mDia1 and mDia3 are dominantly expressed in Sertoli cells and indispensable for spermatogenesis.

(A) PAS-stained stage VIII seminiferous tubule sections from adult WT and mDia1/3 DKO mice. The red (apical region) and black (basal region) boxes are magnified images of the corresponding boxed areas of the lower magnification. Note that in mDia1/3 DKO mice, differentiated sperms are decreased in number and not properly orientated at the lumen of the seminiferous tubule (black arrows, red box), and elongated spermatids are ectopically localized proximal to the basal lamina (black arrow, black box). Scale bars, 50 μm (left) and 10 μm (right). (B) Immunofluorescence staining for mDia1 or mDia3 (green) and vimentin (magenta), a marker for Sertoli cells, in testis sections from WT adult mice. Note the strong mDia1 and mDia3 immunostaining signals in the cytoplasm of Sertoli cells. Scale bar, 100 μm. The red and blue boxes are magnified images of the corresponding boxed areas of the lower magnification. (C) Schematic representation of testicular germ cell suspensions from WT or mDia1/3 DKO mice transplanted into the Sertoli cell-only testes of sterile W/WV mutant mice. (D) HE-stained testis sections from W/Wv mice transplanted with WT or mDia1/3 DKO testicular germ cell suspensions. Note that the histology of W/Wv mouse testes transplanted with mDia1/3 DKO testicular cell suspensions exhibits apparently normal spermatogenesis. Scale bars, 25 μm. The red and black boxes are magnified images of the corresponding boxed areas of the lower magnification. (E) Quantification of the number of spermatozoa per seminiferous tubule in W/Wv mice transplanted with WT testicular germ cell suspensions (13 tubules of two mice) or mDia1/3 DKO testicular germ cell suspensions (17 tubules of two mice). Data are represented as mean ± SEM. (F) Schematic representation of the transplantation of testicular germ cell suspensions from acro/act-EGFP mice into the busulfan-treated Sertoli cell-only testes of WT or mDia1/3 DKO mice. (G) HE-stained testis sections from WT or mDia1/3 DKO mice transplanted with acro/act-EGFP mice-derived testicular germ cell suspensions. Note that mDia1/3 DKO mouse testis transplanted with acro/act-EGFP mice-derived testicular germ cell suspensions exhibits histologically abnormal spermatogenesis, similar to mDia1/3 DKO mouse testis. Scale bars, 25 μm. The red and black boxes are magnified images of the corresponding boxed areas of the lower magnification. Yellow arrowhead indicates ectopic localization of sperm near the basal lamina of mDia1/3 DKO seminiferous tubule. (H) Quantification of the number of spermatozoa per seminiferous tubule in busulfan-treated testes of WT mice (20 tubules of three mice) or mDia1/3 DKO mice (15 tubules of two mice) transplanted with acro/act-EGFP mice-derived testicular germ cell suspensions. Data are represented as mean ± SEM. ***P t test). acro/act-EGFP, acrosin/actin-enhanced green fluorescent protein; DKO, double knockout; GFP, green fluorescent protein; HE, hematoxylin–eosin; mDia1, mammalian diaphanous homolog1; mDia3, mammalian diaphanous homolog3; n.s., not significant (P = 0.2675, Student t test); PAS, Periodic acid-Schiff; WT, wild-type.