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Sox2 (SRY box2) is an essential transcription factor that plays a vital role in spermatogenesis and regulates the genes in this process. Sox2 is important for pluripotency, self-renewal, and even spermatogonial stem cell differentiation. This gene is found in pluripotent and specialized cells, and it is involved in their biological activities.

Protein-protein interaction (PPI) network analysis was performed during spermatogenesis using NCBI, STRING, and Cytoscape databases. Then, after isolating spermatogonial stem cells from 6 C57BL/6 mice, mouse embryonic stem cells and ES-like cells were prepared. In the following, Sox2 expression was examined in differentiated and undifferentiated spermatogonia by immunohistochemistry (IMH), immunocytochemistry (ICC), and Fluidigm PCR (polymerase chain reaction). Finally, the results were compared using the Kruskal-Wallis and Dunn tests at the significance level of p<0.05.

The results of this experiment showed that contrary to expectations, Sox2 has cytoplasmic expression in undifferentiated cells and nuclear expression in differentiated cells in in vitro conditions. In addition, the expression of Sox2 increased during differentiation. Fluidigm PCR showed a significantly higher expression of Sox2 (p<0.05) in differentiated compared to undifferentiated spermatogonia. Sox2 has an interaction with other genes during spermatogenesis such as Oct4, Nanog, Klf4, Stra8, Smad1, Tcf3, and Osm.

Sox2, which is known as a pluripotency marker, has a vital role in spermatogenesis and could be a differential marker. Sox2 has strong connections with other genes such as Oct4, Nanog, Klf4, Tcf3, Osm, Stra8, Lim2, Smad1, Gdnf, and Kit.

In mammals, spermatogenesis is the main process for male fertility that is initiated by spermatogonial stem cells (SSCs) proliferation. SSCs are unipotent progenitor cells accountable for transferring the genetic information to the following generation by differentiating to haploid cells during spermato-and spermiogenesis. DEAD-box helicase 4 (DDX4) is a specific germ cell marker and its expression pattern is localized to, spermatocytes, and spermatids. The expression in the SSCs on the basement membrane of the seminiferous tubules is low.

Immunohistochemistry (IHC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze the expression of DDX4 in testis tissue of fertile and sterile mice and human cases with non-obstructive azoospermia.

Our immunohistochemical findings of fertile and busulfan-treated mice showed expression of DDX4 in the basal and luminal compartment of seminiferous tubules of fertile mice whereas no expression was detected in busulfan-treated mice. The immunohistochemical analysis of two human cases with different levels of nonobstructive azoospermia revealed more luminal DDX4 positive cells.

Our findings indicate that DDX4 might be a valuable germ cell marker for analyzing the pathology of germ cell tumors and infertility as global urological problems.

The glial cell-derived neurotrophic factor (GDNF) family plays essential roles in the maintenance,growth, regulatory and signalling pathways of spermatogonial stem cells (SSCs). In this study, we analysed the expressionof anti-GDNF family receptor alpha 1 antibody (GFRa1) by immunohistochemistry (IHC), immunocytochemistry(ICC), Fluidigm real-time polymerase chain reaction (RT-PCR) and flow cytometry analyses. In this experiment study, ICC, IHC, Fluidigm RT-PCR and flow cytometry were used toanalyse the expression of the germ cell marker GFRa1 in testis tissue and SSC culture. IHC analysis showed that there were two groups of GFRa1 positive cells in the seminiferous tubules basedon their location and expression shape - a small round punctuated shape on the basal compartment donut shape anda C-shaped expression located between the basal and the luminal compartments of the seminiferous tubules. OCT4and PLZF positive cells may have similar patterns of expression as the first group. Assessment of the seminiferoustubule sections demonstrated that about 27% of the SSCs were positive for GFRa1. Fluidigm RT-PCR confirmed thesignificant expression (p <0.001) of GFRa1 in the SSCs compared to testicular stromal cells (TSCs). Flow cytometryanalysis demonstrated that about 75% of the isolated SSCs colonies were positive for GFRa1. The results indicated that GFRa1 had a specific expression pattern both In Vivo and In Vitro . This finding could be helpful for understanding the proliferation, maintenance and signalling pathways of SSCs, and differentiation
of meiotic and haploid germ cells.

We aimed to examine the expression levels of the VASA gene and protein in testis sections of neonate and adult mice as well as testicular cell cultures.

In this experimental study, in order to investigate the expression of this germ cell marker gene in more detail, we analyzed the expression of VASA by immunocytochemistry, immunohistochemistry and fluidigm reverse transcription-polymerase chain reaction (RT-PCR).

The immunohistochemical assays showed that the VASA protein was exclusively expressed in germ cells in the seminiferous tubules of the neonate and adult testis and not in somatic cells. VASA was not detectable in PLZF positive spermatogonial stem cells (SSCs), was weakly expressed in proliferating spermatogonia, and became abundant in spermatocytes and round spermatozoa. Counting VASA-positive cells in the seminiferous tubules of the neonate and adult testis depicted significant higher expression (P<0.05) of VASA in the adult testis in comparison to its neonate counterpart. SSC colonies were established in vitro after digestion of the testis and characterized by immunocytochemistry for CD90 and stage-specific embryonic antigens 3 (SSEA3). Immunocytochemistry confirmed that in contrast to the not detectable signal in vivo, VASA protein was strongly localized in the cytoplasm of both neonate and adult mouse SSCs under in vitro conditions. The results of Fluidigm RT-PCR revealed a significant higher expression of the germ cell gene VASA in adult SSCs in comparison to neonate SSCs in cell culture (P<0.05).

The VASA protein is, therefore, an extremely specific marker of testicular germ cell differentiation in vivo and mostly expressed in the adult testis in spermatocytes and round spermatids. The immunohistochemical signal in spermatogonia is very low. So, PLZF positive SSCs are negative for VASA in vivo, while in contrast, once isolated from the testicular niche VASA is also strongly expressed in SSCs under in vitro conditions.

Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis.

In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR).

IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in the sections of seminiferous tubules of adult and neonate testis revealed significant expression of positive cells in adult testis compared to the neonate (P<0.05). Under in vitro conditions, isolated SSC colonies were strongly ICC-positive for the PLZF germ cell marker, while ES cells and ES-like cells were negative for PLZF. Fluidigm Real-Time-PCR analysis demonstrated a significant expression of the PLZF germ cell gene in the neonate and adult SSCs, compared to ES cells and ES-like cells (P<0.05).

These results indicate that PLZF is a specific transcription factor of testicular germ cell proliferation, but it is downregulated in pluripotent germ cells. This can be supportive for the analysis of germ cells development both in vitro and in vivo.

During the cultivation of spermatogonial stem cells (SSCs) and their conversion into embryonic stem-like (ES-like) cells, transitional ES-like colonies and epiblast-like cells were observable. In the present experimental study, we aimed to analyze the efficiency of the multipotency or pluripotency potential of ES-like cells, transitional colonies and epiblast-like cells. In this experimental study, SSCs were isolated from transgenic octamer-binding transcription factor 4 (Oct4)-green fluorescent protein (GFP)-reporter mice. During cell culture ES-like, transitional and epiblast- like colonies developed spontaneously. The mRNA and protein expression of pluripotency markers were analyzed by Fluidigm real-time polymerase chain reaction (RT-PCR) and immunocytochemistry, respectively. Efficiency to produce chimera mice was evaluated after injection of ES and ES-like cells into blastocysts. Microscopic analyses demonstrated that the expression of Oct4-GFP in ES-like cells was very strong, in epiblast-like cells was not detectable, and was only partial in transitional colonies. Fluidigm RT-PCR showed a higher expression of the germ cell markers Stra-8 and Gpr-125 in ES-like cells and the pluripotency genes Dppa5, Lin28, Klf4, Gdf3 and Tdgf1 in ES-like colonies and embryonic stem cells (ESCs) compared to the epiblast-like and transitional colonies. No significant expression of Oct-4, Nanog, Sox2 and c-Myc was observed in the different groups. We showed a high expression level of Nanog and Klf4 in ES-like, while only a partial expression was observed in transitional colonies. We generated chimeric mice after blastocystic injection from ES and ES-like cells, but not from transitional colonies. We observed that the efficiency to produce chimeric mice in ES cells was more efficient (59%) in comparison to ES-like cells (22%). This new data provides more information on the pluripotency or multipotency potentials of testis-derived ES-like cells in comparison to transitional colonies and epiblast-like cells.

Spermatogonial stem cells (SSCs) provide the cellular basis for sperm production transforming the male’s genetic information to the next generation. We aimed to examine the effect of different feeder layer on proliferation of SSCs. In this experimental study, we compared the in vitro effects of the co-culture of mouse SSCs with mouse embryonic fibroblasts (MEFs), sandos inbred mice (SIM) embryo-derived thioguanine- and ouabain- resistant (STO) feeders, and neonate and adult testicular stroma cell (TSC) feeders on the efficiency of mouse SSC proliferation and colony formation. Cells were cultivated on top of MEFs, STO, and neonate and adult TSCs feeder layers for 30 days. The number and diameter of colonies and also the number of cells were evaluated during day 7, 15, 25, and 30 of culture. The mRNA expression of germ cells and somatic cells were analyzed. In our study, we observed a significant difference in the proliferation rates and colony size of SSCs among the groups, especially for MEFs (P<0.05). SSCs can proliferate on MEFS, but not on STO, neonate or adult TSCs. Using immunocytochemistry by KI67 the proliferative activities of SSC colonies on MEFs were confirmed. The results of Fluidigm real-time polymerase chain reaction (RT-PCR) showed a high expression of the germ cell genes the promyelocytic leukemia zinc finger protein (PLZF), deleted in azoospermia-like (DAZL), octamer-binding transcription factor 4 (OCT4), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4 or VASA) in SSCs, and a low expression of these genes in the feeder layers. Furthermore, we observed a higher expression of vimentin and integrin-B1 in feeder layers than in SSCs (P<0.05). Based on the optimal effect of MEFs for better colonization of SSCs, these feeder cells seem to be appropriate candidates for SSC cultures prior to transplantation. Therefore, it is suggested using these feeder cells for SSC cultivation.

The properties of self-renewal and divisionin spermatogonial stem cells (SSCs) support spermatogenesis. There are a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and self-renewal ofgermline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research andas potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system.
In this system, testicular cells from neonate mice have been cultured in agarose coated plates in the presence of the DMEM (CTRL group) medium, 10% FBS (10% group) and growth factor (G group), containing 2% FBS, GDNF, EGF, and FGF. Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, RT-PCR and flow cytometry against the germ cell markers (α6, β1, c-Kit, Thy-1, C-Ret, Plzf and Oct4). The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA).
Spermatogonial stem-like colonies were established in both G and 10% groups, but not in the CTRL group. Immunocytochemistry, flow cytometry and RT-PCR confirmed the expressions of germ cells markers in these spermatogonial stem-like cells. In the spermatogonial stem-like cells, we observed a significant expression (p According to these results,a non-adherent culture system could provide a favorable methodfor in vitro short term culture of spermatogonial stem-like cell colonies.