Stimulated by retinoic acid gene 8 (STRA8) interacts with the germ cell specific bHLH factor SOHLH1 and represses c‐KIT expression in vitro

Formation of organotypic testicular organoids in microwell culture†

Biology of Reproduction ◽

10.1093/biolre/ioz053 ◽

2019 ◽

Vol 100 (6) ◽

pp. 1648-1660 ◽

Cited By ~ 15

Author(s):

Sadman Sakib ◽

Aya Uchida ◽

Paula Valenzuela-Leon ◽

Yang Yu ◽

Hanna Valli-Pulaski ◽

...

Keyword(s):

Retinoic Acid ◽

Germ Cell ◽

Germ Cells ◽

Sertoli Cells ◽

Cell Interactions ◽

Dependent Manner ◽

Tissue Architecture ◽

Cell Cell

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.

Download Full-text

Retinoic acid promotes the proliferation of primordial germ cell–like cells differentiated from mouse skin-derived stem cells in vitro

Theriogenology ◽

10.1016/j.theriogenology.2015.09.002 ◽

2016 ◽

Vol 85 (3) ◽

pp. 408-418 ◽

Cited By ~ 15

Author(s):

Hui Tan ◽

Jun-Jie Wang ◽

Shun-Feng Cheng ◽

Wei Ge ◽

Yuan-Chao Sun ◽

...

Keyword(s):

Stem Cells ◽

Retinoic Acid ◽

Germ Cell ◽

Primordial Germ Cell ◽

Mouse Skin

Download Full-text

Overexpression of DAZL, STRA8, and BOULE Genes and Treatment With BMP4 or Retinoic Acid Modulate the Expression of MSC Overexpressing Germ Cell Genes

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.667547 ◽

2021 ◽

Vol 8 ◽

Author(s):

Paloma Cordero ◽

Alejandra Guerrero-Moncayo ◽

Monica De los Reyes ◽

Manuel Varas-Godoy ◽

Jahaira Cortez ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Retinoic Acid ◽

Germ Cell ◽

Transgenic Animals ◽

Mrna Levels ◽

Adult Testis ◽

Specific Distribution

In vitro gamete derivation from stem cells has potential applications in animal reproduction as an alternative method for the dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSCs) may be suitable candidates for in vitro gamete derivation considering their differentiative capacity and their potential for cell therapy. Due to its relevance in gametogenesis, it has been reported that retinoic acid (RA) and bone morphogenetic protein (BMP) 4 are able to upregulate the expression of specific markers associated to the early stages of germ cell (GCs) differentiation in bovine fetal MSCs (bfMSCs). In the present study, we used polycistronic vectors containing combinations of GC genes DAZL, STRA8, and BOULE followed by exposure to BMP4 or RA to induce GC differentiation of bovine fetal adipose tissue-derived MSC (AT-MSCs). Cells samples at Day 14 were analyzed according to the expression of pluripotent genes NANOG and OCT4 and GC genes DAZL, STRA8, BOULE, PIWI, c-KIT, and FRAGILIS using Q-PCR. Fetal and adult testis and AT-MSCs samples were also analyzed for the expression of DAZL, STRA8, and NANOG using immunofluorescence. Increased gene expression levels in the adult testis and cell-specific distribution of DAZL, STRA8, and NANOG in the fetal testis suggest that these markers are important components of the regulatory network that control the in vivo differentiation of bovine GCs. Overexpression of DAZL and STRA8 in bi-cistronic and DAZL, STRA8, and BOULE in tri-cistronic vectors resulted in the upregulation of OCT4, NANOG, and PIWIL2 in bovine fetal AT-MSCs. While BMP4 repressed NANOG expression, this treatment increased DAZL and c-KIT and activated FRAGILIS expression in bovine fetal AT-MSCs. Treatment with RA for 14 days increased the expression of DAZL and FRAGILIS and maintained the mRNA levels of STRA8 in bovine fetal AT-MSCs transfected with bi-cistronic and tri-cistronic vectors. Moreover, RA treatment repressed the expression of OCT4 and NANOG in these cells. Thus, overexpression of DAZL, STRA8, and BOULE induced the upregulation of the pluripotent markers and PIWIL2 in transfected bovine fetal AT-MSCs. The partial activation of GC gene expression by BMP4 and RA suggests that both factors possess common targets but induce different gene expression effects during GC differentiation in overexpressing bovine fetal AT-MSCs.

Download Full-text

170. TGFβ SIGNALING IN AN IN VITRO SEMINOMA MODEL

Reproduction Fertility and Development ◽

10.1071/srb09abs170 ◽

2009 ◽

Vol 21 (9) ◽

pp. 88

Author(s):

J. C. Young ◽

A. Jaiprakash ◽

S. Mithraprabhu ◽

C. Itman ◽

S. Kitazawa ◽

...

Keyword(s):

Cell Proliferation ◽

Retinoic Acid ◽

Testicular Cancer ◽

Germ Cell ◽

Germ Cells ◽

Type I ◽

Tgfβ Signaling ◽

Cell Markers ◽

Germ Cell Proliferation

Testicular cancer, the second most common malignancy in young men, has a 95% cure rate but can result in infertility or subfertility. Its incidence has increased significantly in recent decades (1). This cancer is thought to arise during embryogenesis, based on the persistence of embryonic germ cell markers such as Blimp1 (2), Oct3/4 (3) and Nanog (3) in adult seminoma cells. TCam2 cells are a recently characterised in vitro seminoma model (4). We show by Q-PCR and immunofluorescence that they also express these early germ cell markers. TGFβ signaling plays a key role during germ cell development, and is implicated in the development of testicular cancers (5, 6). To investigate this further, we first determined whether the pathway is active in TCam2 cells. By Q-PCR we demonstrate expression of the TGFβ downstream transcription factors Smad 2, 3 and 4, and Activin type I and II receptors. Importantly, ActRIIA, which is undetectable in adult testicular germ cells, but readily detected in human foetal germ cells (7) and clinical seminoma samples (6), is readily detectable at both the mRNA and protein level in TCam2 cells. Furthermore, 24 hour treatment with Activin (5 and 50ng/ml) or BMP4 (5 and 50ng/ml) induces a 3-4 fold increase in ActRIIA mRNA levels, but not ActRIA, ActRIB or ActRIIB. Strikingly, in TCam2 cells BMP4 and to a lesser extent retinoic acid, but not activin, support survival and proliferation of TCam2 cells in the absence of serum. This is consistent with known roles of BMP4 and retinoic acid in enhancing murine foetal germ cell proliferation/self-renewal and survival (8, 9), and activin inhibition of foetal murine germ cell proliferation (10). This study is the first to demonstrate a functional response in seminoma cells consistent with their foetal germ cell-like identity and forms the basis for future mechanistic analyses of the role of TGFβ signaling in human testicular cancer.

Download Full-text