Skip to main content
NCBI home page
Biology of Reproduction logoLink to Biology of Reproduction
. 2015 May 27;93(1):24. doi: 10.1095/biolreprod.115.129759

Ghrelin Prevents Cisplatin-Induced Testicular Damage by Facilitating Repair of DNA Double Strand Breaks Through Activation of p53 in Mice1

Jose M Garcia 4,6,7,8,2, Ji-an Chen 4,5, Bobby Guillory 4,8, Lawrence A Donehower 9, Roy G Smith 7,8,10, Dolores J Lamb 6,7,9,11
PMCID: PMC4706309  PMID: 26019260

Abstract

Cisplatin administration induces DNA damage resulting in germ cell apoptosis and subsequent testicular atrophy. Although 50 percent of male cancer patients receiving cisplatin-based chemotherapy develop long-term secondary infertility, medical treatment to prevent spermatogenic failure after chemotherapy is not available. Under normal conditions, testicular p53 promotes cell cycle arrest, which allows time for DNA repair and reshuffling during meiosis. However, its role in the setting of cisplatin-induced infertility has not been studied. Ghrelin administration ameliorates the spermatogenic failure that follows cisplatin administration in mice, but the mechanisms mediating these effects have not been well established. The aim of the current study was to characterize the mechanisms of ghrelin and p53 action in the testis after cisplatin-induced testicular damage. Here we show that cisplatin induces germ cell damage through inhibition of p53-dependent DNA repair mechanisms involving gamma-H2AX and ataxia telangiectasia mutated protein kinase. As a result, testicular weight and sperm count and motility were decreased with an associated increase in sperm DNA damage. Ghrelin administration prevented these sequelae by restoring the normal expression of gamma-H2AX, ataxia telangiectasia mutated, and p53, which in turn allows repair of DNA double stranded breaks. In conclusion, these findings indicate that ghrelin has the potential to prevent or diminish infertility caused by cisplatin and other chemotherapeutic agents by restoring p53-dependent DNA repair mechanisms.

Keywords: GHSR, growth hormone secretagogue, infertility, testis

INTRODUCTION

Cisplatin is a chemotherapeutic agent that is part of the standard of care for the treatment of cancers that are very prevalent, including bladder, testicular, lung, and head and neck cancer [1]. Male patients receiving cisplatin-based chemotherapy for the treatment of cancer sustain severe and sometimes irreversible damage to the germ cell epithelium leading to infertility [2–4]. After recovery, infertility caused by this treatment becomes a major issue that affects their quality of life. Administration of cisplatin induces DNA cross-links that result in the toxic effects on germ cells and Sertoli cells [5, 6]. Double strand breaks (DSBs) in DNA induced by cisplatin cause inhibition of DNA transcription and replication leading to apoptosis, which plays a key role in the development of cisplatin-induced infertility [7, 8].

Expression of the p53 tumor suppressor gene is generally associated with preventing the development of cancer and tumor progression in cells damaged by noxious agents. Sensing of DNA damage enhances p53 expression, resulting in the production of proteins that block cell division to allow damaged DNA to be repaired, or in extreme cases, p53 expression activates apoptosis to remove damaged cells. Besides preventing tumor cells from proliferating, p53 plays a role in normal physiology [9, 10]. In the testis, p53 predominantly acts as a cell cycle regulator. During spermatogenesis, p53 induces cell cycle arrest to allow DNA meiotic reshuffling and to correct DNA damage [11].

Ghrelin is a novel hormone that has growth hormone (GH)-secreting and orexigenic properties [12–14]. It is mainly produced in the stomach but is also secreted by Leydig cells [15]. Ghrelin receptors are expressed in the testis [16–18], and ghrelin has recently been shown to prevent testicular damage in different settings [19–21] and to have antiapoptotic properties in other tissues [22–24]. However, the mechanisms mediating these testicular and nontesticular effects are not fully understood; p53 was recently suggested as a key mediator of ghrelin's effects on food intake but not on its GH secretagogue activity [9].The aim of the current study was to characterize the mechanism of ghrelin and p53 action in the testis after cisplatin-induced testicular damage.

MATERIALS AND METHODS

Experimental Protocol

Adult C57bl/6j male mice were used for all the experiments (n = 8/group). Animals were randomized to receive vehicle (saline), cisplatin, ghrelin + cisplatin, and ghrelin. Clinical-grade cisplatin was purchased from APP Pharmaceuticals. Rodent ghrelin was synthesized by Baylor College of Medicine Department of Immunology, and its purity checked by mass spectrometry. The dose of cisplatin was 2.5 mg/kg daily given at 0830 intraperitoneally, and the dose for ghrelin was 0.8 mg/kg twice daily given intraperitoneally at 0800 and 1700. The morning dose of ghrelin was given 30 min before cisplatin. Animals were treated for 3 days and killed on the fourth day, 24 h after the last ghrelin injection. This regimen of cisplatin was selected based on published work showing it was compatible with complete survival and not overt toxicity [7, 8], inducing long-term failure of spermatogenesis and germ cell apoptosis in adult C57bl/6j mice. The regimen for ghrelin was selected based on our previous work showing that this regimen prevents fat and muscle atrophy induced by cisplatin in rodents [25, 26].

Animals were individually housed, acclimated to their cages and human handling for 5 days before the experiments were started, and maintained on a 12L:12D (lights on at 0600). Food and water were given ad libitum. All the experiments were conducted with the approval of the Institutional Animal Care and Use Committee at Baylor College of Medicine and were in compliance with the National Institutes of Health Guidelines for Use and Care of Laboratory Animals.

Immunofluorescence

Testes were collected and fixed overnight in CHO fixative (3% paraformaldehyde, 0.2% glutaraldehyde, and 2% sucrose in PBS at pH 7.5), dehydrated in 70% ethanol, and embedded in paraffin. Tissue was sectioned at 7 μm, mounted on charged slides, and stained for p53 using a rabbit polyclonal primary antibody for phospho-(Ser15)-p53 (Cell Signaling); for ataxia telangiectasia mutated (ATM) protein kinase using a mouse monoclonal primary antibody for phospho-ATM (pS1981) (Rockland); for γ-H2AX using a rabbit polyclonal primary antibody for phospho (Ser139)-γ-H2AX (Thermo Scientific), and for p21 using a rabbit polyclonal primary antibody (Santa Cruz Biotechnology). Tissue sections were deparaffinized, rehydrated, blocked with 10% normal rabbit serum, and incubated overnight with the following antibodies: phospho-p53 (1:200 final dilution in blocking buffer), with phospho-ATM (1:200 final dilution in blocking buffer), with phospho-γ-H2AX (1:200 final dilution in blocking buffer), or with p21 (1:200 final dilution in blocking buffer). All of the primary antibodies were diluted in 1% PBS and 0.1% bovine serum albumin (BSA). Afterwards, the slides were washed three times with 0.1% BSA-Tween. Sections were incubated 1 h at room temperature with Alexa Fluor 488 (Abcam) conjugated to IgG. Secondary antibodies were diluted 1:10 000. Sections were washed three times in 1% PBS and 0.1% BSA prior to being incubated in 4′,6-diamidino-2-phenylindole dilactate for nuclear visualization (Cell Signaling Technology). A Nikon microscope and camera (Eclipse TE2000-E) were used for image acquisition, and all the images for each antibody were taken with the same parameters (magnification, exposure, and intensity) and on the same day for all the groups. Representative images of each group are shown in the figures below.

RNA Analysis Using Real-Time PCR

Total RNA was isolated from 30-60 mg of testicular tissue using Trizol (15596-018; Invitrogen). Transcript levels were measured by real-time PCR (7000 Sequence Detection System; Applied Biosystems). Total RNA (500 ng) was reverse transcribed using the QuantiTect Reverse Transcription Kit (205311; Qiagen) to cDNA. Primers and probes for real-time PCR amplification used were selected using Primer Express Software (Applied Biosystems) (Supplemental Table S1; Supplemental Data are available online at www.biolreprod.org). The probe for target genes was labeled at the 5′ end with a reporter dye 6′-carboxyfluorescein and at the 3′ end with a quencher dye 6′-carboxytetramethylrhodamine. The reporter and quencher dyes are in close proximity on the probe, resulting in suppression of reporter fluorescence. The probe is designed to hybridize to a specific sequence within the PCR product. The 5′ to 3′ exonuclease activity of the Taq DNA polymerase allows for separation of the reporter from the close proximity of the quencher dye, resulting in fluorescence of the reporter dye. The resulting signal is measured at each amplification cycle on the ABI Sequence Detection System (Applied Biosystems), thus allowing the measurement of sample abundance in the linear phase of amplification. Target genes were amplified using aliquots of the same cDNA sample, and final quantitation of each sample was achieved by a coamplified relative standard curve.

Sperm Assays

Epididymides were dissected, the caudal region minced in Modified BWW Medium (Irvine Scientific), and incubated for 30 min at 37°C. For total sperm counts, sperm were immobilized by dilution with water and counted in a hemocytometer. Live sperm were spread onto a slide and classified as motile or immotile. Results were expressed as percent motile sperm.

Comet Assay

Alkaline comet assays were performed according to the manufacturer's protocol (Trevigen) with minor revisions. Specifically, 40 mM dithiothreitol was added to the lysis solution during initial incubation, without proteinase K, for 1 h. Slides were then further incubated in lysis solution containing 10 μg/ml of proteinase K for an additional 2.5 h at 37°C. Following electrophoresis, slides were air dried overnight and then stained in SYBR-Green before microscopic analysis. Only sperm with clearly extended Comet tails (at least 2-fold greater than the average comet tail size) were scored as positive.

Hormone Assays

Testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured by radioimmunoassay as previously described. These assays had a sensitivity of 0.088 ng/ml, 0.04 ng/ml, and 6.2 ng/ml, respectively, and were performed at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core.

Statistical Analysis

SPSS 12.00 software for Windows (SPSS Inc.) was used for all the statistical analyses. Parameters are expressed as mean ± SEM. Statistical comparisons for continuous variables were performed using ANOVA. P values of 0.05 or smaller were considered statistically significant.

RESULTS

Ghrelin Prevents Cisplatin-Induced Testicular Damage and Sperm Alterations

Cisplatin administration induced an acute decrease in testicular weight, epididymal sperm count, and sperm motility. Although ghrelin alone had no significant effects on these parameters, the changes induced by cisplatin were partially prevented by coadministration of ghrelin and cisplatin (Fig. 1, A–C).

FIG. 1.

FIG. 1

Open in a new tab

Testicular damage induced by cisplatin is prevented by ghrelin. Testicular weight (A), sperm density (B) and motility (C), and comet assay (D). Animals were treated with vehicle (V), ghrelin (0.8 mg/kg twice a day, G), cisplatin (2.5 mg/kg daily, C), or ghrelin+cisplatin (GC). Data represent mean ± SEM. P < 0.05 compared to V (*) and to GC (§).

Ghrelin Prevents Sperm DNA Damage Induced by Cisplatin

Given that cisplatin induces DNA damage by causing DSBs [27], we evaluated the effect of cisplatin and ghrelin administration on epididymal sperm DNA damage using a comet assay. This technique detects DNA DSBs and is a well-standardized and accepted method of assessing DNA damage. The comet assay was selected because of its greater sensitivity and higher reproducibility compared to other assays; moreover, it was previously used to detect cisplatin-induced sperm DNA damage in rodents [28]. As shown in Figure 1D, cisplatin administration caused a significant increase in comet-positive cells when compared to vehicle-treated animals. Ghrelin coadministration prevented this increase in the percentage of comet-positive cells.

Ghrelin Restores a Normal Pattern of Testicular γ-H2AX Phosphorylation

DNA DSBs induce histone γ-H2AX phosphorylation that in turn activate DNA repair. The importance of this pathway to genomic stability is highlighted by the fact that γ-H2AX−/− mice show genomic instability and infertility [29], and γ-H2AX is particularly important in physiologic DSB repair in germ cells [30]. To assess the activation of γ-H2AX in the testis following testicular damage, we performed immunostaining with a phosphorylated γ-H2AX-specific antibody of testicular samples after cisplatin ± ghrelin administration. Phosphorylated γ-H2AX-positive spermatogonia were evident in vehicle-treated mouse testis, and this staining pattern was not modified by ghrelin administration (Fig. 2, A and B). However, after cisplatin-induced testicular damage, there was a significant decrease in testicular γ-H2AX activation, an effect prevented by ghrelin coadministration. This latter group displayed a normal pattern of γ-H2AX expression after cisplatin+ghrelin cotreatment (Fig. 2, C and D).

FIG. 2.

FIG. 2

Open in a new tab

Ghrelin restores normal expression pattern of testicular γ-H2AX phosphorylation. Phospho-γ-H2AX immunostaining in vehicle (A), ghrelin (B), cisplatin (C), and ghrelin+cisplatin (G+C; D)-treated animals. Animals were treated with vehicle (V), ghrelin (0.8 mg/kg twice a day, G), or cisplatin (2.5 mg/kg daily, C), or ghrelin+cisplatin (G+C). Arrows indicate spermatogonia, arrowheads indicate spermatocytes. No 1ry Ab indicates the section that was stained without primary antibody to provide a negative control. Representative images of each group are shown in the figure.

Ghrelin Restores the Normal Pattern of Testicular p53 Phosphorylation and ATM Expression

Because the DNA damage induced by cisplatin and prevented by ghrelin was associated with changes in γ-H2AX activation, we postulated that the activity of its downstream mediator p53 would be downregulated by cisplatin and restored by ghrelin. Activation of p53 was assessed by immunostaining using an antibody against ser15-phospho-p53. As shown in Figure 3, A–D, phospho-p53 was present in vehicle-, ghrelin-, and cisplatin+ghrelin-treated animals. However, phospho-p53 was not present in cisplatin-treated animals.

FIG. 3.

FIG. 3

Open in a new tab

Ghrelin restores normal expression pattern of testicular p53 phosphorylation. Phospho-p53 immunostaining in vehicle (A), ghrelin (B), cisplatin (C), and ghrelin+cisplatin (G+C; D)-treated animals. Arrows indicate spermatogonia, arrowheads indicate spermatocytes. Animals were treated with vehicle (V), ghrelin (0.8 mg/kg twice a day, G), cisplatin (2.5 mg/kg daily, C), or ghrelin+cisplatin (G+C). No 1ry Ab indicates section stained without primary antibody and used as negative controls. Representative images of each group are shown in the figure.

The downregulation of p53 and γ-H2AX activation induced by cisplatin suggested an upstream loss of the ATM kinase function. ATM is a serine/threonine protein kinase that is recruited and activated by DNA DSBs and is a key regulator of DSB formation during meiosis [31]. As predicted, the expression of phospho-ATM was decreased in spermatogonia of animals treated with cisplatin, and this decreased immunostaining was again prevented by ghrelin coadministration (Fig. 4, A–D).

FIG. 4.

FIG. 4

Open in a new tab

Ghrelin restores normal expression pattern of testicular ATM phosphorylation. Phospho-ATM immunostaining in vehicle (A), ghrelin (B), cisplatin (C), and ghrelin+cisplatin (G+C; D)-treated animals. Arrows indicate spermatogonia. Animals were treated with vehicle (V), ghrelin (0.8 mg/kg twice a day, G), cisplatin (2.5 mg/kg daily, C), or ghrelin+cisplatin (G+C). No 1ry Ab indicates section stained without primary antibody to provide a negative control. Representative images of each group are shown in the figure.

Proapoptotic p53 Targets Are Not Affected by Cisplatin or Ghrelin

In other cell types, one of the functions of p53 is to induce transcription of apoptosis-related target genes. To determine if involvement of p53 in this setting causes changes in testicular expression of these genes, we measured mRNA levels of initial apoptosis signal (Fas), Fas-Ligand (Fas-l), B-cell lymphoma (Bcl)-2, Bcl-2-associated X (Bax), p53 upregulated modulator of apoptosis (Puma), and p53 effector related to PMP-22 (Perp) by quantitative RT-PCR in testicular homogenates. No significant treatment-dependent differences in expression of these genes were observed (Supplemental Fig. S1, A and B). Immunostaining for p21, a major transcriptional target of p53 revealed no difference in expression between groups (Supplemental Fig. S2, A–D). Taken together, these data are consistent with the hypothesis that testicular p53 activation after cisplatin/ghrelin administration does not alter proapoptotic gene expression. Rather, the data implies that coadministration of ghrelin with cisplatin is protective of spermatogenesis by enhancing the repair of DNA DSBs induced by cisplatin.

Testosterone, FSH, LH, and Androgen Receptor Expression Levels Are Not Acutely Affected by Cisplatin or Ghrelin

High doses of cisplatin and exogenous ghrelin administration affect circulating testosterone concentration [32, 33]. Hormonal modulation affects spermatogenesis, and lowered testosterone levels induce testicular apoptosis [34]. Accordingly, testosterone and gonadotropin levels were measured. Testosterone, LH, and FSH levels between groups were not significantly altered (Supplemental Table S2). Androgen receptor (Ar) expression, measured by quantitative RT-PCR, was also unaffected by cisplatin treatment. Hence, the protective effects of ghrelin on spermatogenesis are apparently not mediated by altered production of LH, FSH, or testosterone.

DISCUSSION

In the United States, 17 000 15–45-yr-old men are diagnosed yearly with lymphoma, bone and soft tissue sarcomas, or leukemia [35]. Most of these patients are treated with alkylating agents, platinum drugs, and/or radiation at dosages sufficient to induce prolonged and sometimes permanent azoospermia [36]. When cancer is controlled, the resulting infertility profoundly affects these patients' quality of life. Cisplatin-based chemotherapy results in azoospermia in men treated for lung cancer [37] and osteosarcoma [38, 39]. Prolonged infertility or permanent azoospermia occurs in more than 50% of patients receiving a cumulative dose of more than 600 mg/m2 of cisplatin [2–4]. Despite the significance of chemotherapy-induced gonadal damage, preventive treatments are lacking.

Cisplatin induction of DNA cross-links in germ cells [5, 6] leads to DSBs in DNA and subsequently to infertility [7, 8]. Recently, this dose-dependent effect has been shown to be the result of direct damage to spermatogonial stem cells and its microenvironment [40], and our recent report confirms this as we showed a selective loss of spermatogonia with preservation of Sertoli cells in response to cisplatin administration [20].

Besides its well-characterized role as a proapoptotic transcription factor, the tumor suppressor gene p53 plays an important function in DNA repair mechanisms during spermatogenesis by allowing sufficient time for DNA reshuffling and repair events to take place after DNA damage [11]. DNA DSBs trigger autophosphorylation of the ATM complex that in turn phosphorylates histone γ-H2AX [41]. Activation of p53 in the testis is dependent on histone γ-H2AX phosphorylation, a step thought to be critical in initiating DNA repair [42]. Cisplatin causes DNA DSBs, but the role of this pathway had not been previously explored in cisplatin-induced testicular damage. The administration of cisplatin not only caused DNA damage in sperm, it inhibited ATM-, γ-H2AX-, and p53-phosphorylation, suggesting that alterations in this pathway at least partially mediate the toxic effects of cisplatin on the testis.

Ghsr-1a mRNA expression is augmented following FSH injection, and expression of the Ghsr-1a gene in rat testis occurs in a developmental, stage-specific, hormonally regulated manner [43], suggesting that testicular function may be modulated by ghrelin. Recent reports suggest that ghrelin prevents testicular damage induced by heat, cadmium, or ionizing radiation, and a reduction of oxidative stress has been postulated as a possible mechanism, although the specific pathway remains to be fully elucidated [19, 21, 44, 45]. Ghrelin prevents apoptosis in a number of tissues, including adipocytes, endothelial cells, and others [22–24, 46], but whether these actions are mediated through p53 has not been well-established. Elegant studies recently suggest that p53 is a key mediator of ghrelin's orexigenic actions in the hypothalamus, where it acts as a nutrient sensor, but not of its GH-secretagogue activity that involves direct action on GH-releasing hormone neurons in the arcuate nucleus [9, 47]. On the other hand, in vitro experiments on porcine ovarian granulosa cell cultures and in neurons suggest that ghrelin decreases activation of p53 and its downstream proapoptotic mediator Bax [48, 49]. Ghrelin prevents the increase in Bax expression following heat-induced testicular damage [50] but increases Bax expression in spermatocytes during normal spermatogenesis in rats [51]. Nevertheless, the effects of ghrelin on p53 activity or level of expression were not reported. More recently Li et al. [19] reported that ghrelin antagonism increases radiation-induced damage to spermatogonia in a p53-dependent manner.

We recently reported that ghrelin prevents cisplatin-induced testicular atrophy through its only identified receptor to date—Ghsr-1a—although the specific mechanism of action was not characterized [20]. These are important findings because testicular weight is highly correlated with the degree of atrophy in different clinical scenarios, and sperm count and motility are two parameters used to predict fertility in rodents and humans [7, 8, 52], although neither parameter can reliably distinguish fertility from infertility in humans unless the male is azoospermic [53]. In this study, we show that exogenous administration of ghrelin, when given along with cisplatin, prevents DNA damage in sperm and it does so by preventing the inhibition of ATM-, γ-H2AX-, and p53-phosphorylation caused by cisplatin. Moreover, our data also suggest that endogenous, physiological ghrelin production is not sufficient to prevent cisplatin-induced testicular toxicity, and that pharmacological doses of this hormone are necessary to protect against these effects.

Despite enhancing p53 activity, the proapoptotic transcriptional activity of p53 was not induced by ghrelin or cisplatin, suggesting activation of this pathway is cell type-specific. This failure of activated p53 to enhance apoptosis in the testis is perhaps not surprising because the primary role for testicular p53 is to arrest meiosis and to allow damaged DNA to be repaired, as indicated earlier [11]. Our results also suggest that ghrelin may activate different protective mechanisms according to the specific setting or insult (i.e. normal spermatogenesis vs. heat-induced or cisplatin-induced testicular damage). One potential limitation when assessing the impact of ghrelin and cisplatin on proapoptotic pathways is the relative small sample size we used (n = 8/group) that could potentially fail to identify a small effect. However, given the magnitude of the differences seen between groups for all gene transcripts, it is unlikely that an increase in sample size would expose a clinically relevant effect.

Although the current model does not address the question of whether ghrelin might affect tumor growth by acting directly on the tumor or diminishing the efficacy of cisplatin, this is not likely. All tumor-bearing animal models reported to date where ghrelin or its mimetics have been used have not shown an increase in tumor growth [54, 55], and more recently, a trial of ghrelin treatment in patients with esophageal cancer undergoing cisplatin-based chemotherapy showed ghrelin treatment did not reduce the effectiveness of chemotherapy [56]. Long-term studies using systemic ghrelin mimetics for up to 1 yr in humans have shown good tolerability with small increases in body weight and IGF-1 levels that remained well within the physiologic range [57].

Induction of central hypogonadism is proposed to provide a mechanism for protecting the testis from damage induced by chemotherapy [36]. However, hypogonadism does not occur with cisplatin treatment [58, 59]. In our experiments, cisplatin caused damage to the testis without significantly changing the circulating levels of testosterone, LH, or FSH. Although both the endocrine and exocrine compartments of the testis are affected by cisplatin, the effect of this chemotherapeutic agent on testosterone production is dose-dependent because only high cumulative doses of chemotherapy cause a significant and persistent impairment of Leydig cell function [32]. Hence, it is not surprising that the regimen used in our animals did not suppress testosterone levels given that hormone levels were checked only after 3 doses of cisplatin (total dose 7.5 mg/kg). Ghrelin is synthesized in Leydig cells and its receptor is located in other testicular cell types [15, 60]. Chronic or central ghrelin administration suppresses LH and testosterone levels in prepubertal and adult rats [33, 61]. However, changes in testosterone, AR expression, or gonadotrophins with the current regimen of peripheral ghrelin administration (0.8 mg/kg, twice a day) for 3 days did not occur. Different regimens of ghrelin administration, different species, or the short-term duration of our experiment may explain the differences between our results and those previously published [33, 61].

In summary, cisplatin induces germ cell damage by inhibiting ATM-, γ-H2AX-, and p53-dependent DNA repair mechanisms, and ghrelin prevents these changes induced by cisplatin and restores normal expression of these mediators, thereby decreasing sperm DNA damage by enhancing DNA repair mechanisms. Establishing the mechanisms and effects of ghrelin on fertility, germ cell apoptosis, and p53 activity in this and other settings may be important to develop methods to prevent or reverse the infertility caused by cisplatin and other agents. The potential role of ghrelin as a therapeutic intervention aiming at preventing cisplatin-induced DNA DSBs in the testis should be further explored. The findings will have relevance for protecting the future fertility in men exposed to chemotherapeutic agents, chemicals, or radiation.

ACKNOWLEDGMENT

We thank the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core (supported by NICHD U54-HD28934) and Baylor DERC (P30 DK079638) for their help.

Footnotes

1

This work was supported by the U.S. Dept. of Veterans Affairs (MERIT grants BX000507 and CX000174, MREP, and a SHCNCDA), Dan L. Duncan Pilot Project Award, and NIH Grants AG040583, HD060870 to J.M.G. and P01HD36289 to D.J.L. from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. B.G. is supported by a training grant from the NIA (T32AG000183). J.C. is supported by National Natural Science Foundation of China (81072262, 81372944).

3

These authors contributed equally to this work.

REFERENCES

  1. Verstappen CC, Heimans JJ, Hoekman K, Postma TJ. Neurotoxic complications of chemotherapy in patients with cancer: clinical signs and optimal management. Drugs. 2003;63:1549–1563. doi: 10.2165/00003495-200363150-00003. [DOI] [PubMed] [Google Scholar]
  2. Brennemann W, Stoffel-Wagner B, Helmers A, Mezger J, Jager N, Klingmuller D. Gonadal function of patients treated with cisplatin based chemotherapy for germ cell cancer. J Urol. 1997;158:844–850. doi: 10.1097/00005392-199709000-00041. [DOI] [PubMed] [Google Scholar]
  3. Pont J, Albrecht W. Fertility after chemotherapy for testicular germ cell cancer. Fertil Steril. 1997;68:1–5. doi: 10.1016/s0015-0282(97)81465-3. [DOI] [PubMed] [Google Scholar]
  4. Howell SJ, Shalet SM. Testicular function following chemotherapy. Hum Reprod Update. 2001;7:363–369. doi: 10.1093/humupd/7.4.363. [DOI] [PubMed] [Google Scholar]
  5. Meistrich ML, Finch M, da Cunha MF, Hacker U, Au WW. Damaging effects of fourteen chemotherapeutic drugs on mouse testis cells. Cancer Res. 1982;42:122–131. [PubMed] [Google Scholar]
  6. Huang HF, Pogach LM, Nathan E, Giglio W. Acute and chronic effects of cisplatinum upon testicular function in the rat. J Androl. 1990;11:436–445. [PubMed] [Google Scholar]
  7. Seaman F, Sawhney P, Giammona CJ, Richburg JH. Cisplatin-induced pulse of germ cell apoptosis precedes long-term elevated apoptotic rates in C57/BL/6 mouse testis. Apoptosis. 2003;8:101–108. doi: 10.1023/a:1021734604913. [DOI] [PubMed] [Google Scholar]
  8. Sawhney P, Giammona CJ, Meistrich ML, Richburg JH. Cisplatin-induced long-term failure of spermatogenesis in adult C57/Bl/6J mice. J Androl. 2005;26:136–145. [PubMed] [Google Scholar]
  9. Velasquez DA, Martinez G, Romero A, Vazquez MJ, Boit KD, Dopeso-Reyes IG, Lopez M, Vidal A, Nogueiras R, Dieguez C. The central Sirtuin 1/p53 pathway is essential for the orexigenic action of ghrelin. Diabetes. 2011;60:1177–1185. doi: 10.2337/db10-0802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Vousden KH, Ryan KM. p53 and metabolism. Nat Rev Cancer. 2009;9:691–700. doi: 10.1038/nrc2715. [DOI] [PubMed] [Google Scholar]
  11. Rotter V, Schwartz D, Almon E, Goldfinger N, Kapon A, Meshorer A, Donehower LA, Levine AJ. Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome. Proc Natl Acad Sci U S A. 1993;90:9075–9079. doi: 10.1073/pnas.90.19.9075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 1999;402:656–660. doi: 10.1038/45230. [DOI] [PubMed] [Google Scholar]
  13. Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshimoto A, Harada M, Mori K, Komatsu Y, Usui T, Shimatsu A, Ogawa Y, Hosoda K, et al. Ghrelin strongly stimulates growth hormone release in humans. J Clin Endocrinol Metab. 2000;85:4908–4911. doi: 10.1210/jcem.85.12.7167. [DOI] [PubMed] [Google Scholar]
  14. Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR. Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab. 2001;86:5992. doi: 10.1210/jcem.86.12.8111. [DOI] [PubMed] [Google Scholar]
  15. Tena-Sempere M, Barreiro ML, Gonzalez LC, Gaytan F, Zhang FP, Caminos JE, Pinilla L, Casanueva FF, Dieguez C, Aguilar E. Novel expression and functional role of ghrelin in rat testis. Endocrinology. 2002;143:717–725. doi: 10.1210/endo.143.2.8646. [DOI] [PubMed] [Google Scholar]
  16. Gaytan F, Barreiro ML, Caminos JE, Chopin LK, Herington AC, Morales C, Pinilla L, Paniagua R, Nistal M, Casanueva FF, Aguilar E, Dieguez C, et al. Expression of ghrelin and its functional receptor, the type 1a growth hormone secretagogue receptor, in normal human testis and testicular tumors. J Clin Endocrinol Metab. 2004;89:400–409. doi: 10.1210/jc.2003-031375. [DOI] [PubMed] [Google Scholar]
  17. Sun Y, Butte NF, Garcia JM, Smith RG. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology. 2008;149:843–850. doi: 10.1210/en.2007-0271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Barreiro ML, Gaytan F, Caminos JE, Pinilla L, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M. Cellular location and hormonal regulation of ghrelin expression in rat testis. Biol Reprod. 2002;67:1768–1776. doi: 10.1095/biolreprod.102.006965. [DOI] [PubMed] [Google Scholar]
  19. Li W, Zeng Y, Zhao J, Zhu CJ, Hou WG, Upregulation Zhang S. and nuclear translocation of testicular ghrelin protects differentiating spermatogonia from ionizing radiation injury. Cell Death Dis. 2014;5:e1248. doi: 10.1038/cddis.2014.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Whirledge SD, Garcia JM, Smith RG, Lamb DJ. Ghrelin partially protects against cisplatin-induced male murine gonadal toxicity in a GHSR-1a-dependent manner Biol Reprod 2015. 92 76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kheradmand A, Alirezaei M, Dezfoulian O. Biochemical and histopathological evaluations of ghrelin effects following cadmium toxicity in the rat testis. Andrologia. 2015;47:634–643. doi: 10.1111/and.12311. [DOI] [PubMed] [Google Scholar]
  22. Rak-Mardyla A, Gregoraszczuk EL. ERK. 1/2 and PI-3 kinase pathways as a potential mechanism of ghrelin action on cell proliferation and apoptosis in the porcine ovarian follicular cells. J Physiol Pharmacol. 2010;61:451–458. [PubMed] [Google Scholar]
  23. Deng B, Fang L, Chen X, Chen M, Xie X. Effect of ghrelin on angiotensin II induced human umbilicus vein endothelial cell oxidative stress and endothelial cell injury. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2010;35:1037–1047. doi: 10.3969/j.issn.1672-7347.2010.10.003. [DOI] [PubMed] [Google Scholar]
  24. Slomiany BL, Slomiany A. Role of constitutive nitric oxide synthase S-nitrosylation in Helicobacter pylori-induced gastric mucosal cell apoptosis: effect of ghrelin. Inflammopharmacology. 2010;18:233–240. doi: 10.1007/s10787-010-0051-7. [DOI] [PubMed] [Google Scholar]
  25. Garcia JM, Cata JP, Dougherty PM, Smith RG. Ghrelin prevents cisplatin-induced mechanical hyperalgesia and cachexia. Endocrinology. 2008;149:455–460. doi: 10.1210/en.2007-0828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Garcia JM, Scherer T, Chen JA, Guillory B, Nassif A, Papusha V, Smiechowska J, Asnicar M, Buettner C, Smith RG. Inhibition of cisplatin-induced lipid catabolism and weight loss by ghrelin in male mice. Endocrinology. 2013;154:3118–3129. doi: 10.1210/en.2013-1179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hooser SB, van Dijk-Knijnenburg WC, Waalkens-Berendsen ID, Smits-van Prooije AE, Snoeij NJ, Baan RA, Fichtinger-Schepman AM. Cisplatin-DNA adduct formation in rat spermatozoa and its effect on fetal development. Cancer Lett. 2000;151:71–80. doi: 10.1016/s0304-3835(99)00415-2. [DOI] [PubMed] [Google Scholar]
  28. Delbes G, Hales BF, Robaire B. Effects of the chemotherapy cocktail used to treat testicular cancer on sperm chromatin integrity. J Androl. 2007;28:241–249. doi: 10.2164/jandrol.106.001487. [DOI] [PubMed] [Google Scholar]
  29. Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, et al. Genomic instability in mice lacking histone H2AX. Science. 2002;296:922–927. doi: 10.1126/science.1069398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mahadevaiah SK, Turner JM, Baudat F, Rogakou EP, de Boer P, Blanco-Rodriguez J, Jasin M, Keeney S, Bonner WM, Burgoyne PS. Recombinational DNA double-strand breaks in mice precede synapsis. Nat Genet. 2001;27:271–276. doi: 10.1038/85830. [DOI] [PubMed] [Google Scholar]
  31. Lange J, Pan J, Cole F, Thelen MP, Jasin M, Keeney S. ATM controls meiotic double-strand-break formation. Nature. 2011;479:237–240. doi: 10.1038/nature10508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Gerl A, Muhlbayer D, Hansmann G, Mraz W, Hiddemann W. The impact of chemotherapy on Leydig cell function in long term survivors of germ cell tumors. Cancer. 2001;91:1297–1303. doi: 10.1002/1097-0142(20010401)91:7<1297::aid-cncr1132>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  33. Fernandez-Fernandez R, Navarro VM, Barreiro ML, Vigo EM, Tovar S, Sirotkin AV, Casanueva FF, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M. Effects of chronic hyperghrelinemia on puberty onset and pregnancy outcome in the rat. Endocrinology. 2005;146:3018–3025. doi: 10.1210/en.2004-1622. [DOI] [PubMed] [Google Scholar]
  34. Sun YT, Wreford NG, Robertson DM, de Kretser DM. Quantitative cytological studies of spermatogenesis in intact and hypophysectomized rats: identification of androgen-dependent stages. Endocrinology. 1990;127:1215–1223. doi: 10.1210/endo-127-3-1215. [DOI] [PubMed] [Google Scholar]
  35. Howlader N, Noone AM, Krapcho M, Garshell J, Miller D, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Freuer EJ, Cronin KA. SEER Cancer Statistics Review, 1975-2012, National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/csr/1975_2012/, based on November 2014 SEER data submission, posted to the SEER web site, April 2015. Accessed 1 May 2015. (eds) [Google Scholar]
  36. Shetty G, Meistrich ML. Hormonal approaches to preservation and restoration of male fertility after cancer treatment. J Natl Cancer Inst Monogr. 2005;34:36–39. doi: 10.1093/jncimonographs/lgi002. [DOI] [PubMed] [Google Scholar]
  37. Aasebo U, Slordal L, Aanderud S, Aakvaag A. Chemotherapy and endocrine function in lung cancer. Acta Oncol. 1989;28:667–669. doi: 10.3109/02841868909092291. [DOI] [PubMed] [Google Scholar]
  38. Siimes MA, Elomaa I, Koskimies A. Testicular function after chemotherapy for osteosarcoma. Eur J Cancer. 1990;26:973–975. doi: 10.1016/0277-5379(90)90623-2. [DOI] [PubMed] [Google Scholar]
  39. Meistrich ML, Chawla SP, Da Cunha MF, Johnson SL, Plager C, Papadopoulos NE, Lipshultz LI, Benjamin RS. Recovery of sperm production after chemotherapy for osteosarcoma. Cancer. 1989;63:2115–2123. doi: 10.1002/1097-0142(19890601)63:11<2115::aid-cncr2820631108>3.0.co;2-a. [DOI] [PubMed] [Google Scholar]
  40. Harman JG, Richburg JH. Cisplatin-induced alterations in the functional spermatogonial stem cell pool and niche in C57/BL/6J mice following a clinically relevant multi-cycle exposure. Toxicol Lett. 2014;227:99–112. doi: 10.1016/j.toxlet.2014.03.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Luo Y, Lin FT, Lin WC. ATM-mediated stabilization of hMutL DNA mismatch repair proteins augments p53 activation during DNA damage. Mol Cell Biol. 2004;24:6430–6444. doi: 10.1128/MCB.24.14.6430-6444.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sengupta S, Harris CC. p53: traffic cop at the crossroads of DNA repair and recombination. Nat Rev Mol Cell Biol. 2005;6:44–55. doi: 10.1038/nrm1546. [DOI] [PubMed] [Google Scholar]
  43. Barreiro ML, Suominen JS, Gaytan F, Pinilla L, Chopin LK, Casanueva FF, Dieguez C, Aguilar E, Toppari J, Tena-Sempere M. Developmental, stage-specific, and hormonally regulated expression of growth hormone secretagogue receptor messenger RNA in rat testis. Biol Reprod. 2003;68:1631–1640. doi: 10.1095/biolreprod.102.008862. [DOI] [PubMed] [Google Scholar]
  44. Taati M, Moghadasi M, Dezfoulian O, Asadian P, Kheradmand A, Abbasi M, Zendehdel M. The effect of ghrelin pretreatment on epididymal sperm quality and tissue antioxidant enzyme activities after testicular ischemia/reperfusion in rats. J Physiol Biochem. 2012;68:91–97. doi: 10.1007/s13105-011-0122-2. [DOI] [PubMed] [Google Scholar]
  45. Kheradmand A, Dezfoulian O, Tarrahi MJ. Ghrelin attenuates heat-induced degenerative effects in the rat testis. Regul Pept. 2011;167:97–104. doi: 10.1016/j.regpep.2010.12.002. [DOI] [PubMed] [Google Scholar]
  46. Zwirska-Korczala K, Adamczyk-Sowa M, Sowa P, Pilc K, Suchanek R, Pierzchala K, Namyslowski G, Misiolek M, Sodowski K, Kato I, Kuwahara A, Zabielski R. Role of leptin, ghrelin, angiotensin II and orexins in 3T3 L1 preadipocyte cells proliferation and oxidative metabolism J Physiol Pharmacol 2007. 58 (Suppl 1): 53 64 [PubMed] [Google Scholar]
  47. Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt MJ, Jr, , Fisher MH, Nargund RP, Patchett AA. Peptidomimetic regulation of growth hormone secretion. Endocr Rev. 1997;18:621–645. doi: 10.1210/edrv.18.5.0316. [DOI] [PubMed] [Google Scholar]
  48. Sirotkin AV, Meszarosova M, Grossmann R, Benco A, Valenzuela F. Effect of inhibitor and activator of ghrelin receptor (GHS-R1a) on porcine ovarian granulosa cell functions. Gen Comp Endocrinol. 2011;173:105–110. doi: 10.1016/j.ygcen.2011.05.001. [DOI] [PubMed] [Google Scholar]
  49. Chung H, Kim E, Lee DH, Seo S, Ju S, Lee D, Kim H, Park S. Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen-glucose deprivation. Endocrinology. 2007;148:148–159. doi: 10.1210/en.2006-0991. [DOI] [PubMed] [Google Scholar]
  50. Kheradmand A, Dezfoulian O, Alirezaei M. Ghrelin regulates Bax and PCNA but not Bcl-2 expressions following scrotal hyperthermia in the rat. Tissue Cell. 2012;44:308–315. doi: 10.1016/j.tice.2012.04.009. [DOI] [PubMed] [Google Scholar]
  51. Kheradmand A, Dezfoulian O, Alirezaei M, Rasoulian B. Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats. Biochem Biophys Res Commun. 2012;419:299–304. doi: 10.1016/j.bbrc.2012.02.014. [DOI] [PubMed] [Google Scholar]
  52. Sue Marty M, Singh NP, Stebbins KE, Ann Linscombe V, Passage J, Bhaskar Gollapudi B. Initial insights regarding the role of p53 in maintaining sperm DNA integrity following treatment of mice with ethylnitrosourea or cyclophosphamide. Toxicol Pathol. 2010;38:244–257. doi: 10.1177/0192623309357947. [DOI] [PubMed] [Google Scholar]
  53. Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK, Nakajima ST, Coutifaris C, Carson SA, Cisneros P, Steinkampf MP, Hill JA, Xu D, Vogel DL, et al. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med. 2001;345:1388–1393. doi: 10.1056/NEJMoa003005. [DOI] [PubMed] [Google Scholar]
  54. Hanada T, Toshinai K, Kajimura N, Nara-Ashizawa N, Tsukada T, Hayashi Y, Osuye K, Kangawa K, Matsukura S, Nakazato M. Anti-cachectic effect of ghrelin in nude mice bearing human melanoma cells. Biochem Biophys Res Commun. 2003;301:275–279. doi: 10.1016/s0006-291x(02)03028-0. [DOI] [PubMed] [Google Scholar]
  55. DeBoer MD, Zhu XX, Levasseur P, Meguid MM, Suzuki S, Inui A, Taylor JE, Halem HA, Dong JZ, Datta R, Culler MD, Marks DL. Ghrelin treatment causes increased food intake and retention of lean body mass in a rat model of cancer cachexia. Endocrinology. 2007;148:3004–3012. doi: 10.1210/en.2007-0016. [DOI] [PubMed] [Google Scholar]
  56. Hiura Y, Takiguchi S, Yamamoto K, Takahashi T, Kurokawa Y, Yamasaki M, Nakajima K, Miyata H, Fujiwara Y, Mori M, Kangawa K, Doki Y. Effects of ghrelin administration during chemotherapy with advanced esophageal cancer patients: a prospective, randomized, placebo-controlled phase 2 study. Cancer. 2012;118:4785–4794. doi: 10.1002/cncr.27430. [DOI] [PubMed] [Google Scholar]
  57. Nass R, Pezzoli SS, Oliveri MC, Patrie JT, Harrell FE, Jr, , Clasey JL, Heymsfield SB, Bach MA, Vance ML, Thorner MO. Effects of an oral ghrelin mimetic on body composition and clinical outcomes in healthy older adults: a randomized trial. Ann Intern Med. 2008;149:601–611. doi: 10.7326/0003-4819-149-9-200811040-00003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Morris ID. Protection against cytotoxic-induced testis damage–experimental approaches. Eur Urol. 1993;23:143–147. doi: 10.1159/000474583. [DOI] [PubMed] [Google Scholar]
  59. Handelsman DJ, Peng S, Sikka S, Swerdloff RS, Rajfer J. Effects of gonadotropin-releasing hormone analogs on cis-platinum-induced spermatogenic damage. Int J Androl. 1988;11:425–435. doi: 10.1111/j.1365-2605.1988.tb01015.x. [DOI] [PubMed] [Google Scholar]
  60. Ishikawa T, Fujioka H, Ishimura T, Takenaka A, Fujisawa M. Ghrelin expression in human testis and serum testosterone level. J Androl. 2007;28:320–324. doi: 10.2164/jandrol.106.000810. [DOI] [PubMed] [Google Scholar]
  61. Wang L, Fang F, Li Y, Zhang Y, Pu Y, Zhang X. Role of ghrelin on testosterone secretion and the mRNA expression of androgen receptors in adult rat testis. Syst Biol Reprod Med. 2011;57:119–123. doi: 10.3109/19396368.2010.529984. [DOI] [PubMed] [Google Scholar]

Articles from Biology of Reproduction are provided here courtesy of Oxford University Press

RESOURCES