Sendai virus-mediated RNA delivery restores fertility to congenital and chemotherapy-induced infertile female mice.

Current infertility treatment strategies focus on mature gametes, leaving a significant proportion of cases with gamete progenitors that stopped complete differentiation. On the other hand, recent advancements in next-generation sequencing have identified many candidate genes that may promote maturation of germ cells. Although gene therapy has shown success in mice, concerns about the integration of DNA vectors into oocytes hinder clinical applications.

Spermatogonial stem cells in the 129 inbred strain exhibit unique requirements for self-renewal.

Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice).

Restoration of fertility in nonablated recipient mice after spermatogonial stem cell transplantation.

Spermatogonial stem cell (SSC) transplantation is a valuable tool for studying stem cell-niche interaction. However, the conventional approach requires the removal of endogenous SSCs, causing damage to the niche. Here we introduce WIN18,446, an ALDH1A2 inhibitor, to enhance SSC colonization in nonablated recipients.

WIN18,446 enhances spermatogonial stem cell homing and fertility after germ cell transplantation by increasing blood-testis barrier permeability.

Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration.

Allogeneic offspring produced by induction of PD-L1 in spermatogonial stem cells via self-renewal stimulation.

The testis is an immune-privileged organ. It is considered that the testis somatic microenvironment is responsible for immune suppression. However, immunological properties of spermatogonial stem cells (SSCs) have remained unknown.

Adenovirus-mediated gene delivery restores fertility in congenitally infertile female mice.

Oogenesis depends on close interactions between oocytes and granulosa cells. Abnormal signaling between these cell types can result in infertility. However, attempts to manipulate oocyte-granulosa cell interactions have had limited success, likely due to the blood-follicle barrier (BFB), which prevents the penetration of exogenous materials into ovarian follicles.

Adeno-associated-virus-mediated gene delivery to ovaries restores fertility in congenital infertile mice.

Oocytes and granulosa cells closely interact with each other during follicular development, and a lack of appropriate signaling between them results in infertility. Attempts to manipulate oocyte microenvironment have been impeded by the impermeability of the blood-follicle barrier (BFB). To establish a strategy for manipulating oogenesis, we use adeno-associated viruses (AAVs), which have a unique ability of transcytosis.

Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids.

Gametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs.

Spermatogonial stem cell transplantation into nonablated mouse recipient testes.

Spermatogonial transplantation has been used as a standard assay for spermatogonial stem cells (SSCs). After transplantation into the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) between Sertoli cells and settle in a niche. Unlike in the repair of other self-renewing systems, SSC transplantation is generally performed after complete destruction of endogenous spermatogenesis.

An interplay of NOX1-derived ROS and oxygen determines the spermatogonial stem cell self-renewal efficiency under hypoxia.

Reactive oxygen species (ROS) produced by NADPH1 oxidase 1 (NOX1) are thought to drive spermatogonial stem cell (SSC) self-renewal through feed-forward production of ROS by the ROS-BCL6B-NOX1 pathway. Here we report the critical role of oxygen on ROS-induced self-renewal. Cultured SSCs proliferated poorly and lacked BCL6B expression under hypoxia despite increase in mitochondria-derived ROS.

Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells†.

Spermatogonial stem cells (SSCs) undergo continuous self-renewal division in response to self-renewal factors. The present study identified ephrin type-A receptor 2 (EPHA2) on mouse SSCs and showed that supplementation of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), which are both SSC self-renewal factors, induced EPHA2 expression in cultured SSCs. Spermatogonial transplantation combined with magnetic-activated cell sorting or fluorescence-activated cell sorting also revealed that EPHA2 was expressed in SSCs.

ROS amplification drives mouse spermatogonial stem cell self-renewal.

Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here, we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification.

Reversible inhibition of the blood-testis barrier protein improves stem cell homing in mouse testes.

Stem cell homing is a complex phenomenon that involves multiple steps; thus far, attempts to increase homing efficiency have met with limited success. Spermatogonial stem cells (SSCs) migrate to the niche after microinjection into seminiferous tubules, but the homing efficiency is very low. Here we report that reversible disruption of the blood-testis barrier (BTB) between Sertoli cells enhances the homing efficiency of SSCs.

Transfer of a Mouse Artificial Chromosome into Spermatogonial Stem Cells Generates Transchromosomic Mice.

The introduction of megabase-sized large DNA fragments into the germline has been a difficult task. Although microcell-mediated chromosome transfer into mouse embryonic stem cells (ESCs) allows the production of transchromosomic mice, ESCs have unstable karyotypes and germline transmission is unreliable by chimera formation. As spermatogonial stem cells (SSCs) are the only stem cells in the germline, they represent an attractive target for germline modification.

Myc/Mycn-mediated glycolysis enhances mouse spermatogonial stem cell self-renewal.

Myc plays critical roles in the self-renewal division of various stem cell types. In spermatogonial stem cells (SSCs), Myc controls SSC fate decisions because Myc overexpression induces enhanced self-renewal division, while depletion of Max, a Myc-binding partner, leads to meiotic induction. However, the mechanism by which Myc acts on SSC fate is unclear.

Fertility of Male Germline Stem Cells Following Spermatogonial Transplantation in Infertile Mouse Models.

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis. Earlier studies have shown that the transplantation of SSCs restores fertility to infertile recipients. However, most of the previously described experiments have depended on transplantation using sexually immature animals, and the effectiveness of spermatogonial transplantation in mature animals has not been examined in detail.

Enrichment of Mouse Spermatogonial Stem Cells by the Stem Cell Dye CDy1.

Spermatogonial stem cells (SSCs) comprise a small population of germ cells with self-renewal potential. Previous studies have shown that SSCs share several common features with stem cells in other self-renewing tissues, including surface markers and proliferative machinery. However, studies of SSCs are severely handicapped by the small number of SSCs and the lack of SSC-specific markers.

ROS-Generating Oxidase Nox3 Regulates the Self-Renewal of Mouse Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) represent a unique population of germ cells with self-renewal potential. Although reactive oxygen species (ROS) are considered toxic to germ cells, we recently showed that moderate levels of ROS are required for SSC self-renewal and that Nox1 is involved in ROS generation. In this study, we showed that self-renewal factor treatment induces Nox3 to trigger SSC self-renewal.

Functional differences between GDNF-dependent and FGF2-dependent mouse spermatogonial stem cell self-renewal.

Spermatogonial stem cells (SSCs) are required for spermatogenesis. Earlier studies showed that glial cell line-derived neurotrophic factor (GDNF) was indispensable for SSC self-renewal by binding to the GFRA1/RET receptor. Mice with mutations in these molecules showed impaired spermatogenesis, which was attributed to SSC depletion.

The Trp53-Trp53inp1-Tnfrsf10b pathway regulates the radiation response of mouse spermatogonial stem cells.

Germ cells are thought to exhibit a unique DNA damage response that differs from that of somatic stem cells, and previous studies suggested that Trp53 is not involved in the survival of spermatogonial stem cells (SSCs) after irradiation. Here, we report a critical role for the Trp53-Trp53inp1-Tnfrsf10b pathway during radiation-induced SSC apoptosis. Spermatogonial transplantation revealed that Trp53 deficiency increased the survival of SSCs after irradiation.

Improved serum- and feeder-free culture of mouse germline stem cells.

Spermatogonial stem cells (SSCs) undergo self-renewal division, which can be recapitulated in vitro. Attempts to establish serum-free culture conditions for SSCs have met with limited success. Although we previously reported that SSCs can be cultured without serum on laminin-coated plates, the growth rate and SSC concentration were relatively low, which made it inefficient for culturing large numbers of SSCs.

ROS are required for mouse spermatogonial stem cell self-renewal.

Reactive oxygen species (ROS) generation is implicated in stem cell self-renewal in several tissues but is thought to be detrimental for spermatogenesis as well as spermatogonial stem cells (SSCs). Using cultured SSCs, we show that ROS are generated via the AKT and MEK signaling pathways under conditions where the growth factors glial cell line-derived neurotrophic factor and fibroblast growth factor 2 drive SSC self-renewal and, instead, stimulate self-renewal at physiological levels. SSCs depleted of ROS stopped proliferating, but they showed enhanced self-renewal when ROS levels were increased by the addition of hydrogen peroxide, which induced the phosphorylation of stress kinases p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK).

Enrichment of mouse spermatogonial stem cells by melanoma cell adhesion molecule expression.

Spermatogonial stem cells (SSCs) provide the foundation of spermatogenesis, but studies are hampered by their scarcity. Although the cryptorchid operation is often used to obtain an enriched SSC population, making cryptorchid testes is time-consuming and the technique is not applicable to many animal species. In the present study, we screened for a new surface antigen on SSCs using germline stem (GS) cells (i.

Reconstitution of mouse spermatogonial stem cell niches in culture.

Spermatogonial stem cells (SSCs) reside in specific niches within seminiferous tubules. These niches are thought to secrete chemotactic factors for SSCs, because SSCs migrate to them upon transplantation. However, the identity of these chemotactic molecules remains unknown.