Roles of CD34+ cells and ALK5 signaling in the reconstruction of seminiferous tubule-like structures in 3-D re-aggregate culture of dissociated cells from neonatal mouse testes.

Shin-Ichi Abe,Haruhiko Akiyama,Tomoaki Harada,Kazuko Abe,Hiroki Oshikawa,Kenji Shimamura,Go Mizumoto,Jidong Zhang

doi:10.1371/journal.pone.0188705

Shin-Ichi Abe, Haruhiko Akiyama + Show 6 more

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https://doi.org/10.1371/journal.pone.0188705

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Abstract

Tissue reconstruction in vitro can provide, if successful, a refined and simple system to analyze the underlying mechanisms that drive the morphogenesis and maintain the ordered structure. We have recently succeeded in reconstruction of seminiferous cord-like and tubule-like structures using 3-D re-aggregate culture of dissociated testicular cells. In testis formation, endothelial cells that migrated from mesonephroi to embryonic gonads have been shown to be critical for development of testis cords, but how endothelial cells contribute to testis cord formation remains unknown. To decipher the roles of endothelial and peritubular cells in the reconstruction of cord-like and tubule-like structures, we investigated the behavior of CD34+ endothelial and p75+ cells, and peritubular myoid cells (PTMCs) in 3-D re-aggregate cultures of testicular cells. The results showed that these 3 types of cells had the capacity of re-aggregation on their own and with each other, and of segregation into 3 layers in a re-aggregate, which were very similar to interstitial and peritubular tissues in vivo. Observation of behaviors of fluorescent Sertoli cells and other non-fluorescent types of cells using testes from Sox9-EGFP transgenic mice showed dynamic cell movement and segregation in re-aggregate cultures. Cultures of testicular cells deprived of interstitial and peritubular cells resulted in dysmorphic structures, but re-addition of them restored tubule-like structures. Purified CD34+ cells in culture differentiated into p75+ cells and PTMCs. These results indicate that CD34+ cells differentiate into p75+ cells, which then differentiate into PTMCs. TGFβ signaling inhibitors, SB431542 and ALK5i, disturbed the reconstruction of cord-like and tubule-like structures, and the latter compromised re-construction of interstitial-like and peritubular-like structures, as well as the proliferation of CD34+, p75+, PTMCs, and Sertoli cells, and their movement and differentiation. These results indicate that CD34+ cells and signaling through ALK5 play pivotal roles in the morphogenesis of interstitial-like, peritubular-like and cord-like structures.

Highlights

Testicular morphogenesis is initiated from bipotential gonads at 10.5 dpc by the expression of Sry, a testis-determining gene in pre-Sertoli cells (SCs), followed by sequential steps toward formation of seminiferous cords and tubules [1,2,3]
In order to determine the roles of endothelial cells and peritubular cells in the reconstruction of seminiferous tubule-like structures we observed the behavior of CD34+ cells, p75+ cells, and peritubular myoid cells (PTMCs) by immunofluorescent staining with antibodies specific for each cell type on day 0, 1, 3, 5 and 7 of culture (Figs 1 and 2)
Since we have shown that interstitial and peritubular cells are required for the reconstruction of seminiferous cord-like structures, we assessed the effect of ALK5i on the behavior of CD34+ cells, p75+ cells and PTMCs during re-aggregate culture of testicular cells

Summary

Introduction

Testicular morphogenesis is initiated from bipotential gonads at 10.5 dpc (days post coitum) by the expression of Sry, a testis-determining gene in pre-Sertoli cells (SCs), followed by sequential steps toward formation of seminiferous cords and tubules [1,2,3]. Critical roles of mesonephroi in the formation of the cord have been reported by many investigators [1,2,3]. Some studies suggested that PTMCs are derived from the migrating population and contribute to the formation of the cords. Important roles of co-operative interactions between SCs and PTMCs in the formation of the cords in in vitro cultures were reported [9,10,11]. Cool et al [14] showed that PTMCs labeled with EYFP do not migrate from mesonephroi to the XY gonad, and suggested that endothelial cells migrate and are required for epithelialization of testis cords. How endothelial cell migration contributes to the formation of the cords remains to be clarified

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