Data on in vivo phenotypes of GFR?1-positive spermatogonia stimulated by interstitial GDNF signals in mouse testes.
ABSTRACT: This article contains the data related to the research article "in vivo dynamics of GFR?1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay" (Uchida et al., 2016) . A novel transplantation assay of growth factor-soaked beads into the mammalian testicular interstitium was developed, in order to examine the effects of various soluble factors on in vivo dynamics of the spermatogonia including spermatogonial stem cells (SSC). Here we provide the image data of GFR?1-positive stem/progenitor spermatogonia in mouse seminiferous tubules near the beads soaked in GDNF (glial cell-derived neurotrophic factor), one of the SSC niche factors. The data provide various phenotypes of GFR?1-positive spermatogonia induced by bead-derived GDNF signals, which are useful to understand the active state of GFR?1-positive stem/progenitor spermatogonia in vivo.
Project description:BACKGROUND AND AIMS: In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor ?1 (GFR?1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFR?1-positive spermatogonia in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFR?1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFR?1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFR?1-positive spermatogonia. In both species, GFR?1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. CONCLUSION/SIGNIFICANCE: Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFR?1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.
Project description:Chromodomain helicase/ATPase DNA binding protein 1-like gene (Chd1l) participates in chromatin-dependent processes, including transcriptional activation and DNA repair. In this study, we have found for the first time that Chd1l is mainly expressed in the testicular tissues of prepubertal and adult mice and colocalized with PLZF, OCT4, and GFR?1 in the neonatal mouse testis and THY1+ undifferentiated spermatogonia or spermatogonial stem cells (SSCs). Knockdown of endogenous Chd1l in cultured mouse undifferentiated SSCs inhibited the expression levels of Oct4, Plzf, Gfr?1, and Pcna genes, suppressed SSC colony formation, and reduced BrdU incorporation, while increasing SSC apoptosis. Moreover, the Chd1l gene expression is activated by GDNF in the cultured mouse SSCs, and the GDNF signaling pathway was modulated by endogenous levels of Chd1l; as demonstrated by the gene expression levels of GDNF, inducible transcripts Etv5, Bcl6b, Pou3f, and Lhx1, but not that of GDNF-independent gene, Taf4b, were significantly downregulated by Chd1l knockdown in mouse SSCs. Taken together, this study provides the first evidence to support the notion that Chd1l is an intrinsic and novel regulator for SSC survival and self-renewal, and it exerts such regulation at least partially through a GDNF signaling pathway.
Project description:BACKGROUND: In vitro culture of spermatogonial stem cells (SSCs) is important for exploration of SSCs self-renewal, differentiation, and manipulation. There are several reports on rodent SSC cultures; however, data on SSC cultures in domestic animals are limited. To provide basic scientific information on canine SSC cultures, we report canine testes development, and the development of spermatogonia-derived colonies (SDCs) for in vitro cultures. METHODOLOGY/PRINCIPAL FINDINGS: Testes from 2-, 3-, and 12-month-old beagles were used for histology, immunohistochemistry, in vitro culture, immunocytochemistry, and PCR. Protein gene product 9.5 (PGP9.5)-positive spermatogonia, both single and paired, were found to be abundant in the testes of 2-month-old beagles. stempro-34 and Dulbecco's modified Eagle medium with 5% fetal bovine serum provided as useful substrates for culture of SDCs, and fibroblast growth factor (FGF) played a key role in colony formation. Colonies were positive for alkaline phosphatase and anti-PGP9.5 staining. The early spermatogonia and stem cell markers such as octamer binding protein 4 (Oct4), Nanog homeobox (Nanog), promyelocytic leukemia zinc finger (PLZF), PGP9.5, and GDNF family receptor alpha-1 (GFR?-1) were expressed in the colonies at higher levels than in the testis tissue. CONCLUSIONS: Testes of the 2-month-old beagles had abundant single and paired spermatogonia, which can be used for derivation of SDCs, and FGF was important for colony formation.
Project description:The identification and physical isolation of testis stem cells, a subset of type A spermatogonia, is critical to our understanding of their growth regulation during the first steps of spermatogenesis. These stem cells remain poorly characterized because of the paucity of specific molecular markers that permit us to distinguish them from other germ cells. Thus, the molecular mechanisms driving the first steps of spermatogenesis are still unknown. We show in the present study that GFR alpha-1, the receptor for GDNF (glial cell line-derived neurotrophic factor), is strongly expressed by a subset of type A spermatogonia in the basal part of the seminiferous epithelium. Using this characteristic, we devised a method to specifically isolate these GFR alpha-1-positive cells from immature mouse testes. The isolated cells express Ret, a tyrosine kinase transmembrane receptor that mediates the intracellular response to GDNF via GFR alpha-1. After stimulation with rGDNF, the isolated cells proliferated in culture and underwent the first steps of germ cell differentiation. Microarray analysis revealed that GDNF induces the differential expression of a total of 1124 genes. Among the genes upregulated by GDNF were many genes involved in early mammalian development, differentiation, and the cell cycle. This report describes the first isolation of a pure population of GFR alpha-1-positive cells in the testis and identifies signaling pathways that may play a crucial role in maintaining germ-line stem cell proliferation and/or renewal.
Project description:In the human testis, beginning at approximately 2 months of age, gonocytes are replaced by adult dark (Ad) and pale (Ap) spermatogonia that make up the spermatogonial stem cell (SSC) pool. In mice, the SSC pool arises from gonocytes approximately 6 days after birth. During puberty in both species, complete spermatogenesis is established by cells that differentiate from SSCs. Essentially pure populations of prepubertal human spermatogonia and mouse gonocytes were selected from testis biopsies and validated by confirming the presence of specific marker proteins in cells. Stem cell potential of germ cells was demonstrated by transplantation to mouse testes, following which the cells migrated to the basement membrane of the seminiferous tubule and were maintained similar to SSCs. Differential gene expression profiles generated between germ cells and testis somatic cells demonstrated that expression of genes previously identified as SSC and spermatogonial-specific markers (e.g., zinc-finger and BTB-domain containing 16, ZBTB16) was greatly elevated in both human spermatogonia and mouse gonocytes compared to somatic cells. Several genes were expressed at significantly higher levels in germ cells of both species. Most importantly, genes known to be essential for mouse SSC self-renewal (e.g., Ret proto-oncogene, Ret; GDNF-family receptor alpha1, Gfr alpha1; and B-cell CLL/lymphoma 6, member B, Bcl6b) were more highly expressed in both prepubertal human spermatogonia and mouse gonocytes than in somatic cells. The results indicate remarkable conservation of gene expression, notably for self-renewal genes, in these prepubertal germline cells between two species that diverged phylogenetically approximately 75 million years ago.
Project description:Undifferentiated spermatogonia comprise a pool of stem cells and progenitor cells that show heterogeneous expression of markers, including the cell surface receptor GFR?1. Technical challenges in isolation of GFR?1+ versus GFR?1- undifferentiated spermatogonia have precluded the comparative molecular characterization of these subpopulations and their functional evaluation as stem cells. Here, we develop a method to purify these subpopulations by fluorescence-activated cell sorting and show that GFR?1+ and GFR?1- undifferentiated spermatogonia both demonstrate elevated transplantation activity, while differing principally in receptor tyrosine kinase signaling and cell cycle. We identify the cell surface molecule melanocyte cell adhesion molecule (MCAM) as differentially expressed in these populations and show that antibodies to MCAM allow isolation of highly enriched populations of GFR?1+ and GFR?1- spermatogonia from adult, wild-type mice. In germ cell culture, GFR?1- cells upregulate MCAM expression in response to glial cell line-derived neurotrophic factor (GDNF)/fibroblast growth factor (FGF) stimulation. In transplanted hosts, GFR?1- spermatogonia yield GFR?1+ spermatogonia and restore spermatogenesis, albeit at lower rates than their GFR?1+ counterparts. Together, these data provide support for a model of a stem cell pool in which the GFR?1+ and GFR?1- cells are closely related but show key cell-intrinsic differences and can interconvert between the two states based, in part, on access to niche factors.
Project description:Spermatogonial stem cells (SSCs) are unique male germline stem cells that support spermatogenesis and male fertility. Long non-coding RNAs (lncRNA) have been identified as key regulators of stem cell fate; however, their role in SSCs has not been explored. Here, we report that a novel spermatogonia-specific lncRNA (lncRNA033862) is essential for the survival of murine SSCs. LncRNA033862 is expressed in early spermatogonia including SSC and was among 805 lncRNAs identified by global expression profiling as responsive to glial cell-derived neurotrophic factor (GDNF), a growth factor required for SSC self-renewal and survival. LncRNA033862 is an antisense transcript of the GDNF receptor alpha1 (Gfra1) that lacks protein coding potential and regulates Gfra1 expression levels by interacting with Gfra1 chromatin. Importantly, lncRNA033862 knockdown severely impairs SSC survival and their capacity to repopulate recipient testes in a transplantation assay. Collectively, our data provide the first evidence that long non-coding RNAs (lncRNAs) regulate SSC fate.
Project description: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. Here we show that SSCs undergo GDNF-independent self-renewal. A small number of spermatogonia formed colonies when testis fragments from a Ret mutant mouse strain were transplanted into heterologous recipients. Moreover, fibroblast growth factor 2 (FGF2) supplementation enabled in vitro SSC expansion without GDNF. Although GDNF-mediated self-renewal signaling required both AKT and MAP2K1/2, the latter was dispensable in FGF2-mediated self-renewal. FGF2-depleted testes exhibited increased levels of GDNF and were enriched for SSCs, suggesting that the balance between FGF2 and GDNF levels influences SSC self-renewal in vivo. Our results show that SSCs exhibit at least two modes of self-renewal and suggest complexity of SSC regulation in vivo.
Project description:In the murine testis, self-renewal of spermatogonial stem cells (SSCs) requires glial cell line-derived neurotrophic factor (GDNF) secreted from neighboring somatic cells. However, it not clear how GDNF promotes self-renewal in vivo or what downstream signaling pathways are required for SSC maintenance. We found that GDNF is normally expressed cyclically during spermatogenesis. Stage-specific ectopic expression of GDNF caused the accumulation of a GFRA1+ LIN28- Asingle population, which has enhanced SSC activity compared with wild type, suggesting that GDNF normally limits self-renewal to specific stages. Despite the increase in SSC cell number, EdU labeling during steady-stage spermatogenesis, and during recovery after busulfan-mediated spermatogonial depletion, indicated that GDNF promotes self-renewal by blocking differentiation and not by promoting proliferation. Increased GDNF signaling led to increased phosphorylation of AKT3 in undifferentiated spermatogonia, but not of AKT1 or AKT2, and was independent of RPS6 phosphorylation, suggesting that AKT3 functions in SSC self-renewal or progenitor cell expansion.
Project description:The development of a stem cell culture system would expedite our understanding of the biology of tissue regeneration. Spermatogonial stem cell (SSC) is the foundation for lifelong male spermatogenesis and the SSC culture has been optimized continuously in recent years. However, there have been many inconveniences to reconstruct SSC self-renewal and proliferation in vitro, such as the frequent refreshment of recombinant cytokines, including GDNF, the essential growth factor for SSC maintenance. In the present study, we observed that both STO and MEF cells, which were previously used as feeders for SSC growth, did not express GDNF, but a GDNF-expressing STO feeder could support undifferentiated mouse spermatogonia propagation in vitro for three months without the refreshment of recombinant growth factor GDNF. The cell morphology, growth rate and SSC-associated gene expression remained identical to the SSCs cultured using previous methods. The transplantation of SSCs growing on these GDNF-expressing STO feeders could generate extensive colonies of spermatogenesis in recipient testes, functionally validating the stemness of these cells. Collectively, our data indicated that the further modification of feeder cells might facilitate the self-renewal and propagation of SSCs in vitro.