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Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone.

ABSTRACT: Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dppa3, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dppa3 compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells.

SUBMITTER: Silva C

PROVIDER: S-EPMC2664383 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone.

Silva Celso C Wood Jennifer R JR Salvador Lisa L Zhang Zhibing Z Kostetskii Igor I Williams Carmen J CJ Strauss Jerome F JF

Molecular reproduction and development 20090101 1

Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The e ...[more]

PMID: 18425777

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Project description:BackgroundAdipose-derived mesenchymal stem cells (ADMSCs) are considered an efficient and important candidate for male infertility treatment because they contain pluripotent stem cells, which can differentiate into all cells from three germ layers. However, the efficient generation of male germ-like cell (MGLCs) is one of the key issues, and little is known about the mechanisms underlying generation of MGLCs. Herein, we attempt to improve the efficient generation of MGLCs derived during co-culturing of rat ADMSCs with SCs under retinoic acid (RA) and testosterone (T) treatment.MethodsADMSCs isolated from male SD rat were induced into generation of MGLCs by using respective methods in vitro. Transwell insert system was used for co-culturing. Busulfan-induced non-obstructive azoospermia rat mode was used to evaluate spermatogenic recovery ability of treated ADMSCs. Besides, the relative gene expression level was detected by reverse transcription PCR, quantitative RT-PCR. The relative protein expression level was detected by western blot (WB) and immunostaining analysis.ResultsThe results showed that ADMSCs co-cultured with TM4 cells under RA and T induction enhanced the formation of bigger and tightly packed MGLCs feature colonies in vitro. Moreover, the expression of male germ cell-related markers (Oct4, Stella, Ddx4, Dazl, PGP9.5, Stra8, and ITGα6) is significantly upregulated in TM4 cell-co-cultured ADMSCs in vitro and in busulfan-treated rat testis after injecting TM4 cell-treated ADMSCs for 2 months. Comparatively, the ADMSCs treated by TM4 cell with RA and T exhibited the highest expression of male germ cell-related markers. RA- and T-treated TM4 cell showed fewer dead cells and higher cytokine secretion than untreated groups. The protein expression level of TGFβ-SMAD2/3, JAK2-STAT3, and AKT pathways in ADMSCs co-cultured with TM4 cells under RA and T was higher than others. Whereas, downregulation of male germ cell-related marker expression subsequently inhibited the phosphorylation of SMAD2/3, JAK2, STAT3, and AKT.ConclusionThese results suggested that TM4 cells could efficiently stimulate in vitro generation of MGLCs during co-culturing of ADMSCs under RA and T treatment. Conclusively, the ADMSCs co-cultured with TM4 cell under RA and T induction stimulate the efficient generation of MGLCs in vitro through activating TGFβ-SMAD2/3, JAK2-STAT3, and AKT pathways. Among them, JAK2-STAT3 and AKT pathways are being first reported to show involvement of in vitro generation of MGLCs during ADMSC co-culturing with SCs.

Dataset Information

Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone.

Publications

Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone.

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