The influence of retinoic acid-induced differentiation on the radiation response of male germline stem cells

Abstract

Lifelong mammalian male fertility is maintained through an intricate balance between spermatogonial proliferation and differentiation. DNA damage in spermatogonia, for instance caused by chemo- or radiotherapy, can induce cell cycle arrest or germ cell apoptosis, possibly resulting in male infertility. Spermatogonia are generally more radiosensitive and prone to undergo apoptosis than somatic cells. Among spermatogonial subtypes the response to DNA damage is differentially modulated; undifferentiated spermatogonia, including the spermatogonial stem cells (SSCs), are relatively radio-resistant, whereas differentiating spermatogonia are very radiosensitive. To investigate the molecular mechanisms underlying this difference, we used an in vitro system consisting of mouse male germline stem (GS) cells that can be induced to differentiate. Using RNA-sequencing analysis, we analyzed the response of undifferentiated and differentiating GS cells to ionizing radiation (IR). At the RNA expression level, both undifferentiated and differentiating GS cells showed a very similar response to IR. Protein localization of several genes found to be involved in either spermatogonial differentiation or radiation response was investigated using mouse testis sections. For instance, we found that the transcription factor PDX1 was specifically expressed in undifferentiated spermatogonia and thus may be a novel marker for these cells. Interestingly, also at the protein level, undifferentiated GS cells showed a more pronounced upregulation of p53 in response to IR than differentiating GS cells. The higher p53 protein level in undifferentiated spermatogonia may preferentially induce cell cycle arrest, thereby giving these cells more time to repair inflicted DNA damage and increase their radio-resistance.

In Vitro Meiosis of Male Germline Stem Cells

“…In line with previous reports (Dann et al, 2008;Wang et al, 2016), we also recently characterized RA-induced spermatogonial differentiation (Zheng et al, 2018). Western blot, qPCR, and RNA-sequence analyses showed substantial downregulation of the SSC self-renewal genes Plzf and Oct4, while RNA and protein levels of the differentiation marker STRA8 markedly increased after 3 days of RA exposure.…”

“…As described (Kanatsu-Shinohara et al, 2003), in our laboratory these GSCs also retain their stem cell capacity and are able to undergo full spermatogenesis and generate healthy offspring after transplantation into the testes of recipient mice (Mulder et al, 2017). Moreover, by using retinoic acid (RA) treatment, we are able to induce spermatogonial differentiation in vitro, which is an important early step of spermatogenesis that irrevocably pushes the spermatogonia away from self-renewal in the direction of meiosis (Zheng et al, 2018). In vivo, spermatogonial differentiation and meiosis are supported by growth factors and hormones that are secreted by the Sertoli cells (De Gendt et al, 2004;Ramaswamy and Weinbauer, 2014).…”

“…The culture system consisted of three stages, with three different culture media, that represent (1) spermatogonial self-renewal/proliferation, (2) SSC differentiation and early meiosis, and (3) meiosis and formation of spermatid-like cells ( Figures 1A and 1B). During the first stage the GSCs were cultured with the factors known to be necessary for maintaining SSC self-renewal, glial-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) (Kanatsu-Shinohara et al, 2003), and behaved exactly as described previously (Zheng et al, 2018). Also, spermatogonial differentiation was induced exactly as reported previously by us (Zheng et al, 2018) by replacing the medium with medium containing RA, activin A, and bone morphogenetic protein 4 (BMP4).…”

“…During the first stage the GSCs were cultured with the factors known to be necessary for maintaining SSC self-renewal, glial-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) (Kanatsu-Shinohara et al, 2003), and behaved exactly as described previously (Zheng et al, 2018). Also, spermatogonial differentiation was induced exactly as reported previously by us (Zheng et al, 2018) by replacing the medium with medium containing RA, activin A, and bone morphogenetic protein 4 (BMP4). Finally, to establish meiotic progression, the medium was replaced with medium containing follicle-stimulating hormone (FSH), testosterone, and bovine pituitary extract (BPE) to induce meiotic progression and subsequent spermatid formation.…”

Di-n-butyl phthalate epigenetically induces reproductive toxicity via the PTEN/AKT pathway

“…In a state of oxidative stress, excess reactive species can damage various cellular components, including membrane lipids and DNA. The response of germ cells to DNA damage include initiating DNA-PK and ATM/ATR to activate DNA repair, cell cycle arrest, and/or apoptosis 21,22 . Hence, we hypothesized that DNA damage could generate in GC-1 and GC-2 cells after treated with DBP.…”