A key step in studying the biology of spermatogonia is to determine their global gene expression profile. However, disassociation of these cells from the testis may alter their profile to a considerable degree. To characterize the molecular phenotype of human spermatogonia, including spermatogonial stem cells (SSCs), within their cognate microenvironment, a rare subtype of human defective spermatogenesis was exploited in which spermatogonia were the only germ cell type. The global expression profile of these samples was assessed on the Affymetrix microarray platform and compared with tissues showing homogeneous Sertoli-cell-only appearance; selected genes were validated by quantitative real-time PCR and immunohistochemistry on disparate sample sets. Highly significant differences in gene expression levels correlated with the appearance of spermatogonia, including 239 best candidates of human spermatogonially expressed genes. Specifically, fibroblast growth factor receptor 3 (FGFR3), desmoglein 2 (DSG2), E3 ubiquitin ligase c-CBL (casitas B-cell lymphoma), cancer/testis antigen NY-ESO-1 (CTAG1A/B), undifferentiated embryonic cell transcription factor 1 (UTF1) and synaptosomal-associated protein, 91 kDa homolog (SNAP91) were shown to represent specific biomarkers of human spermatogonia. These biomarkers, specifically the surface markers FGFR3 and DSG2, may facilitate the isolation and enrichment of human stem and/or progenitor spermatogonia and thus lay a foundation for studies of long-term maintenance of human SSCs/progenitor cells, spermatogonial self-renewal, clonal expansion and differentiation.

It is unclear whether the distinct nuclear morphologies of human A_dark (Ad) and A_pale (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear ‘vacuolar spaces’ representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex- and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing. Abstract It is unclear whether the distinct nuclear morphologies of human A_dark (Ad) and A_pale (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear ‘vacuolar spaces’ representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex- and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing.

In this review, we provide an up-to-date compilation of published human spermatogonial markers, with focus on the three nuclear subtypes Adark, Apale and B. In addition, we have extended our recently published list of putative spermatogonial markers with protein expression and RNA-sequencing data from the Human Protein Atlas and supported these by literature evidence. Most importantly, we have put substantial effort in acquiring a comprehensive list of new and potentially interesting markers by refiltering the raw data of 15 published germ cell expression datasets (four human, eleven rodent) and subsequent building of intersections to acquire a robust, cross-species set of spermatogonia-enriched or -specific transcripts.

Mutations in the cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), serine protease inhibitor Kazal type 1 (SPINK1), and chymotrypsin C (CTRC) genes are associated with an elevated risk for chronic pancreatitis, which is a known risk factor for pancreatic cancer (PC). Therefore, we analyzed whether PRSS1, CFTR, SPINK1, and/or CTRC mutations are associated with pancreatic adenocarcinoma. The study cohort was composed of 121 PC patients, of whom 74 were classified as having chronic pancreatitis, 102 patients with idiopathic chronic pancreatitis, and 130 as healthy controls. Mutation analyses for the CFTR, SPINK1, PRSS1, and CTRC genes were performed for the presence of the most common mutations. The frequency of CFTR mutations in patients with PC was not significantly different in comparison with healthy controls and controls with pancreatitis. The SPINK1 mutation frequency was significantly decreased in patients with PC in comparison with patients with idiopathic pancreatitis but varied not significantly in comparison with healthy controls. None of the selected 121 PC samples showed a pancreatitis-predisposing mutation in the PRSS1 or CTRC gene. Mutations in the genes CFTR, SPINK1, PRSS1, and CTRC do not seem to significantly increase the risk for pancreatic adenocarcinoma.

Is it possible to induce in vitro reorganization of primary human testis cells from testicular sperm extraction (TESE) biopsies, maintain their long-term cultivation in a 2D system and identify cellular compositions? In vitro reorganization of primary human testis cells from TESE biopsies and their long-term cultivation on uncoated cell culture dishes is feasible and the cellular compositions can be uncovered through gene expression and microscopic analyses. It has been shown in the rodent model that mixtures of testicular cell types are able to reassemble into clusters when cultivated on different kinds of surfaces or three-dimensional matrices. Two recent publications demonstrated the ability of primary human testicular cells to assemble into testicular organoids and their cultivation for a period of 3–4 weeks. Primary human testis cells from TESE biopsies from 16 patients were reorganized in vitro and the clusters were cultivated long term on uncoated cell culture dishes, providing a solid ground for in vitro spermatogenesis. Gene expression analysis as well as fluorescence/transmission electron microscopy (TEM) were employed to uncover the cellular composition of the clusters. Testis biopsies from adult, normogonadotropic patients displaying full spermatogenesis (n = 11), hypospermatogenesis (n = 2), predominantly full spermatogenesis with some hypospermatogenic tubules (n = 1), meiotic arrest (n = 1) or mixed atrophy (n = 1) were enzymatically digested and dispersed cells were cultivated on 96-well plates or chamber dishes as aggregate-free cell suspensions. Time-lapse imaging of cluster formation was performed over a period of 48 h. For receptor tyrosine kinase inhibition of cluster formation, cells were treated twice with K252a within 2–3 days. Immunofluorescence staining and confocal microscopy was carried out on clusters after 1–3 weeks of cultivation to identify the presence of Sertoli cells (SC) (SOX9), peritubular myoid cells (SMA), Leydig cells (LC) (STAR), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Single clusters from four patients and a pool of eight larger clusters from another patient were manually picked and subjected to quantitative real-time PCR to evaluate the presence of SC (SOX9, AR), LC (INSL3, STAR, HSD3B1), peritubular myoid cells (ACTA2), fibroblasts (FSP1), endothelial cells (CD34), macrophages (CD68), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Finally, an ultrastructural investigation was conducted based on TEM of clusters from six different patients, among them 3-month cultivated large clusters from two patients. Quantitative PCR-based analysis of single-picked testicular cell clusters identified SC, peritubular myoid cells, endothelial cells, fibroblasts, macrophages, spermatids and LC after 1, 2 or 3 weeks or 3 months of cultivation. Immunofluorescence positivity for SC and peritubular myoid cells corroborated the presence of these two kinds of testis niche cells. In addition, round as well as elongated spermatids were frequently encountered in 1 and 2 weeks old clusters. Transmission electron microscopical classification confirmed all these cell types together with a few spermatogonia. Macrophages were found to be of the proinflammatory M1 subtype, as revealed by CD68⁺/CD163⁻/IL6⁺ expression. Time-lapse imaging uncovered the specific dynamics of cluster fusion and enlargement, which could be prevented by addition of protein kinase inhibitor K252a. N/A. Cell composition of the clusters varied based on the spermatogenic state of the TESE patient. Although spermatids could be observed with all applied methods, spermatogonia were only detected by TEM in single cases. Hence, a direct maintenance of these germ cell types by our system in its current state cannot be postulated. Moreover, putative dedifferentiation and malignant degeneration of cells in long-term cluster cultivation needs to be investigated in the future. This work demonstrates that the reorganization of testicular cells can be achieved with TESE biopsies obtained from men enroled in a standard clinical assisted reproduction program. The formed clusters can be cultivated for at least 3 months and are composed, to a large extent, of the most important somatic cell types that are essential to support spermatogenesis. These findings may provide the cellular basis for advances in human in vitro spermatogenesis and/or the possibility for propagation of spermatogonia within a natural stem cell niche-like environment. The project was funded by a DFG grant to K.v.K. (KO 4769/2-1). The authors declare they have no conflicts of interest.