Formation of organotypic testicular organoids in microwell culture†

Abstract

Three-dimensional (3D) organoids can serve as an in vitro platform to study cell–cell interactions, tissue development, and toxicology. Development of organoids with tissue architecture similar to testis in vivo has remained a challenge. Here, we present a microwell aggregation approach to establish multicellular 3D testicular organoids from pig, mouse, macaque, and human. The organoids consist of germ cells, Sertoli cells, Leydig cells, and peritubular myoid cells forming a distinct seminiferous epithelium and interstitial compartment separated by a basement membrane. Sertoli cells in the organoids express tight junction proteins claudin 11 and occludin. Germ cells in organoids showed an attenuated response to retinoic acid compared to germ cells in 2D culture indicating that the tissue architecture of the organoid modulates response to retinoic acid similar to in vivo. Germ cells maintaining physiological cell–cell interactions in organoids also had lower levels of autophagy indicating lower levels of cellular stress. When organoids were treated with mono(2-ethylhexyl) phthalate (MEHP), levels of germ cell autophagy increased in a dose-dependent manner, indicating the utility of the organoids for toxicity screening. Ablation of primary cilia on testicular somatic cells inhibited the formation of organoids demonstrating an application to screen for factors affecting testicular morphogenesis. Organoids can be generated from cryopreserved testis cells and preserved by vitrification. Taken together, the testicular organoid system recapitulates the 3D organization of the mammalian testis and provides an in vitro platform for studying germ cell function, testicular development, and drug toxicity in a cellular context representative of the testis in vivo.

Primary cilia: biosensors of the male reproductive tract

“…Two types of cilia are found in the testis: primary cilia and solitary motile cilia (the sperm flagellum). In the immature testis, primary cilia are present in somatic cells, where they have been shown to contribute to ex vivo testicular tubule formation through Hh signaling pathway (Ou et al ., ; Nygaard et al ., ; Dores et al ., ; Sakib et al ., ). In addition, Ponzio et al .…”

“…Furthermore, since immature TM3 Leydig cells are responsive to Hh agonists in vitro through the recruitment of Smo to the cilium, and mature Leydig cells lose their primary cilium as they differentiate, this suggests a potential role for primary cilia in testicular cell differentiation via the Hh signaling pathway. This hypothesis is further supported by the impaired ability of neonatal testicular cells from pig to form testicular‐like tubules and spheroid structures in vitro following primary cilia disruption with ciliobrevin D dynein inhibitor, and the loss of Gli‐2 and Smo Hh signaling factors (Dores et al ., ; Sakib et al ., ).…”

Three-dimensional testicular organoids as novel in vitro models of testicular biology and toxicology

“…1). The resulting organoids have a tissue architecture that is similar to testis in vivo [89]. These organoids have a clearly delineated exterior (seminiferous epithelium) and interior compartment (interstitial) separated by the basement membrane.…”

“…Environmental toxicants can also trigger autophagy [93, 96] and autophagy as a biomarker for toxicity within the male reproductive tract has been described [97–99]. Exposure to increasing doses of di-(2-ethylhexyl) phthalate, a commonly used plasticizer, induced an increase in the number of autophagosomes in germ cells in a dose-dependent manner in 2D culture [99] and this observation could be replicated in organoids [89]. Similarly, exposure of cells to a small molecule inhibitor of primary cilia led to a loss of morphogenic capacity.…”

“…Similarly, exposure of cells to a small molecule inhibitor of primary cilia led to a loss of morphogenic capacity. These initial observations point towards the utility of testicular organoid systems for screening the effects of drugs and toxins on morphogenesis and cell function [89].…”