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The blood-testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role of Cldn11 , a major component of the BTB, in spermatogenesis using spermatogonial transplantation. Cldn11 -deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage. Cldn11 -deficient SSCs failed to colonize wild-type testes efficiently, and Cldn11 -deficient SSCs that underwent double depletion of Cldn3 and Cldn5 showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However, Cldn11 -deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intact Cldn11 -deficient testes, those from WT or Cldn11 -deficient testes regenerated sperm in Cldn11 -deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion of Cldn3 / 5 regenerated endogenous spermatogenesis in Cldn11 -deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue., Competing Interests: The authors declare no competing interest.

Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased Wnt7b expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant Wnt7b expression activated c- jun N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Ppargc1a Down-regulation of Ppargc1a probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the Klotho -deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging., Competing Interests: The authors declare no conflict of interest.

Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis throughout life. Although several positive regulators of SSC self-renewal have been discovered, little is known about the negative regulators. Here, we report that F-box and WD-40 domain protein 7 (FBXW7), a component of the Skp1-Cullin-F-box-type ubiquitin ligase, is a negative regulator of SSC self-renewal. FBXW7 is expressed in undifferentiated spermatogonia in a cell cycle-dependent manner. Although peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1), essential for spermatogenesis, is thought to destroy FBXW7, Pin1 depletion decreased FBXW7 expression. Spermatogonial transplantation showed that Fbxw7 overexpression compromised SSC activity whereas Fbxw7 deficiency enhanced SSC colonization and caused accumulation of undifferentiated spermatogonia, suggesting that the level of FBXW7 is critical for self-renewal and differentiation. Screening of putative FBXW7 targets revealed that Fbxw7 deficiency up-regulated myelocytomatosis oncogene (MYC) and cyclin E1 (CCNE1). Although depletion of Myc/Mycn or Ccne1/Ccne2 compromised SSC activity, overexpression of Myc, but not Ccne1, increased colonization of SSCs. These results suggest that FBXW7 regulates SSC self-renewal in a negative manner by degradation of MYC.

Spermatogonial stem cells (SSCs) undergo self-renewal division to support spermatogenesis. Although several positive regulators of SSC self-renewal have been identified, little is known about the mechanisms that negatively regulate SSCs. Here we developed a novel transplantation assay for SSCs and demonstrate that p21 and p27 cyclin-dependent kinase inhibitors play critical roles in SSC self-renewal and differentiation. Overexpression of p21 or p27 abrogated proliferation of cultured SSCs in vitro, and their expression levels were downregulated by exogenous self-renewal signals. In contrast, no apparent defects were found in p21 or p27-deficient SSCs by spermatogonial transplantation. However, competitive spermatogonial transplantation with WT SSCs revealed that the loss of either gene causes distortion of germline transmission: p21-deficiency facilitated mutant offspring production, whereas germline transmission was limited by p27-deficiency. Serial transplantation also showed that the loss of p27, but not p21, decreases secondary colony formation, suggesting that appropriate amounts of p27 are necessary for sustaining SSC self-renewal. Thus, p21 and p27 cyclin-dependent kinase inhibitors play critical roles in germline transmission by regulating the balance between SSC self-renewal and differentiation, and competitive spermatogonial transplantation technique will be useful for analyzing subtle defects in spermatogenesis that are not evident by traditional spermatogonial transplantation.

Although ovarian theca cells play an indispensable role in folliculogenesis by providing follicular structural integrity and steroid substrates for estrogen production, little information is available about their recruitment, growth, and differentiation because their immature forms have not been identified. We have isolated putative thecal stem cells with the ability to self-renew and differentiate in vivo and in vitro. They are similar to fibroblasts in morphology and proliferate in vitro as round colonies with a homogenous cell population. They were induced to differentiate into early precursors and steroidogenic cells in a stepwise manner after treatment with serum, luteinizing hormone, and paracrine factors from granulosa cells. At each differentiation step, these cells displayed appropriate gene expression and morphological markers and later secreted androstenedione. The fully mature morphology was achieved by coculture with isolated granulosa cells. When transplanted into the ovaries, the putative thecal stem cells colonized exclusively in the ovarian interstitium and the thecal layer of follicles as differentiated cells. Thus, thecal stem cells appear to be present in neonatal ovaries and can be isolated, purified, and induced to differentiate in vitro. Thecal stem cells could provide an invaluable in vitro experimental system to study interactions among the oocytes, granulosa cells, and theca cells during normal folliculogenesis and to study ovarian pathology caused by theca cell dysfunction.

Spermatogonial stem cells represent a self-renewing population of spermatogonia, and continuous division of these cells supports spermatogenesis throughout the life of adult male animals. Previous attempts to introduce adenovirus vectors into spermatogenic cells, including spermatogonial stem cells, have failed to yield evidence of infection, suggesting that male germ cells may be resistant to adenovirus infection. In this study we show the feasibility of transducing spermatogonial stem cells by adenovirus vectors. When testis cells from ROSA26 Cre reporter mice were incubated in vitro with a Cre-expressing adenovirus vector, Cre-mediated recombination occurred at an efficiency of 49-76%, and the infected spermatogonial stem cells could reinitiate spermatogenesis after transplantation into seminiferous tubules of infertile recipient testes. No evidence of germ-line integration of adenovirus vector could be found in offspring from infected stem cells that underwent Cre-mediated recombination, which suggests that the adenovirus vector infected the cells but did not stably integrate into the germ line. Nevertheless, these results suggest that adenovirus may inadvertently integrate into the patient's germ line and indicate that there is no barrier to adenovirus infection in spermatogonial stem cells.

Spermatogonial transplantation has demonstrated a unique opportunity for studying spermatogenesis and provided an assay for spermatogonial stem cells. However, it has remained unknown whether germ cells that matured in a xenogeneic environment are functionally normal. In this investigation, we demonstrate the successful production of xenogeneic offspring by using spermatogonial transplantation. Rat spermatogonial stem cells were collected from immature testis and transplanted into the seminiferous tubules of busulfan-treated nude mouse testis. Using rat spermatids or spermatozoa that developed in xenogeneic surrogate mice, rat offspring were born from fresh and cryopreserved donor cells after microinsemination with rat oocytes. These offspring were fertile and had a normal imprinting pattern. The xenogeneic offspring production by interspecies germ cell transplantation and in vitro microinsemination will become a powerful tool in animal transgenesis and species conservation.

Stem cells represent a unique population of cells with self-renewal capacity. Although they are important therapeutic targets, the genetic manipulation of tissue-specific stem cells has been limited, which complicates the study and practical application of these cells. Here, we demonstrate successful gene trapping and homologous recombination in spermatogonial stem cells. Cultured spermatogonial stem cells were transfected with gene trap or gene targeting vectors. Mutagenized stem cells were expanded clonally by drug selection. These cells underwent spermatogenesis and produced heterozygous offspring after transplantation into the seminiferous tubules of infertile mouse testes. Heterozygous mutant mice were intercrossed to produce homozygous gene knockouts. Using this strategy, the efficiency of homologous recombination for the occludin gene locus was 1.7% using a nonisogenic DNA construct. These results demonstrate the feasibility of altering genes in tissue-specific stem cells in a manner similar to embryonic stem cells and have important implications for gene therapy and animal transgenesis.

Sertoli cells play a pivotal role in spermatogenesis through their interactions with germ cells. To set up a strategy for treating male infertility caused by Sertoli cell dysfunction, we developed a Sertoli cell gene transfer system by using an adenovirus vector, which maintained long-term transgene expression in the testes of infertile mice. Introduction of an adenovirus carrying the mouse Steel (Sl) gene into Sertoli cells restored partial spermatogenesis in infertile Steel/Steel(dickie) (Sl/Sl(d)) mutant mouse testes. Although these males remained infertile, round spermatids and spermatozoa from the testes produced normal fertile offspring after intracytoplasmic injection into oocytes. None of the offspring showed evidence of germ line transmission of adenoviral DNA. Thus, we demonstrate a successful treatment for infertility by using a gene therapy vector. Therefore, adenovirus-mediated gene delivery into Sertoli cells not only provides an efficient and convenient means for studying germ cell-Sertoli cell interactions through manipulation of the germ cell microenvironment in vivo, but also is a useful method to treat male infertility resulting from a Sertoli cell defect.