype were used. Images are representative of at least three independent experiments. The results are presented as the mean ± S.D., and two-sided unpaired Student’s t-tests were used to calculate the P-values (*P < 0.05, **P < 0.01, ***P < 0.001).

Tmc7 deficiency leads to severe defects in spermiogenesis.

(a, b) Hematoxylin staining in testis and caudal epididymis sections of 9-week-old WT and Tmc7−/− male mice. Scale bar, 50 μm. (c) The total number of sperm in the caudal epididymis from 9-week-old WT and Tmc7−/− male mice. Three mice were used. (d) Hematoxylin-eosin staining of the spermatozoa isolated from the caudal epididymis in 9-week-old WT and Tmc7−/− male mice. H: head, F: flagella. Scale bar, 10 μm, 2 μm. (e) The percentage of spermatozoa with abnormal heads in the caudal epididymis from WT and Tmc7−/− male mice. Three mice were used. (f) PAS and hematoxylin staining in 9-week-old WT and Tmc7−/− mouse seminiferous tubules. The numbers represent the stages of spermatogenesis. P: pachytene spermatocyte, PL: preleptotene spermatocyte, L: leptotene spermatocyte, Z: zygotene spermatocyte, D: diplotene spermatocyte, RS: round spermatid, ES: elongating spermatid. Scale bar, 5 μm. (g) Quantification of spermatid numbers in seminiferous tubules from spermatid stages 1–16. N = 15 tubules per stage and per genotype; 3 mice per genotype were used and 5 tubules per stage and per mouse were counted. (h) Mitochondrial sheath staining by Mito-tracker in sperm collected from the caudal epididymis. Scale bar, 5 μm. (i, j) The percent of progressive and motile spermatozoa in 9-week-old WT and Tmc7−/− mice. Three mice were used. Images are representative of at least three independent experiments. The results are presented as the mean ± S.D., and two-sided unpaired Student’s t-tests were used to calculate the P-values. (*P < 0.05, **P < 0.01, ***P < 0.001).

Acrosome biogenesis is impaired starting from the Golgi phase in Tmc7−/− spermatids.

(a) Immunofluorescence staining of PNA (green) in the phases of acrosome biogenesis in 9-week-old WT and Tmc7−/−spermatids. Nuclei were stained with DAPI (blue). The phases of acrosome biogenesis and corresponding spermiogenesis steps are the Golgi phase (steps 1–3), cap phase (steps 4–7), acrosome phase (steps 8–12), and maturation phase (steps 13–16). (b) TEM images of testicular sections of 9-week-old WT and Tmc7−/− mice. The red star represents the abnormal plasma membrane space and accumulated vesicles. N: nucleus, Ac: acrosome; Apx: acroplaxome; pm: plasma membrane. G: Golgi complex; Scale bar, 2 μm. (c) Immunofluorescence staining with the antibody against Gm130 in spermatids from the testes of 9-week-old WT and Tmc7−/− mice. Scale bar, 2 μm. (d) Calculation of the aspect ratio (height [X] over width [Y]) of 9-week-old WT and Tmc7−/− Golgi apparatuses based on TEM images. N = 27, and 9 tubules in each testis were counted from 3 mice. (e) Western blot analysis of protein levels of Golgi stress-associated proteins (Hsp47, Tfe3) in 9-week-old WT and Tmc7−/− testes (n = 3 per group). Image J was used to quantify the protein level. (f) Immunofluorescence staining of Gopc (red) and PNA (green) and the quantitative analysis of Gopc and PNA lectin colocalization in squashed tubules of 9-week-old WT and Tmc7−/− mice. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Four mice were used, and 200 cells were counted per mouse. Images are representative of at least three independent experiments. The results are presented as the mean ± S.D., and two-sided unpaired Student’s t-tests were used to calculate the P-values. (*P < 0.05, **P < 0.01, ***P < 0.001).

Tmc7 localizes to the cis-Golgi, and this is required for maintaining Golgi integrity.

(a) Immunohistochemical staining with an antibody against Tmc7 in seminiferous tubules of 9-week-old mouse testes. Scale bar, 50 μm. (b) Immunofluorescence staining of Tmc7 (red) and Gm130 (green, a marker of the cis-Golgi), with DAPI indicating the nuclei in seminiferous tubules of 9-week-old WT and Tmc7−/− testis. Scale bar, 10 μm. Co-localization was quantified by Image J, and 20 cells were analyzed for the Pearson’s R-value. (c) Immunofluorescence staining of Tmc7 (red) and Tgn46 (green, a marker of the trans-Golgi), with DAPI indicating the nuclei in the seminiferous tubule. Scale bar, 10 μm. (d) Immunofluorescence staining of Tmc7 (red) and PNA (green, a marker of the acrosome), with DAPI indicating the nuclei in the seminiferous tubule. Scale bar, 10 μm. (e) Western blot was used to analyze the results of co-immunoprecipitation and showed the interaction between Tmc7 and Gm130, P115, and Grasp65 in 9-week-old WT testes. (f, g) Immunofluorescence staining and quantification of Gm130 (green), P115 (red) and Grasp65 (red) in testicular sections of 9-week-old WT and Tmc7−/− mice. Scale bar, 50 μm. (h) Western blots of the protein levels of Gm130 and the Gm130-interacting proteins P115 and Grasp65 in 9-week-old WT and Tmc7−/− testes. Image J was used to quantify the protein level. Images are representative of at least three independent experiments. The results are presented as the mean ± S.D., and two-sided unpaired Student’s t-tests were used to calculate the P-values. (*P < 0.05, **P < 0.01, ***P < 0.001).

Tmc7 depletion impairs cellular homeostasis and leads to spermatid apoptosis.

(a) The Ca2+ levels were measured by flow cytometry in WT and Tmc7−/− germ cells isolated from PD30 testes. (b) The pH levels were measured by flow cytometry in WT and Tmc7−/− germ cells isolated from PD30 testes. (c) The ROS levels were measured by flow cytometry in WT and Tmc7−/− germ cells isolated from PD30 testes. (d) Western blot analysis of protein levels of ER chaperone-associated proteins (P-Eif2α, Eif2α, Chop, and Grp78) in 9-week-old WT and Tmc7−/− testes. Image J was used to quantify the protein level. Each value was detected in three mice of each group. (e) TUNEL assay and the number of TUNEL-positive cells per tubule in seminiferous tubules and cauda epididymis of 9-week-old WT and Tmc7−/− male mice. Scale bar, 50 μm. N = 60, 20 tubules in each testis and cauda epididymis were counted, and three mice were used. (f) Apoptosis-related proteins (Cleaved-caspase3, Caspase3, Bax, Bcl2) were detected by western blotting in 9-week-old WT and Tmc7−/− testes. Image J was used to quantify the protein level. Images are representative of at least three independent experiments. The results are presented as the mean ± S.D., and two-sided unpaired Student’s t-tests were used to calculate the P-values. (*P < 0.05, **P < 0.01, ***P < 0.001).