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Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

A new type of mutation in the lrp gene of Salmonella typhimurium which paves the way for D-histidine utilization is described. This mutation was found to exert a suppressor effect on other mutations in genes coding for histidine permease. However, suppression was not found to occur through an increase in permease level. Instead, D-histidine utilization takes place in suppressor mutants through higher racemization levels.

Image analysis of subcellular structures and biological processes relies on specific, context-dependent pipelines, which are labor-intensive, constrained by the intricacies of the specific biological system, and inaccessible to broader applications. Here we introduce the application of dispersion indices, a statistical tool traditionally employed by economists, to analyze the spatial distribution and heterogeneity of subcellular structures. This computationally efficient high-throughput approach, termed GRID (Generalized Readout of Image Dispersion), is highly generalizable, compatible with open-source image analysis software, and adaptable to diverse biological scenarios. GRID readily quantifies diverse structures and processes to include autophagic puncta, mitochondrial clustering, and microtubule dynamics. Further, GRID is versatile, applicable to both 2D cell cultures and 3D multicellular organoids, and suitable for high-throughput screening and performance metric measurements, such as half-maximal effective concentration (EC50) values. The approach enables mechanistic analysis of critical subcellular structure processes of relevance for diseases ranging from metabolic and neuronal diseases to cancer as well as a first-pass screening method for identifying biologically active agents for drug discovery., Statement of Significance: Current methods for image analysis in microscopy are tailored to specific biological contexts, which creates challenges in implementation and efficiency for researchers studying a diverse range of subcellular processes. Our application of dispersion indices, traditionally used in economics, offers a universal framework for high throughput quantification of biological structures, enabling easier and more consistent analysis across various biological contexts. By transforming pixel intensity and count into meaningful statistical measures, our method simplifies the quantification of subcellular structures such as autophagic puncta, microtubule dynamics, and mitochondrial clustering. This approach accelerates the quantitative analysis of sub-cellular processes in disease as well as imaged-based drug discovery. Classification: Bioengineering, Cell Biology, Applied Biological Sciences.