Organotypic Rat Testicular Organoids for the Study of Testicular Maturation and Toxicology

Abstract

An in vitro system to study testicular maturation in rats, an important model organism for reproductive toxicity, could serve as a platform for high-throughput drug and toxicity screening in a tissue specific context. In vitro maturation of somatic cells and spermatogonia in organ culture systems has been reported. However, this has been a challenge for organoids derived from dissociated testicular cells. Here, we report generation and maintenance of rat testicular organoids in microwell culture for 28 days. We find that rat organoids can be maintained in vitro only at lower than ambient O2 tension of 15% and organoids cultured at 34°C have higher somatic cell maturation and spermatogonial differentiation potential compared to cultures in 37°C. Upon exposure to known toxicants, phthalic acid mono-2-ethylhexyl ester and cadmium chloride, the organoids displayed loss of tight-junction protein Claudin 11 and altered transcription levels of somatic cell markers that are consistent with previous reports in animal models. Therefore, the microwell-derived rat testicular organoids described here can serve as a novel platform for the study of testicular cell maturation and reproductive toxicity in vitro.

Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations

“…In the present study, bovine Leydig, Sertoli and peritubular myoid cells were isolated from testes and cultured separately, mixed at a concentration of 1 × 10 6 cells/mL, cultured for 7 days and then transferred to Matrigel for further 3D organization. This protocol differs from those commonly used in 3D testicular models, where testicular cells are obtained through enzymatic digestion and the remotion of connective tissue [ 6 , 8 , 30 , 32 , 34 , 48 ]. These cell isolation protocols may unintentionally provide other cell types to the testicular cell mix, including endothelial-like or uncharacterized cell types that may interfere with further experiments [ 8 , 30 , 31 , 32 , 49 ].…”

“…The use of Matrigel during culture increases the area where cells can reorganize and permits the formation of larger TOs. Despite the fact that the comparison of diameters and areas of TOs among studies is difficult due to the lack of measurements reported, previous studies have indicated diameters between 100 and 600 μm and average areas around 2966–4628 μm 2 [ 7 , 33 , 34 , 50 , 59 ]. In our study, TOs presented diameters that ranged between 555.15 μm and 1272.88 μm.…”

“…In the present study, COL1A + peritubular myoid cells were located in the central area at day 3 and randomly distributed with lower intensity from days 7 to 28 of culture. The main marker used for the detection of peritubular myoid cells is α-SMA; however, COL1A is usually used as a complement for the detection of these cells [ 8 , 30 , 31 , 32 , 33 , 34 , 40 , 58 , 59 , 69 ]. However, some reports have used only COL1A as a marker for the detection of peritubular myoid cells in TOs [ 70 ].…”

“…Despite the exponential increase in the number of reports on organoid derivation for in vitro studies, limited information has been published on TOs. To date, TOs have been mainly reported in humans, rodents and some domestic animal species including pigs and monkeys [ 31 , 32 , 33 , 34 ]. Nevertheless, to the best of our knowledge, currently there are no reports of TOs in the bovine species.…”

Effect of Temperature on the Development of Stages of Spermatogenesis and the Functionality of Sertoli Cells In Vitro

“…Furthermore, recent studies showed that somatic cell maturation and spermatogonial potential differentiation of rat testicular organoids were higher when incubated at 34 • C than when incubated at 37 • C [19]. Additional studies using in vitro culture models of spermatogonial stem cells (SSC) have demonstrated that high temperatures (37 • C or 43 • C) significantly inhibited SSC differentiation through P53, ribosomes and carbon metabolism signaling pathways, changing their transcriptome [20].…”