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

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.

Treatment of semen samples with α-chymotrypsin alters the expression pattern of sperm functional proteins-a pilot study.

Semen hyperviscosity delays the liquefaction of semen sample and is subjected to limited proteolysis by addition of α-chymotrypsin to reduce the viscosity. α-Chymotrypsin is a proteolytic enzyme involved in degradation of the proteins and polypeptides. Even though α-chymotrypsin improves the handling of hyperviscous samples, its effect on the sperm proteins is not clear. This study was aimed to evaluate the alteration in the expression of sperm functional proteins in samples treated with α-chymotrypsin. Among all the proteins examined in both donor and patient samples, HSPA2 (70 KDa), BAG6 (150 KDa), HIST1H2BA (14 KDa), SPA17 (17 KDa formed after cleavage of C-terminal calmodulin-binding domain), and OXPHOS complexes were undetectable in α-chymotrypsin-treated samples, while the expression of the native SPA17 (20 KDa) was significantly decreased in the α-chymotrypsin-treated samples in comparison with controls. The use of α-chymotrypsin for liquefaction of hyperviscous samples degrades functional proteins of spermatozoa. Intracellular proteins, such as OXPHOS complexes and HIST1H2BA, and sperm surface proteins (HSPA2, BAG6, and SPA17) were degraded in all treated samples. Whether treatment of samples with α-chymotrypsin affects the global proteomic outcome is unclear. More in-depth calibration studies are required to determine the appropriate concentration of α-chymotrypsin for processing hyperviscous semen samples without compromising its protein expression and function. Similarly, the effects of altered protein function on assisted reproductive techniques (ART), such as intrauterine insemination (IUI) and in vitro fertilization (IVF) outcome, are not known and require further research.

Calibration of redox potential in sperm wash media and evaluation of oxidation-reduction potential values in various assisted reproductive technology culture media using MiOXSYS system.

Oxidation-reduction potential describes the balance between the oxidants and antioxidants in fluids including semen. Various artificial culture media are used in andrology and IVF laboratories for sperm preparation and to support the development of fertilized oocytes under in vitro conditions. The composition and conditions of these media are vital for optimal functioning of the gametes. Currently, there are no data on the status of redox potential of sperm processing and assisted reproduction media. The purpose of this study was to compare the oxidation-reduction potential values of the different media and to calibrate the oxidation-reduction potential values of the sperm wash medium using oxidative stress inducer cumene hydroperoxide and antioxidant ascorbic acid. Redox potential was measured in 10 different media ranging from sperm wash media, freezing media and assisted reproductive technology one-step medium to sequential media. Oxidation-reduction potential values of the sequential culture medium and one-step culture medium were lower and significantly different (p < 0.05) from the sperm wash media. Calibration of the sperm wash media using the oxidant cumene hydroperoxide and antioxidant ascorbic acid demonstrated that oxidation-reduction potential and the concentration of oxidant or antioxidant are logarithmically dependent. This study highlights the importance of calibrating the oxidation-reduction potential levels of the sperm wash media in order to utilize it as a reference value to identify the physiological range of oxidation-reduction potential that does not have any adverse effect on normal physiological sperm function.