Spermatogenesis is a well planned process that relies on a strictly controlled stem cell niche. Spermatogonia and somatic cells such as Sertoli cells, peritubular myoid cells, and Leydig cells make up most of this spermatogonial stem cell niche. These somatic cells exert strong control on the destiny of spermatogonial cells. To better understand developmental processes, including spermatogonial differentiation and reproductive toxicity in vitro, a three-dimensional organotypic culture method can fill the gap between cell cultures and whole animal models. With this objective in mind, several three-dimensional testicular organoid systems have been proposed. In a new study published in Frontiers in Endocrinology, Canadian scientists from the University of Calgary: Dr. Sadman Sakib, Dr. Nathalia Lara, Brandon Huynh, and led by Professor Ina Dobrinski recently described the creation of robust and complex in vitro models which better mimic in vivo conditions and allow researchers to address more challenging scientific questions. The new technique allow the adaptation of microwell-derived testicular organoids to rat testicular organoids and highlighted the species-specific challenges encountered during this endeavour. The new developed rat testicular organoids were shown to be a useful tool for studying reproductive toxicity who provided proof of concept using substances that are known to be environmentally harmful.
The research team demonstrated that rat organoids can only be maintained in vitro at a 15% lower O2 tension than ambient, and that organoids cultured at 34°C have a higher capacity for somatic cell maturation and spermatogonial differentiation than those grown at 37°C. The authors fully characterized the organoid model and showed that sex hormone-binding globulin (Shbg) was highly expressed, Early meiotic cells were presented, and cells with an elongated SYCP3 staining pattern were presented at 34°C culture conditions, all of which point to the advantage on Sertoli cell growth and spermatogonial division. Although zygotene spermatocytes, which are distinguished by the characteristic elongated staining pattern of SYCP3, were no longer adherent to the organoids, the quantity of germ cells dropped over the course of the culture. Numerous media changes made over the course of the 28-day culture are probably to blame for the gradual loss or dislodging of weakly attached germ cells.
The authors conducted a proof of concept study to assess the utility of rat organoids for toxicological evaluation of medicines and environmental toxicants after determining the best conditions for fostering development and early spermatogonial differentiation. Fshr and Star were expressed more when exposed to MEHP, but Cyp17a1 was expressed less. Through the overexpression of Star and the downregulation of Fshr, Shbg, Hsd17b3 and Cyp17a1, they saw the cadmium-mediated disruption of Sertoli and Leydig cell function. The steroidogenic machinery is clearly disrupted by the level of concentrations tested. This proof-of-concept study demonstrates the viability of using rat testicular organoids as platforms for simulating male reproductive toxicity.
In summary, Professor Ina Dobrinski and her research group described the rat testicular organoid system that can promote early testicular maturation and reflects the unique morphology of the testis. Additionally, this system aids in the development of germ cells through early meiosis and to the zygotene stage. To support complete in vitro spermatogenesis, additional tweaking of the differentiation conditions may be necessary. As a result, the rat testicular organoids made from microwells described here can be used as a cutting-edge platform for the in vitro investigation of testicular cell maturation and reproductive toxicity.
Sadman Sakib, Nathalia de Lima e Martins Lara, Brandon Christopher Huynh and Ina Dobrinski. Organotypic rat testicular organoids for the study of testicular maturation and toxicology. Frontiers in endocrinology. 2022;13.