Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice Mito Kanatsu-Shinoharaa,b, Narumi Ogonukic, Shogo Matobac, Atsuo Ogurac, and Takashi Shinoharaa,1 aDepartment of Molecular Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan; bAgency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), AMED, Chiyodaku, 100-0004 Tokyo, Japan; and cInstitute for Physical and Chemical Research (RIKEN), BioResource Research Center, 305-0074 Tsukuba, Japan Edited by Ryuzo Yanagimachi, University of Hawaii System, Honolulu, HI, and approved March 2, 2020 (received for review September 4, 2019) The blood–testis barrier (BTB) is thought to be indispensable for for meiosis and also protects haploid germ cells from the immune spermatogenesis because it creates a special environment for mei- system (5). Thus, the BTB is unique among blood–tissue barriers osis and protects haploid cells from the immune system. The BTB in the body in terms of its cell biology and immunological aspects, divides the seminiferous tubules into the adluminal and basal com- and understanding the molecular mechanism underlying the germ partments. Spermatogonial stem cells (SSCs) have a unique ability cell–Sertoli cell interaction has important implications for our to transmigrate from the adluminal compartment to the basal understanding of infertility. compartment through the BTB upon transplantation into the sem- Research over the last decade has revealed the molecular iniferous tubule. Here, we analyzed the role of Cldn11, a major structure of the BTB. Although the tight junctions of the BTB component of the BTB, in spermatogenesis using spermatogonial are formed between Sertoli cells, the functional BTB is composed transplantation. Cldn11-deficient mice are infertile due to the ces- of the Sertoli cell tight junctions, a physiological permeability Cldn11 sation of spermatogenesis at the spermatocyte stage. - barrier, and an immunological barrier (5, 9). Several tight junction deficient SSCs failed to colonize wild-type testes efficiently, and proteins (TJPs) are identified, and the phenotypes of knockout Cldn11 Cldn3 -deficient SSCs that underwent double depletion of (KO) mice for these components vary from normal, as seen in Cldn5 and showed minimal colonization, suggesting that claudins F11r KO mice, to slowly degenerative, as seen in Ocln KO mice on SSCs are necessary for transmigration. However, Cldn11- > (10), to sterile in Cldn11 KO mice (11). All animals with BTB deficient Sertoli cells increased SSC homing efficiency by 3-fold, defects are infertile because these defects likely cause immuno- suggesting that CLDN11 in Sertoli cells inhibits transmigration of CELL BIOLOGY logical or other types of damages to the meiotic and postmeiotic SSCs through the BTB. In contrast to endogenous SSCs in intact cells (5, 9). Although the BTB is formed between Sertoli cells, Cldn11-deficient testes, those from WT or Cldn11-deficient testes spermatogonia and spermatocytes also express several TJPs (12). regenerated sperm in Cldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endog- However, the roles of these TJPs are unknown because germ enous germ cells for recipient preparation, which reprogrammed cells do not form tight junction by themselves. Because germ claudin expression patterns in Sertoli cells. Consistent with this cells also express TJPs, defective spermatogenesis in TJP KO idea, in vivo depletion of Cldn3/5 regenerated endogenous sper- mice may be a result of defects in both the germ cells and matogenesis in Cldn11-deficient mice. Thus, coordinated claudin ex- Sertoli cells. pression in both SSCs and Sertoli cells expression is necessary for Here, we used spermatogonial transplantation to analyze the SSC homing and regeneration of spermatogenesis, and autolo- role of TJPs in Cldn11 KO mice, which completely lack the BTB. gous stem cell transplantation can rescue congenital defects of a self-renewing tissue. Significance claudin | Sertoli cell | spermatogenesis Stem cell transplantation is widely used to rescue defects in stem cell-derived cells. However, it is generally impossible to permatogenesis is maintained by close interactions between rescue tissue dysfunction caused by defective microenviron- Sgerm cells and somatic cells. Any defects in this interaction ment. In this study, we report that autologous spermatogonial result in defective spermatogenesis, leading to infertility. Sper- stem cell (SSC) transplantation rescues congenital male in- matogonial stem cells (SSCs) undergo self-renewal divisions (1, fertility caused by Cldn11 deficiency. Cldn11-deficient mice lack 2) and can recolonize empty seminiferous tubules and regenerate spermatogenesis due to defects in the blood–testis barrier. Cldn11 spermatogenesis (3). SSCs from a congenitally defective micro- However, WT or -deficient SSC transplantation allowed environment can rescue SSC defects caused by Kit mutations development of fertile sperm from the donor cells in chemically Cldn11 such that normal offspring are born (4). However, spermatogenic castrated -deficient mice. Because in vivo depletion of Cldn3 Cldn5 defects can occur due to a number of mutations, most of which or restored endogenous spermatogenesis, com- cannot be explained at the molecular level. This is particularly plete spermatogenesis may be inhibited by the imbalance of claudin expression caused by Cldn11 deficiency. Our result true in clinical cases, in which many responsible genes have not suggests that some forms of male infertility can be rescued by yet been identified. autologous SSC transplantation. The blood–testis barrier (BTB) forms between the Sertoli cells ∼ – at 12 14 d postpartum (dpp) in mice, when they stop mitotic Author contributions: M.K.-S. and T.S. designed research; M.K.-S., N.O., S.M., A.O., and proliferation (5, 6). The BTB divides the seminiferous tubules T.S. performed research; M.K.-S., N.O., S.M., A.O., and T.S. contributed new reagents/ into adluminal and basal compartments (7). After mitotic divi- analytic tools; M.K.-S. and T.S. analyzed data; and M.K.-S. and T.S. wrote the paper. sion of SSCs, a clone of an interconnected spermatocyte trans- The authors declare no competing interest. migrates through the BTB by continuous dynamic restructuring, This article is a PNAS Direct Submission. and haploid cells eventually develop in the adluminal compart- Published under the PNAS license. ment (8). These spermatocytes are temporarily enclosed in an 1To whom correspondence may be addressed. Email: [email protected]. intermediate compartment and transported into the adluminal This article contains supporting information online at https://www.pnas.org/lookup/suppl/ side. It is considered that the integrity of the BTB is essential for doi:10.1073/pnas.1914963117/-/DCSupplemental. normal spermatogenesis because it creates a special environment First published March 30, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1914963117 PNAS | April 7, 2020 | vol. 117 | no. 14 | 7837–7844 Downloaded by guest on October 1, 2021 SSCs have the unique ability to transmigrate through the BTB, Results and SSCs regenerated from the transplanted SSCs can complete Immunohistochemical Analysis of Cldn11 KO Mice. Cldn11 KO testes normal spermatogenesis (3). Therefore, this technique has been were significantly smaller than wild-type (WT) mouse testes used to analyze the germ cell–Sertoli cell interaction. Of the when the testes were collected from 11-wk-old mice (Fig. 1 A and several TJP-related KO mice, Cldn11 KO mice show the most B). Although germ cells were found in the seminiferous tu- prominent effects, because spermatogenesis in Cldn11 KO mice bules (Fig. 1C, SI Appendix, Fig. S1A), no spermatozoa were does not proceed beyond the spermatocyte stage (11, 13). In our found in the epididymis (SI Appendix, Fig. S1B). Close exami- attempt to analyze germ cell–Sertoli cell interaction using this model, nation by lectin immunostaining showed a lack of peanut ag- we found that autologous SSC transplantation restores fertility. glutinin (PNA)-expressing haploid cells in the mutant testes (Fig. Fig. 1. Characterization of Cldn11 KO mouse testes. (A and B) Appearance (A) and testis weight (B; n = 4–8) of Cldn11 KO mouse testis. (C and D) Histological appearance of testis (C). Sertoli cell clusters (arrow) are shown in Inset.(D) Lectin (PNA) immunostaining. (E and F) Immunostaining of + CLDN3 (E) and CLDN5 (F) in busulfan-treated Cldn11 KO mouse testes. (G) Immunohistochemical analysis of KIT spermatogonia. (H) Quantification of + + + KIT cells. At least 11 tubules were counted. (I and J) Immunohistochemical analysis of apoptotic CLGN (I) and SYCP (J) cells by TUNEL staining. Ar- rowheads indicate apoptotic cells. (Scale bars: A,1mm;C,200μm; D, G, I,andJ,50μm; E and F,20μm.) Stain: hematoxylin & eosin (C), Hoechst 33342 (D–G, I,andJ). 7838 | www.pnas.org/cgi/doi/10.1073/pnas.1914963117 Kanatsu-Shinohara et al. Downloaded by guest on October 1, 2021 1D). Interestingly, we occasionally found clusters of Sertoli cells in SSC Activity of Cldn11 KO Mice. In the first set of transplantation thetubulelumen(Fig.1C, Inset) (14). Terminal deoxynucleotidyl experiments, Cldn11 KO mice were used as donors to examine transferase-mediated dUTP nick end-labeling (TUNEL) staining whether Cldn11 deficiency influences SSC activity. To introduce showed no evidence of increased