Elsevier

Toxicology in Vitro

Volume 43, September 2017, Pages 47-57
Toxicology in Vitro

Increased Sat2 expression is associated with busulfan-induced testicular Sertoli cell injury

https://doi.org/10.1016/j.tiv.2017.05.023Get rights and content

Highlights

  • Busulfan upregulated Sat2 expression both in vivo and in vitro.

  • Sat2 overexpression triggered significant changes in protein expression profiles of Sertoli cells.

  • Sat2 overexpression affected cell cycle of Sertoli cells.

  • Sat2 overexpression can be detrimental to protein metabolism of Sertoli cells.

  • Sat2 may alter mRNA translation of Sertoli cells through ribosomal modification.

Abstract

Busulfan is a chemotherapeutic agent used to treat chronic myelogenous leukemia and other myeloproliferative disorders. Increasing evidence has demonstrated that busulfan may induce testicular dysfunction by targeting genes that are expressed in the testis. Here, we showed that spermidine/spermine N1-acetyltransferase 2 (Sat2) was present in testicular Sertoli cells, and its expression was significantly increased by busulfan treatment. To investigate the implications of Sat2 upregulation for cell growth and function, a Sat2-overexpressing TM4 Sertoli cell model was established. Increased Sat2 expression led to inhibited cell proliferation and arrested cell cycle. Based on iTRAQ proteomics analysis, we revealed that Sat2 overexpression is detrimental to cell cycle progression and cell communication, and notably, Sat2 may disturb protein metabolic processes by altering translation regulation and protein complex subunit organization. In summary, the present study provides evidence that Sat2 upregulation induces alterations in the growth and function of Sertoli cells. In testis tissue subjected to busulfan, increased expression of Sat2 can cause cellular injury and subsequent organ damage, which could lead to male infertility. Therefore, Sat2 may be a novel molecular target for treating busulfan-induced testicular toxicity.

Introduction

In industrialized countries over the past five decades, populations have experienced a decline in total fertility rates (TFR) (Skakkebaek et al., 2016). Cultural, social, economic and biological factors have contributed substantially to decreasing TFR. Nowadays, infertility is recognized as a global public health problem, approximately 15% of couples fail to achieve a pregnancy after 12 months or more of regular, unprotected sexual intercourse (Inhorn and Patrizio, 2015). Male factor infertility is responsible for 50% of infertile couples (Gurunath et al., 2011). Many factors such as lifestyle, chemotherapy drugs, infection, and environmental toxicants lead to an increased risk of male infertility (Cuypers et al., 2010, Fossato da Silva et al., 2011, Mocarelli et al., 2011).

Busulfan (1,4-bis [methanesulfonyl-y] butane) is a chemotherapeutic agent that is used to treat various malignant diseases, such as chronic myeloid leukemia and polycythemia vera (Buggia et al., 1994, Galaup and Paci, 2013), and has also been used prior to hematopoietic stem cell transplantation (Buggia et al., 1994).

Busulfan is a highly toxic agent that can induce adverse effects on the hematologic, nervous, and reproductive systems (Mirhoseini et al., 2014, Westerhof et al., 2000). Testis could be particularly sensitive to the toxic effect of busulfan, implicated by the fact that busulfan treatment leads to reduced testicular size and weight, with testicular atrophy and germ cell loss, and decreased sperm count, motility, and viability (Brinster and Zimmermann, 1994, Bucci and Meistrich, 1987, Choi et al., 2004, Vasiliausha et al., 2016). Thus, busulfan may cause male reproductive dysfunction, and recovery of normal reproductive function subsequent to treatment, especially in boys and young men, has become an important area of research consideration (Dehghani et al., 2013, Udagawa et al., 2001, Wallace et al., 2005).

Busulfan may induce testicular damage through various molecular mechanisms. Bucci reported that the cytotoxicity of busulfan is believed to be due to the formation of DNA adducts, which in turn can be detrimental to the survival of germ cells, Sertoli cells, and other somatic cells (Bucci and Meistrich, 1987). Choi et al. showed that busulfan may induce germ cell apoptosis through the loss of c-kit signaling in a Fas/FasL- and p53-independent manner (Choi et al., 2004). Additionally, O'Shaughnessy et al. showed that busulfan treatment altered the transcript levels of several Sertoli cell markers, such as sex hormone binding globulin, clusterin, and interleukin 1 alpha (O'Shaughnessy et al., 2008).Spermidine/spermine N1-acetyltransferase 2 (Sat2) was originally identified based on its homology to Sat1. In mammals, polyamines are essential for normal cell growth and proliferation (Gerner and Meyskens, 2004). SAT1 is a rate-limiting enzyme in polyamine catabolism, critically involved in the regulation of the intracellular concentration of polyamines and their transport out of cells (Gerner and Meyskens, 2004, Pegg, 2008). Although Sat2 is related to Sat1, with 46% sequence identity and 64% sequence similarity, Sat2 does not appear to play a role in polyamine catabolism (Coleman et al., 2004). Baek et al. demonstrated that Sat2 is an essential component of the ubiquitin ligase complex that regulates the rapid ubiquitination and degradation of hypoxia-inducible factor 1 alpha (Baek et al., 2007). A detailed expression analysis and functional characterization studies of Sat2 in the testis have not yet been reported. In our preliminary screening study, a significant increase in Sat2 transcript levels was observed in testes with impaired or absent sperm production, implying that Sat2 may be involved in testicular function. Therefore, in the present study, we have characterized the expression and cellular localization of Sat2 in the mouse testis, and attempted to determine whether Sat2 overexpression is implicated in busulfan-induced testicular Sertoli cell injury.

Section snippets

Materials

DMEM/F-12 and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA), and the protein extraction kit and BeyoECL Plus western blotting detection reagent were purchased from Beyotime Biotechnology (Jiangsu, China). The antibody for SAT2 (16246-1-AP) was purchased from Proteintech (Wuhan, China). The SYBR PrimeScript RT-PCR Kit (Perfect Real Time) and SYBR Premix Ex Taq II were purchased from TaKaRa Biotech (Liaoning, China). Busulfan was purchased from Sigma (St Louis, MO, USA).

Animal experiments

Expression of SAT2 in mouse testis

Immunohistochemical staining was performed to elucidate the spatial distribution of SAT2 in the mouse testis. As shown in Fig. 1A, positive signals were detected in the Sertoli cells and the interstitial area.

We further investigated the subcellular location of SAT2 in TM4 Sertoli cells by immunofluorescence staining. Fig. 1C shows that SAT2 was distributed throughout the cytoplasm.

Effect of busulfan on the expression of Sat2 in mouse testis and TM4 Sertoli cells

We next examined possible changes in Sat2 expression in a reproductive dysfunction mouse model with busulfan

Discussion

Busulfan can alter testicular function, leading to testicular atrophy and a decline in fertility (Anjamrooz et al., 2007, Brinster and Zimmermann, 1994, Bucci and Meistrich, 1987, Choi et al., 2004). However, the precise molecular mechanisms underlying the testicular toxic effects of busulfan have not been fully elucidated. As a cytotoxic drug, busulfan maybe exert direct effects on the somatic cells of the testis.

Sertoli cells are somatic cells that provide structural and nutritional support

Conclusions

In conclusion, our study provides evidence that Sat2 is expressed in Sertoli cells, and its expression is upregulated by busulfan treatment. Overexpression of Sat2 may decrease cell growth and arrest the cell cycle. Proteomics analysis revealed that Sat2 overexpression is detrimental to cell cycle progression, cell communication, and protein metabolic processes through modification of translation and protein complex subunit organization, which in turn causes Sertoli cell injury. It is obvious

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Acknowledgments

This study was supported by the National Natural Science Foundation of China (81270687, 30901602).

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    These authors contributed equally to this work.

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