RESUMEN
While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2-A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.
Asunto(s)
Animales , Masculino , Ratones , Diferenciación Celular , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Inmunohistoquímica , Ratones Endogámicos C57BL , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genética , Proteínas Proto-Oncogénicas c-kit/genética , Túbulos Seminíferos/citología , Espermatogénesis , Espermatogonias/metabolismo , Testículo/citología , Fijación del Tejido , Factores de Transcripción/genéticaRESUMEN
Background: Acute myeloblastic leukemia [AML] is a clonal disorder due to bone marrow failure and uncontrolled proliferation of myeloid lineage. Acute promyelocytic leukemia [APL] is a subtype of AML. Heterocyclic compounds, such as indole, are considered as attractive candidates for cancer therapy, due to their abundance in nature and known biological activity. Sal-like protein [SALL4] is a zinc finger transcription factor involving in the multi-potency of stem cells, in the NB4 cell line. This study was aimed to evaluate the effects of basal indole and its new derivative, 2-[1-[[2, 4-Aril]imino]-2,2,2-trifluoroethyl] phenyl-1H Indole-3- carbaldehyde [TFPHC], on the expression of SALL4
Methods: Cells were cultured and treated with different concentrations [75, 150, and 300 micro g/mL] of the new indole derivative and DMSO, as a vehicle control, for 24 and 48 hours. Cell proliferation was evaluated by using Trypan blue exclusion and MTT assays. The percentage of apoptotic cells was determined by flowcytometry analysis using the Annexin V/PI apoptosis detection kit; mRNA expression of SALL4 was studied using absolute quantitative RT-PCR
Results: Our findings demonstrated the effects of new indole derivatives on SALL4 mRNA expression. Expression of SALL4 mRNA was significantly decreased at 75, 150, and 300 micro g/mL concentrations
Conclusion: SALL4 plays a role in the survival of APL cells. SALL4 expression could be suppressed by the novel indole derivative. Additionally, SALL4 gene suppression can serve as a target in APL therapy