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.

Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure.

Methylation changes of CpG islands can be determined using PCR-based assays. However, the exact impact of the amount of input templates (TAIT) on DNA methylation analysis has not been previously recognized. Using COL2A1 gene as an input reference, TAIT difference between human tissues with methylation-positive and -negative detection was calculated for two representative genes GFRA1 and P16. Results revealed that TAIT in GFRA1 methylation-positive frozen samples (n = 332) was significantly higher than the methylation-negative ones (n = 44) (P < 0.001). Similar difference was found in P16 methylation analysis. The TAIT-related effect was also observed in methylation-specific PCR (MSP) and denatured high performance liquid chromatography (DHPLC) analysis. Further study showed that the minimum TAIT for a successful MethyLight PCR reaction should be ≥ 9.4 ng (CtCOL2A1 ≤ 29.3), when the cutoff value of the methylated-GFRA1 proportion for methylation-positive detection was set at 1.6%. After TAIT of the methylation non-informative frozen samples (n = 94; CtCOL2A1 > 29.3) was increased above the minimum TAIT, the methylation-positive rate increased from 72.3% to 95.7% for GFRA1 and 26.6% to 54.3% for P16, respectively (Ps < 0.001). Similar results were observed in the FFPE samples. In conclusion, TAIT critically affects results of various PCR-based DNA methylation analyses. Characterization of the minimum TAIT for target CpG islands is essential to avoid false-negative results.

A Niche for GFRα1-Positive Spermatogonia in the Terminal Segments of the Seminiferous Tubules in Hamster Testes.

In invertebrate species such as flies and nematodes, germline stem cells are maintained in a niche environment, which is restricted to the terminal end of the tubular structure in the gonads. In mice, spermatogonial stem cells (SSCs), a subpopulation of Asingle GFRα1 (glial cell line-derived neurotrophic factor [GDNF] family receptor-α1)-positive spermatogonia, are widely distributed along the longitudinal axis in the convoluted seminiferous tubules, preferentially juxtaposed to the interstitial vasculature. However, whether this area is the only SSC niche is not known. In this study, we identified a valve-like terminal segment of the seminiferous tubules, the Sertoli valve (SV), adjacent to the rete testis as another niche for GFRα1-positive spermatogonia in hamsters. Here, we show that the SV epithelium is composed of the modified Sertoli cells that are still capable of proliferation and missing most spermatogenic activities in the adult stage. The SV epithelium constitutively expresses GDNF, a major niche factor for SSCs, and supports the stable proliferation and selective maintenance of an Asingle subpopulation of GFRα1-positive spermatogonia in hamsters. The SV region of hamster seminiferous tubules has features that are similar to the stem cell niche in invertebrate gonads. Therefore, we propose that the SV may be a novel niche for Asingle GFRá1-positive spermatogonia potentially including a SSC population, at the terminal segments of the seminiferous tubules in hamsters.

Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon.

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.

Prognostic significance of the expression of GFRα1, GFRα3 and syndecan-3, proteins binding ARTEMIN, in mammary carcinoma.

BACKGROUND: Artemin (ARTN) has been implicated in promoting oncogenicity, tumor growth and invasiveness in diverse human malignancies. However, the clinical and prognostic significance of upstream ligand binding components, potentially mediating ARTN oncogenicity, largely remain to be determined. METHODS: We determined the mRNA and protein expression of three proteins demonstrated to bind ARTN, namely GFRα1, GFRα3 and syndecan-3 (SDC3), in benign breast disease and mammary carcinoma by in situ hybridization and immunohistochemistry, respectively. Their prognostic significance combined with ARTN expression was also investigated in mammary carcinoma. RESULTS: The expression of GFRα1 and GFRα3, but not SDC3, was significantly increased in mammary carcinoma and positively associated with tumor lymph node metastases, higher clinical stage and HER-2 positivity. Moreover, both GFRα1 and GFRα3 expression were significantly associated with survival outcome of patients with mammary carcinoma by univariate and multivariate analyses, whereas expression of SDC3 was not. Co-expression of ARTN with either GFRα1 or GFRα3, but not SDC3, produced synergistic increases in the odds ratio for both relapse-free and overall survival in patients with mammary carcinoma. Furthermore, significant association of GFRα1 and GFRα3 expression with survival outcome observed herein were restricted to ER negative or HER-2 negative mammary carcinoma. CONCLUSIONS: The expression of GFRα1 and/or GFRα3, especially when combined with ARTN expression, may be useful predictors of disease progression and outcome in specific subtypes of mammary carcinoma.

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.

Age affects gene expression in mouse spermatogonial stem/progenitor cells.

Spermatogenesis in man starts with spermatogonial stem cells (SSCs), and leads to the production of sperm in approximately 64 days, common to old and young men. Sperm from elderly men are functional and able to fertilize eggs and produce offspring, even though daily sperm production is more than 50% lower and damage to sperm DNA is significantly higher in older men than in those who are younger. Our hypothesis is that the SSC/spermatogonial progenitors themselves age. To test this hypothesis, we studied the gene expression profile of mouse SSC/progenitor cells at several ages using microarrays. After sequential enzyme dispersion, we purified the SSC/progenitors with immunomagnetic cell sorting using an antibody to GFRA1, a known SSC/progenitor cell marker. RNA was isolated and used for the in vitro synthesis of amplified and labeled cRNAs that were hybridized to the Affymetrix mouse genome microarrays. The experiments were repeated twice with different cell preparations, and statistically significant results are presented. Quantitative RT-PCR analysis was used to confirm the microarray results. Comparison of four age groups (6 days, 21 days, 60 days, and 8 months old) showed a number of genes that were expressed specifically in the older mice. Two of them (i.e. Icam1 and Selp) have also been shown to mark aging hematopoietic stem cells. On the other hand, the expression levels of the genes encoding the SSC markers Gfra1 and Plzf did not seem to be significantly altered by age, indicating that age affects only certain SSC/progenitor properties.

Functional RET G691S polymorphism in cutaneous malignant melanoma.

RET proto-oncogene encodes a receptor tyrosine kinase whose ligand is glial cell line-derived neurotrophic factor (GDNF), and its polymorphism at G691S juxtamembrane region (RETp) is a germline polymorphism. Cutaneous melanomas, particularly the desmoplastic subtype, are highly neurotropic; thus we sought to determine the frequency of RETp in cutaneous melanoma and its functional responsiveness to GDNF. RETp was assessed in 71 non-desmoplastic cutaneous melanomas (non-DMs) and 70 desmoplastic melanomas (DMs). Melanoma cell lines with RETp, RET wild type (RETwt), BRAF V600E mutation (BRAFmt) or BRAF wild type (BRAFwt) were assessed for functional activity. RETp frequency was significantly higher in DMs (61%) than in non-DMs (31%, P<0.001). BRAFmt was detected in only 11% of DMs. GDNF stimulation significantly amplified cell proliferation, migration and invasion in RETp, but not in RETwt melanoma cells. GDNF stimulation of RETp cell lines enhanced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt of the RET-RAS-RAF-ERK and RET-phosphatidylinositol 3-kinase (PI3K)-Akt pathways, respectively. GDNF response of RETp cells in signal transduction and other functional studies were not affected by BRAFmt. The study demonstrates that RETp is frequently found in cutaneous melanoma, particularly desmoplastic subtypes, and responds to GDNF inducing events favorable for tumor progression.

Cellular localization of GDNF and its GFRalpha1/RET receptor complex in the developing pancreas of cat.

Glial cell line-derived neurotrophic factor (GDNF) acts through RET receptor tyrosine kinase and its co-receptor GFRalpha1. In an effort to better understand the possible biological contribution of the GDNF and GFRalpha1/RET complex in pancreatic development, in this study we report the cellular localization of these proteins in the pancreas of domestic cat embryos and fetuses by immunocytochemical methods. In early embryos, GDNF, GFRalpha and RET immunoreactivity (IR) was localized in closely intermingled cells. GDNF and RET immunoreactive cells displayed chromogranin (an endocrine marker) and PGP 9.5 (a neuronal marker) IR, respectively. GFRalpha IR was present in both a few GDNF/chromogranin and RET/PGP 9.5 immunoreactive cells. In elderly fetuses, GDNF and GFRalpha IR were co-localized in glucagon cells and RET IR was detected in few neurons and never co-localized with GFRalpha or GDNF IR. In early embryos, the presence of GDNF IR in chromogranin immunoreactive cells and GFRalpha1/RET complex IR in PGP9.5 immunoreactive cells seems to suggest a paracrine action of GDNF contained in endocrine cell precursors on neuronal cell precursors expressing its receptor complex. The presence in different cell populations of RET and its co-receptor GFRalpha1 IR could be due to independent signaling of GRFalpha1. Thus, the co-presence of GDNF and GFRalpha1 in chromogranin and glucagon cells could lead to the hypothesis that GDNF can act in an autocrinal manner. In fetuses, RET IR was detected only in intrapancreatic ganglia. Because of the lack of GFRalpha1 IR in pancreatic innervation, RET receptor could be activated by other GFR alphas and ligands of GDNF family. In conclusion, these findings suggest that in differently aged embryos and fetuses the GDNF signal is differently mediated by RET and GFRalpha1.

Immunoreactivity to glial cell line-derived neurotrophic factor and its receptors in the trout pancreas: a further endocrine-exocrine relationship?

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor promoting the survival of several neuronal populations in the central, peripheral and autonomous nervous system. Outside the nervous system, GDNF functions as a morphogen in kidney development and regulates spermatogonial differentiation. GDNF exerts its roles by binding to glial cell line-derived neurotrophic factor receptor (GFR) a1, which forms a heterotetramic complex with rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor. In this study we report the presence of GDNF-, RET- and GFRa1-like immunoreactivity in the pancreas of juvenile trout. GDNF immunoreactivity was observed in the islet cells, while GFRa1- and RET- immunoreactivity was observed in the exocrine portion. These findings suggest a paracrine role of GDNF towards exocrine cells showing GDNF receptors GFRa1 and RET. The relationship could reflect physiological interactions, as previously indicated in mammalian pancreas, and/or a trophic role by endocrine cells on exocrine parenchyma, which shows a conspicuous increase during animal growth.

Glial-derived neurotrophic factor promotes ovarian primordial follicle development and cell-cell interactions during folliculogenesis.

Female fertility is determined in part by the size and development of the primordial follicle pool. The current study investigates the role of glial cell-line-derived neurotrophic factor (GDNF) in the regulation of primordial follicle development in the ovary. Ovaries from 4-day-old female rat pups were maintained in organ culture for 10 days in the absence (control) or presence of GDNF or kit ligand (KL)/stem cell factor. Ovaries treated with GDNF contained a significant increase in developing follicles, similar to that observed with KL treatment previously shown to promote follicle development. The actions of GDNF on the ovarian transcriptome were investigated with a microarray analysis. Immunohistochemical studies demonstrated that GDNF is localized to oocyte cytoplasm in follicles of all developmental stages, as well as to cumulus granulosa cells and theca cells in antral follicles. GDNF receptor alpha1 (GFRalpha1) staining was localized to oocyte cytoplasm of primordial and primary follicles, and at reduced levels in the oocytes of antral follicles. GFRalpha1 was present in mural granulosa cells of antral follicles, theca cells, and ovarian surface epithelium. The localization studies were confirmed with molecular analysis. Microarray analysis was used to identify changes in the ovarian transcriptome and further elucidate the signaling network regulating early follicle development. Observations indicate that GDNF promotes primordial follicle development and mediates autocrine and paracrine cell-cell interactions required during folliculogenesis. In contrast to the testis, ovarian GDNF is predominantly produced by germ cells (oocytes) rather than somatic cells.

Gfra1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase.

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation.

Immunolocalization of glial cell-derived neurotrophic factor (GDNF) and its receptor GFR-alpha1 in varicocele-induced rat testis.

The aim of the study was to determine the immunolocalisation of glial cell-derived neurotrophic factor (GDNF) and its receptor (GFRalpha1) in testicular dysfunction induced by experimental left varicocele. Male Wistar rats were divided randomly into two groups: a varicocele-induced group and a sham-operated group for 9, 11 and 13 weeks (each group n=6). After orchiectomy, part of the left testis from each animal was fixed, processed and embedded in paraffin wax for immunohistochemistry and the other part was fixed for ultrastructural investigations. GDNF immunoreactivity was localized in the interstitial space in Leydig cell cytoplasm and there was no significant difference (P=0.5) between the varicocele-induced groups at the various time points. GFRalpha1 localization was perinuclear in spermatids and cytoplasmic in Leydig cells. The decrease of GFRalpha1 immunoreactivity was significant (P=0.001) in varicocele-induced testis at 13 weeks when compared with the age-matched sham group. This is the first study to describe the immunolocalization patterns of GDNF and GFRalpha1 in a rat model of varicocele. Although there was no change in GDNF labelling at the different time points after varicocele, GFRalpha1 was significantly decreased in the 13-week group. Distribution of GDNF and its receptor GFRalpha1 in normal and varicocele-induced rat testes suggests both autocrine and paracrine regulation of spermatogenesis.

Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis.

Glial cell-line-derived neurotropic factor (GDNF) and its receptors glial cell-line-derived neurotropic factor alpha (GFR1alpha) and rearranged during transformation (RET) have been localized in the rat testis during postnatal development. The three mRNAs, and GDNF and GFR1alpha proteins were detected in testis extracts from 1- to 90-day-old rats by reverse transcriptase PCR and Western blotting respectively. The three mRNAs were present in Sertoli cells from 20- and 55-day-old rats, pachytene spermatocytes (PS), and round spermatids (RS). The GDNF and GFR1alpha proteins were detected in PS, RS, and Sertoli cells. GDNF and GFR1alpha were also detected using flow cytometry in spermatogonia and preleptotene spermatocytes, and in secondary spermatocytes. The localization of GDNF and GFR1alpha in germ and Sertoli cells was confirmed by immunocytochemistry. The hypothesis that GDNF may control DNA synthesis of Sertoli cells and/or spermatogonia in the immature rat was addressed using cultures of seminiferous tubules from 7- to 8-day-old rats. Addition of GDNF for 48 h resulted in a twofold decrease in the percentage of spermatogonia able to duplicate DNA, whereas Sertoli cells were not affected. These results are consistent with a role of GDNF in inhibiting the S-phase entrance of a large subset of differentiated type A spermatogonia, together with an enhancing effect of the factor on a small population of undifferentiated (stem cells) spermatogonia. Moreover, the wide temporal and spatial expression of GDNF and its receptors in the rat testis suggest that it might act at several stages of spermatogenesis.

[Neurotrophic factors of the GDNF family and their receptors are detectable in spiral ganglion cells of normal hearing as well as of deafened rats]. / Neurotrophe Faktoren der GDNF-Familie und ihre Rezeptoren lassen sich in Spiralganglienzellen hörender, aber auch ertaubter Ratten nachweisen.

BACKGROUND: Recent studies have shown that neurotrophic factors like BDNF, NT-3 and GDNF induce protective effects on spiral ganglion cells after noise- or drug-induced hearing loss. According to these studies it is suggested that deafness leads to a lack of neurotrophic factor or relating receptor expression in spiral ganglion cells, that has to be compensated by local cochlear application of these factors. METHODS: In the present study we examined the expression pattern of members of the GDNF family (GDNF, Neurturin, Artemin, Persephin) and their relating receptors (Ret, GFRalpha1 - 3) as well as BDNF and trkB on spiral ganglion cells of normal hearing and experimentally deafened rats (10 % neomycine). Indirect immunofluorescence was carried out to determine protein expression of these factors and their receptors 26 days following deafening. RESULTS: Our results demonstrate neurotrophic factor and receptor expression on spiral ganglion cells of normal hearing as well as experimentally deafened animals. CONCLUSIONS: Our data indicate that within a period of 26 days after deafening no detectable reduction of the GDNF-family member expression and their receptors was ascertainable on spiral ganglion cells by immunohistochemistry. Thus, a lack of neurotrophic factor expression is unlikely to be the only cause of spiral ganglion cell loss following deafening.

Immunocytochemical evidence that most sensory neurons of the rat molar pulp express receptors for both glial cell line-derived neurotrophic factor and nerve growth factor.

Most pulpal afferent neurons have cytochemical features in common with the class of nociceptors that express neuropeptides and respond to NGF, while very few bind the plant lectin IB4, a widely used marker for the class of nociceptors that respond to the GDNF family of neurotrophic factors. The present study was undertaken to determine whether the GDNF receptor, GFRalpha-1, is expressed by pulpal afferents, and, further, to determine whether tooth injury evokes changes in expression of the GDNF and NGF receptors among pulpal afferents. The tracer, fluoro-gold (FG), was applied to shallow cavities in dentin of first and second maxillary molars. After 4 weeks, the molars of one side received a test injury consisting of a deeper cavity that exposed pulp horns. Animals were perfusion fixed 2 days later, and sections of the trigeminal ganglia were double immunostained with combinations of antibodies against GFRalpha-1, and TrkA. Under control conditions, GFRalpha-1 immunostaining was observed in 72% of neurons that projected to the molar pulp, TrkA in 78%, and immunostaining for both receptors was observed in 65% of pulpal afferents. Tooth injury evoked up-regulation of GFRalpha-1 expression (to 93%) and a slight down-regulation of TrkA expression (67%) among tooth afferents, while there was no discernable change in the proportion of pulpal afferents that expressed both TrkA and GFRalpha-1 (to 61%).