PTN from Leydig cells activates SDC2 and modulates human spermatogonial stem cell proliferation and survival via GFRA1.

BACKGROUND: Spermatogonial stem cells (SSCs) are essential for the maintenance and initiation of male spermatogenesis. Despite the advances in understanding SSC biology in mouse models, the mechanisms underlying human SSC development remain elusive. RESULTS: Here, we analyzed the signaling pathways involved in SSC regulation by testicular somatic cells using single-cell sequencing data (GEO datasets: GSE149512 and GSE112013) and identified that Leydig cells communicate with SSCs through pleiotrophin (PTN) and its receptor syndecan-2 (SDC2). Immunofluorescence, STRING prediction, and protein immunoprecipitation assays confirmed the interaction between PTN and SDC2 in spermatogonia, but their co-localization was observed only in approximately 50% of the cells. The knockdown of SDC2 in human SSC lines impaired cell proliferation, DNA synthesis, and the expression of PLZF, a key marker for SSC self-renewal. Transcriptome analysis revealed that SDC2 knockdown downregulated the expression of GFRA1, a crucial factor for SSC proliferation and self-renewal, and inhibited the HIF-1 signaling pathway. Exogenous PTN rescued the proliferation and GFRA1 expression in SDC2 knockdown SSC lines. In addition, we found downregulation of PTN and SDC2 as well as altered localization in non-obstructive azoospermia (NOA) patients, suggesting that downregulation of PTN and SDC2 may be associated with impaired spermatogenesis. CONCLUSIONS: Our results uncover a novel mechanism of human SSC regulation by the testicular microenvironment and suggest a potential therapeutic target for male infertility.

Gdnf Acts as a Germ Cell-Derived Growth Factor and Regulates the Zebrafish Germ Stem Cell Niche in Autocrine- and Paracrine-Dependent Manners.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type Aund spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.

A subpopulation of human Adark spermatogonia behaves as the reserve stem cell.

Human spermatogonial stem cells (SSCs) are an essential source to maintain spermatogenesis as an efficient process for daily sperm production with high self-renewal capacity along adulthood. However, the phenotype and the subpopulation that represent the real reserve SSC for the human testis remain unknown. Moreover, although SSC markers have been described for undifferentiated spermatogonia (Adark and Apale), the existence of a specific subtype that could be identified as the actual/true SSC has not yet been fully determined. Herein we evaluated spermatogonial morphology, kinetics, positioning regarding blood vasculature in relation to protein expression (UTF1, GFRA1, and KIT) as well as proliferative activity (MCM7) and identified a small subpopulation of Adark with nuclear rarefaction zone (AdVac) that behaves as the human reserve SSC. We show that AdVac is the smallest human spermatogonial population (10%), staying quiescent (89%) and positioned close to blood vessels throughout most of the stages of the seminiferous epithelium cycle (SEC) and divides only at stages I and II. Within this AdVac population, we found a smaller pool (2% of A undifferentiated spermatogonia) of entirely quiescent cells exhibiting a high expression of UTF1 and lacking GFRA1. This finding suggests them as the real human reserve SSC (AdVac UTF1+/GFRA1-/MCM7-). Additionally, Adark without nuclear vacuole (AdNoVac) and Apale have similar kinetic and high proliferative capacity throughout the SEC (47%), indicating that they are actively dividing undifferentiated spermatogonia. Identification of human stem cells with evident reserve SSC functionality may help further studies intending to sort SSCs to treat male diseases and infertility.

GDNF and GFRα1 Are Required for Proper Integration of Adult-Born Hippocampal Neurons.

The glial cell line-derived neurotrophic factor (GDNF) is required for the survival and differentiation of diverse neuronal populations during nervous system development. Despite the high expression of GDNF and its receptor GFRα1 in the adult hippocampus, the functional role of this system remains unknown. Here, we show that GDNF, acting through its GFRα1 receptor, controls dendritic structure and spine density of adult-born granule cells, which reveals that GFRα1 is required for their integration into preexisting circuits. Moreover, conditional mutant mice for GFRα1 show deficits in behavioral pattern separation, a task in which adult neurogenesis is known to play a critical role. We also find that running increases GDNF in the dentate gyrus and promotes GFRα1-dependent CREB (cAMP response element-binding protein) activation and dendrite maturation. Together, these findings indicate that GDNF/GFRα1 signaling plays an essential role in the plasticity of adult circuits, controlling the integration of newly generated neurons.

Characterization of spermatogonial cells and niche in the scorpion mud turtle (Kinosternon scorpioides).

Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.

GDNF/GFRα1 Complex Abrogates Self-Renewing Activity of Cortical Neural Precursors Inducing Their Differentiation.

The balance between factors leading to proliferation and differentiation of cortical neural precursors (CNPs) determines the correct cortical development. In this work, we show that GDNF and its receptor GFRα1 are expressed in the neocortex during the period of cortical neurogenesis. We show that the GDNF/GFRα1 complex inhibits the self-renewal capacity of mouse CNP cells induced by fibroblast growth factor 2 (FGF2), promoting neuronal differentiation. While GDNF leads to decreased proliferation of cultured cortical precursor cells, ablation of GFRα1 in glutamatergic cortical precursors enhances its proliferation. We show that GDNF treatment of CNPs promoted morphological differentiation even in the presence of the self-renewal-promoting factor, FGF2. Analysis of GFRα1-deficient mice shows an increase in the number of cycling cells during cortical development and a reduction in dendrite development of cortical GFRα1-expressing neurons. Together, these results indicate that GDNF/GFRα1 signaling plays an essential role in regulating the proliferative condition and the differentiation of cortical progenitors.

Role of Estrogens in the Size of Neuronal Somata of Paravaginal Ganglia in Ovariectomized Rabbits.

We aimed to determine the role of estrogens in modulating the size of neuronal somata of paravaginal ganglia. Rabbits were allocated into control (C), ovariectomized (OVX), and OVX treated with estradiol benzoate (OVX + EB) groups to evaluate the neuronal soma area; total serum estradiol (E2) and testosterone (T) levels; the percentage of immunoreactive (ir) neurons anti-aromatase, anti-estrogen receptor (ERα, ERß) and anti-androgen receptor (AR); the intensity of the immunostaining anti-glial cell line-derived neurotrophic factor (GDNF) and the GDNF family receptor alpha type 1 (GFRα1); and the number of satellite glial cells (SGCs) per neuron. There was a decrease in the neuronal soma size for the OVX group, which was associated with low T, high percentages of aromatase-ir and neuritic AR-ir neurons, and a strong immunostaining anti-GDNF and anti-GFRα1. The decrease in the neuronal soma size was prevented by the EB treatment that increased the E2 without affecting the T levels. Moreover, there was a high percentage of neuritic AR-ir neurons, a strong GDNF immunostaining in the SGC, and an increase in the SGCs per neuron. Present findings show that estrogens modulate the soma size of neurons of the paravaginal ganglia, likely involving the participation of the SGC.

Immunolocalization of proteins in the spermatogenesis process of canine.

Spermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine. Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

The GDNF-GFRα1 complex promotes the development of hippocampal dendritic arbors and spines via NCAM.

The formation of synaptic connections during nervous system development requires the precise control of dendrite growth and synapse formation. Although glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are expressed in the forebrain, the role of this system in the hippocampus remains unclear. Here, we investigated the consequences of GFRα1 deficiency for the development of hippocampal connections. Analysis of conditional Gfra1 knockout mice shows a reduction in dendritic length and complexity, as well as a decrease in postsynaptic density specializations and in the synaptic localization of postsynaptic proteins in hippocampal neurons. Gain- and loss-of-function assays demonstrate that the GDNF-GFRα1 complex promotes dendritic growth and postsynaptic differentiation in cultured hippocampal neurons. Finally, in vitro assays revealed that GDNF-GFRα1-induced dendrite growth and spine formation are mediated by NCAM signaling. Taken together, our results indicate that the GDNF-GFRα1 complex is essential for proper hippocampal circuit development.

Treating congenital megacolon by transplanting GDNF and GFRα-1 double genetically modified rat bone marrow mesenchymal stem cells.

We studied the survival and gene expression of glial cell line-derived neurotrophic factor (GDNF) and GDNF receptor α-1 (GFRα-1) double-genetically modified rat bone marrow mesenchymal stem cells (BMSCs) transplanted into the intestinal walls of the rat models with congenital megacolon and determine the feasibility of treatment by transplantation of double-genetically modified rat BMSCs. The rat colorectal intestinal wall nerve plexus was treated with the cationic surface active agent benzalkonium chloride to establish an experimental megacolon model. The rat target genes GDNF and GFRα-1 were extracted and ligated into pEGFP-N1. Eukaryotic fluorescent expression vectors carrying the GDNF and GFRα-1 genes were transfected into BMSCs by in vitro culture. We treated congenital megacolon by transplanting double-genetically modified rat bone marrow mesenchymal stem cells. The pEGFP-EGFP-GDNF-GFRα-1 double-gene co-expressing the eukaryotic expression plasmid vector was successfully established. Protein gene protein 9.5 and vasoactive intestinal peptide-positive ganglion cells showed no positive expression in the phosphate-buffered saline transplantation group based on an immunofluorescence test at 1, 2, and 4 weeks after transplantation of BMSCs. Additionally, compared with the phosphate-buffered saline transplantation group, the expression of rearranged during transfection, GDNF, and GFRα-1 mRNA in the stem cell transplantation group increased gradually. The double-genetically modified BMSCs colonized and survived in the intestinal wall of the experimental megacolon rat model and expressed related genes, partially recovering the colonic neuromuscular regulatory functions and thus providing an experimental basis for treating congenital megacolon by cellular transplantation.

Morphometric evaluation of the spermatogonial stem cell distribution and niche in vertebrates.

Morphometry is a classical quantitative method often used in biology to provide a data basis for functional interpretations/interactions of a particular organ or system. Herein we took advantage of this valuable approach to evaluate the spermatogonial stem cell niche using the horse testis and immunocytochemical localization of GFRA1 [glial cell line-derived neurotrophic factor receptor produced by Sertoli cells)] as an example. Using the NIH ImageJ free software, we describe in detail all the necessary steps to investigate this specific and crucial microenvironment. Based on several recently published papers from our research group, this approach has proved to be fast, simple, and adaptable to a wide range of species and has the potential to be easily reproducible in different laboratories.

Spermatogonial stem cell markers and niche in equids.

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and are located in a highly dynamic microenvironment called "niche" that influences all aspects of stem cell function, including homing, self-renewal and differentiation. Several studies have recently identified specific proteins that regulate the fate of SSCs. These studies also aimed at identifying surface markers that would facilitate the isolation of these cells in different vertebrate species. The present study is the first to investigate SSC physiology and niche in stallions and to offer a comparative evaluation of undifferentiated type A spermatogonia (Aund) markers (GFRA1, PLZF and CSF1R) in three different domestic equid species (stallions, donkeys, and mules). Aund were first characterized according to their morphology and expression of the GFRA1 receptor. Our findings strongly suggest that in stallions these cells were preferentially located in the areas facing the interstitium, particularly those nearby blood vessels. This distribution is similar to what has been observed in other vertebrate species. In addition, all three Aund markers were expressed in the equid species evaluated in this study. These markers have been well characterized in other mammalian species, which suggests that the molecular mechanisms that maintain the niche and Aund/SSCs physiology are conserved among mammals. We hope that our findings will help future studies needing isolation and cryopreservation of equids SSCs. In addition, our data will be very useful for studies that aim at preserving the germplasm of valuable animals, and involve germ cell transplantation or xenografts of equids testis fragments/germ cells suspensions.

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu).

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.

Genetic association of the GDNF alpha-receptor genes with schizophrenia and clozapine response.

GDNF (glial-cell-line derived neurotrophic factor) is a potent neurotrophic factor for dopaminergic neurons. Neuropsychiatric diseases and their treatments are associated with alterations in the levels of both GDNF and its receptor family (GDNF family receptor alpha or GFRA). GFRA1, GFRA2 and GFRA3 are located in chromosomal regions with suggestive linkage to schizophrenia. In this study we analyzed polymorphisms located in all four known GFRA genes and examined association with schizophrenia and clozapine response. We examined SNPs across the genes GFRA1-4 in 219 matched case-control subjects, 85 small nuclear families and 140 schizophrenia patients taking clozapine for 6months. We observed that GFRA3 rs11242417 and GFRA1 rs11197557 variants were significantly associated with schizophrenia after combining results from both schizophrenia samples. Furthermore, we found an overtransmission of the G-C GFRA1 rs7920934-rs730357 haplotype to subjects with schizophrenia and association of A-T-G-G GFRA3 rs10036665-rs10952-rs11242417-rs7726580 with schizophrenia in the case-control sample. On the other hand, GFRA2 variants were not associated with schizophrenia diagnosis but subjects carrying T-G-G rs1128397-rs13250096-rs4567028 haplotype were more likely to respond to clozapine treatment. The statistical significance of results survived permutation testing but not Bonferroni correction. We also found nominally-significant evidence for interactions between GFRA1, 2 and 3 associated with schizophrenia and clozapine response, consistent with the locations of these three genes within linkage regions for schizophrenia.

Gas1 reduces Ret tyrosine 1062 phosphorylation and alters GDNF-mediated intracellular signaling.

The present results show that the expression of Growth Arrest Specific1 (Gas1) in SH-SY5Y neuroblastoma cells significantly inhibits the increased phosphorylation of tyrosine 1062 of the Ret receptor tyrosine kinase induced by glial-cell-line-derived neurotrophic factor (GDNF). We also observed that Gas1 significantly reduces the activation of Akt. GDNF and members of its family of ligands (GFLs), signal through a molecular complex consisting of one of its receptors (GFRalphas) and the Ret receptor tyrosine kinase. GDNF is a key component to preserve several cell populations in the nervous system, including dopaminergic and motor neurons, and also participates in the survival and differentiation of peripheral neurons such as enteric, sympathetic and parasympathetic. On the other hand, Gas1 is a molecule involved in cell arrest that can induce apoptosis when over-expressed in different cell lines, including cells of neuronal and glial origin. Although, Gas1 is widely expressed during development, its role in vivo has not yet been clearly defined. We recently showed the structural homology between Gas1 and GFRalphas, thus suggesting that the physiological role of Gas1 is that of modulating the biological responses induced by GDNF and/or other members of this family of signaling molecules. The results of this work are consistent with the hypothesis of Gas1 acting as a negative modulator of GDNF signaling.

Cultured nestin-positive cells from postnatal mouse small bowel differentiate ex vivo into neurons, glia, and smooth muscle.

Little is known about postnatal enteric nervous system (ENS) development, but some reports suggest that the postnatal bowel may contain neural stem cells. Therefore, we created an in vitro model of desegregation using an enzymatic and mechanical tissue technique. This approach yielded a group of cells from the small intestine of lactating and adult mice, which ex vivo attach to the culture dish; actively proliferate; and express nestin, vimentin, and the pro-neural transcription factors neurogenin-2 (ngn-2), Sox-10, and Mash-1. In the conditions grown, double immunostains suggest that they differentiate into various cell types, particularly neurons, smooth muscle, and glia including 04 protein-positive cells. They also express the neurotrophic-protein tyrosine kinase (Trk) receptors TrkA, TrkB, and TrkC; the low-affinity neurotrophin receptor p75NTR; and the glial-derived neurotrophic factor receptors (GFR)alpha-1, GFRalpha-2, and GFRalpha-3. The neurons expressed several sensory and motor neurotransmitters present in the central and enteric nervous systems, including calcitonin gene-related peptide, neuropeptideY, peptideYY, substance P, vasoactive intestinal polypeptide, and galanin; along with glia, these neurons formed elaborate intercellular connections. They also express c-KIT, CD34, CD20, and CD45RO, suggesting they either have a hematogenous origin or may differentiate toward hematogenous lines. These findings suggest that these cells may be enteric neural stem cells (ENSCs); may normally be present in the small intestine; and may have the capacity to proliferate and differentiate into neurons, glia, and smooth muscle. Further identification and purification of intestinal ENSCs will provide a means to study the regulation of their differentiation and should give insight into the mechanisms involved in development and remodeling of the ENS. The possible therapeutic application of postnatal stem cells such as ENSCs needs to be evaluated, including their use for transplantation in the central nervous system.