Abstract
The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 μM vs SCs and ***p < 0.001 at 3.33μM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 μM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33μM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 μM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 μM vs SCs and ***p < 0.001 at 1 μM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.
Highlights
Pre-pubertal boy’s cancer survival rate has markedly increased in recent decades, with the current 5-year survival rate across all childhood cancers above 80% [1], for both early diagnosis and improved cancer treatment protocols [2]
The gene expression levels of glial cell line-derived neurotrophic factor (GDNF) were significantly downregulated after treatment to all three cisplatin concentrations in a dose-dependent manner compared to untreated SCs
Compared with the control group, the gene and protein expression levels of GDNF were significantly down-regulated following treatment to all three doxorubicin concentrations in a dosedependent manner compared to untreated SCs (Figures 3E–G)
Summary
Pre-pubertal boy’s cancer survival rate has markedly increased in recent decades, with the current 5-year survival rate across all childhood cancers above 80% [1], for both early diagnosis and improved cancer treatment protocols [2]. At present, alkylating and alkylating-like agents such as cyclophosphamide and cisplatin, as well as doxorubicin, are three drugs commonly used in treatment regimens for a wide range of childhood cancer, resulting in more clinical information available, including that of patient serum concentrations. The future fertility in men has been extensively studied and there are clear links between the use of chemotherapy drugs in treatment regimens for childhood cancers and subsequent impairment of their fertility status [7, 8]. Several studies indicate that chemotherapy treatments in pre-pubertal subjects, are able to reduce the overall size of the testis where there is depletion of the germ cells (spermatogonia Ad and Ap), resulting in Sertoli cellonly tubules, as well as interstitial fibrosis and basement membrane thickening [9,10,11]. It is known that treatment with chemotherapeutic drugs induces a reduction of both Sertoli Cell (SCs) viability [12] and function [13] resulting in a complete inhibition of spermatogenesis and, in sterility due to azoospermia [7, 8]
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