Fig 1.
In situ hybridization with Tmco5 probe.
The sections of testis from 1-week to 8-week old mice were hybridized with Tmco5 probe, indicating that the mRNA for Tmco5 is expressed in the round and elongating spermatids in the seminiferous tubes of adult testis (8-week old mouse). In addition, we could not detect the expression of Tmco5 mRNA in the testis until mice were 3-weeks old. Scale bars are 100 μm.
Fig 2.
Immunobloting with the anti-TMCO5 monoclonal antibody (RTm01).
(A) In the adult tissues, TMCO5 is expressed only in the testis. The arrow indicates the corresponding 36 K-band of TMCO5. Molecular weight markers are shown. (B) TMCO5 is detectable in the testis after mice were 4-weeks old. The arrow indicates the bands of TMCO5 (C) Even a highly-sensitive method using chemiluminescent detection could not detect TMCO5 in the epididymis, indicating that TMCO5 is not a component of sperm. Molecular weight markers are shown.
Fig 3.
Immunostaining of adult testis using RTm01 antibody followed by nuclear counterstaining with Hematoxylin.
(A) Not all the seminiferous tubes are stained, only the tubes of stage IX to XII (indicated by arrows) are stained. The indications on the seminiferous tubules show the stages of the seminiferous tubules. Scale bar is 100 μm. (B) Brown-colored positive cells in the enlarged area surrounded by the square are spermatids, indicating that TMCO5 is expressed in step 9 to 12 spermatids. Scale bar is 100 μm.
Fig 4.
Induced expression of Tmco5 in the CHO cells whose Golgi apparatus is tagged with EGFP.
(A) Immunoblotting with the anti-TMCO5 monoclonal antibody (RTm01) using the extract of CHO and TMCO5-induced CHO cell line. The arrow indicates the corresponding 36 K-band of TMCO5. Molecular weight markers are shown. (B) Without induction, green-colored Golgi apparatus is scattered around the nuclei (blue) as shown in the cells surrounded by the white circles. Scale bar is 50 μm. (C) With the induction of TMCO5 (red), Golgi apparatus (green) concentrates to the point at the center of the region, where TMCO5 is distributed, as shown in the red-colored circles. Scale bar is 50 μm. (D) The enlarged picture of the surrounded area by the red circles in C are shown. The fluorescent image of TMCO5 may be fibrous like that of the cytoskeleton. Scale bar is 10 μm.
Fig 5.
Co-localization of TMCO5 and β-tubulin in the CHO cells.
The intracellular localization of TMCO5 and β-tubulin was examined in the TMCO5-induced CHO cells. Localization of TMCO5 (green) and β-tubulin (red) and nuclei (blue) was determined as shown in Fig 5A and 5B. The merged picture in C shows that both are co-localized around the nuclei (blue), suggesting that TMCO5 is a component of the microtubules or that of the vehicles moving on the network. (D) The enlarged pictures of the surrounded area by the square show that coexisting areas are not entirely identical, but the area of tubulin is slightly wider than that of TMCO5. Scale bars are 50 μm for A to C and 10 μm for D.
Fig 6.
Immunofluorescent staining of adult testis.
(A) TMCO5 (red) is localized to the opposite side of the acrosome (green) across the nucleus (blue), indicating that TMCO5 may be localized to the manchette. The bars indicate the position of the acrosome, nucleus, and TMCO5 positive region respectively. Scale bar is 50 μm.
Fig 7.
Co-localization of TMCO5 and β-tubulin in the spermatids.
TMCO5 (red) and β-tubulin (green) are co-localized in the spermatids of adult testes (merged). The enlarged picture indicates that both are almost co-localized. However, in the most posterior region of the manchette, TMCO5-signal is not detectable. Scale of bars are indicated in each figure.
Fig 8.
The schematic diagram of manchette.
Relative position of acrosome, nucleus, TMCO5-positive, and negative manchette is illustrated according to the anterior to posterior direction.