A quantitative global proteomics approach to understanding the functional pathways dysregulated in the spermatozoa of asthenozoospermic testicular cancer patients

Abstract

Background: Testicular cancer (TC) is the most common cancer diagnosed in men of reproductive age group. Sperm banking is recommended in these patients prior to cancer treatment. There is no literature describing the proteins dysregulated in the spermatozoa of TC patients with poor motility. Objective: The primary objective of this study was to compare the differences in the sperm proteome of normozoospermic (motility > 40%) and asthenozoospermic (motility < 40%) TC patients who had cryopreserved semen samples before initiating cancer therapy. Materials and methods: Pooled sperm samples from healthy fertile men (n = 8), normozoospermic (n = 20), and asthenozoospermic (n = 11) TC patients were used for quantitative global proteomic profiling by liquid chromatography-tandem mass spectrometry (LC-MS). The functional bioinformatic analysis was done by ingenuity pathway analysis software. Key differentially expressed proteins (DEPs) associated with binding of zona pellucida (CCT3), mitochondrial dysfunction (ATP5A1 and UQCRC2), sperm motility (ATP1A4), and an exosomal protein involved in the metabolic process of the spermatozoa (MMP9) were validated using Western blot analysis by comparing normozoospermic (n = 10) with asthenozoospermic (n = 10) TC patients. Statistical analysis was conducted using Mann-Whitney test. Results: A total of 813 and 957 proteins were detected in spermatozoa of normozoospermic and asthenozoospermic TC patients, respectively. On the other hand, 1139 proteins were detected in the spermatozoa of healthy fertile men. 198 proteins were identified as DEPs between TC patients with normal and abnormal semen parameters. Validation of DEPs revealed downregulation of key proteins (CCT3, ATP1A4, ATP5A1, and UQCRC2) implicated in the reproductive function in asthenozoospermic TC patients. Discussion: DEPs involved in the reproductive function pathways suggest defective spermatogenesis and maturation process in asthenozoospermic TC patients. Furthermore, dysfunctional exosomal pathway may be a possible cause of infertility in TC patients. Conclusion: The proteins associated with sperm function and fertilization process are compromised in TC patients irrespective of their semen parameters.

Testicular germ cell cancer: recent developments in biology and clinical management

“…A large clinical study from a semen bank confirmed that TGCT patients had decreased sperm concentration and total motile sperm count even before treatment (Xu et al , ). Similar findings are reported in another translational study, which followed these observations by comparing the proteomic profiles of spermatozoa from testicular cancer patients with asthenozoospermia and normospermia, and found quantitative changes in several proteins involved in mitochondrial function, oxidative phosphorylation and fertilisation (Panner Selvam et al , ).…”

Dysregulation of Key Proteins Associated with Sperm Motility and Fertility Potential in Cancer Patients

“…In the current study, molecular functions associated with mitochondria were dysregulated in the spermatozoa of cancer patients, mainly due to the aberrant expression of the mitochondrial proteins. Earlier proteomic studies reported that sperm mitochondrial proteins were affected in cancer conditions such as testicular cancer [22,28] and Hodgkin's disease [23]. Based on our proteomic results, the oxidative phosphorylation, a process linked to mitochondrial function, was defective in the spermatozoa of cancer patients (irrespective of the type of cancer included in this study).…”

“…During the past decade, the proteomics platform was used to profile the proteins in seminal fluids of patients diagnosed with several types of cancers, such as prostate cancer [25][26][27], testicular cancer [21,22,28], Hodgkin's disease [23], and leukemia [24]. Based on their ability to detect the expression of thousands of proteins simultaneously, proteomics techniques are of great interest to unravel the molecular mechanisms leading to infertility in cancer patients.…”

“…Our results show an altered sperm protein profile in cancer patients. Previous proteomic studies conducted on patients before starting any therapy reported 11 and 46 DEPs to be uniquely expressed in normozoospermic testicular cancer patients [22,28], while 15 and 4 proteins were uniquely expressed in asthenozoospermic patients affected by testicular cancer [22,28], suggesting that cancer might affect sperm maturation, even if the conventional semen parameters remain unaltered. Furthermore, an overexpression of the exosomal protein, matrix metalloproteinase 9 (MMP9), has been reported in both normo-and asthenozoospermic cancer patients [28], in agreement with previous reports of its increased expression in infertile men [29].…”

“…Previous proteomic studies conducted on patients before starting any therapy reported 11 and 46 DEPs to be uniquely expressed in normozoospermic testicular cancer patients [22,28], while 15 and 4 proteins were uniquely expressed in asthenozoospermic patients affected by testicular cancer [22,28], suggesting that cancer might affect sperm maturation, even if the conventional semen parameters remain unaltered. Furthermore, an overexpression of the exosomal protein, matrix metalloproteinase 9 (MMP9), has been reported in both normo-and asthenozoospermic cancer patients [28], in agreement with previous reports of its increased expression in infertile men [29]. Similarly, the proteins endothelial lipase precursor, apolipoprotein A-IV precursor, and carcinoembryonic antigen-related cell adhesion molecule 8 precursor were reported to be expressed uniquely in patients affected by Hodgkin's disease, suggesting an impairment of metabolic processes and pathways related to the production and synthesis of reactive oxygen species (ROS) [23].…”