RESEARCHES
Discovery of Fertility-Related Biomarkers
Conventional semen analysis has been used for prognosis and diagnosis of male fertility. Although this tool is essential for providing initial quantitative information about semen, it remains a subject of debate. Therefore, development of new methods for the prognosis and diagnosis of male fertility should be seriously considered for animal species of economic importance as well as for humans. In the present study, we applied a comprehensive proteomic approach to identify global protein biomarkers in boar spermatozoa in order to increase the precision of male fertility prognoses and diagnoses. We determined that L-amino acid oxidase, mitochondrial malate dehydrogenase 2, NAD (MDH2), cytosolic 5 -nucleotidase 1B, lysozyme-like protein 4, and calmodulin (CALM) were significantly and abundantly expressed in high-litter size spermatozoa. We also found that equatorin, spermadhesin AWN, triosephosphate isomerase (TPI), Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2) were significantly and abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A, TPI, and NDUFS2 were negatively correlated with litter size, whereas CALM and MDH2 were positively correlated. This study provides novel biomarkers for the prediction of male fertility. To the best of our knowledge, this is the first work that shows significantly increased litter size using male fertility biomarkers in a field trial. Moreover, these protein markers may provide new developmental tools for the selection of superior sires as well as for the prognosis and diagnosis of male fertility. Molecular & Cellular Proteomics 14: 10.1074/mcp. M114.045369, 1230–1240, 2015.
Conventional semen analyses are used to evaluate male factor fertility/infertility in humans and other animals. However, their clinical value remains controversial. Therefore, new tools that more accurately assess male fertility based on sperm function and fertilization mechanism are of interest worldwide. While protein markers in spermatozoa that might help differentiate fertile and infertile sperm have been identified, studies are in their infancy, and the markers require validation in field trials. In the present study, to discover more sensitive biomarkers in spermatozoa for predicting male fertility, we assessed protein expression in capacitated spermatozoa. The results demonstrated that cytochrome b-c1 complex subunit 2 (UQCRC2) was abundantly expressed in high-litter size spermatozoa (>3-fold). On the other hand, equatorin, beta-tubulin, cytochrome b-c1 complex subunit 1 (UQCRC1), speriolin, Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and seminal plasma sperm motility inhibitor were abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A and UQCRC1 expression negatively correlated with litter size, while UQCRC2 expression positively correlated with litter size. Finally, the putative biomarkers predicted litter size in field trials. Our study suggests that biomarkers present in spermatozoa after capacitation can help differentiate superior male fertility from below-average fertility with high sensitivity.
Physiologycal Studies of Discorvered Biomarkers
Arginine vasopressin (VP) is neurohypophysial hormone has been implicated in stimulating contractile activity of the male reproductive tract in the testis. Higher levels of VP decrease sperm count and motility. However, very little is known about the involvement of VP in controlling mammalian reproductive process. The goal of this study was to confirm that effect of VP receptor (AVPR2) on sperm function in capacitation condition. Deamino [Cys 1, D-ArgS] vasopressin (dDAVP), an AVPR2 agonist that operates only on AVPR2, was used. Also, Mouse spermatozoa were incubated with various concentrations of dDAVP (10-11-10-5 M) and sperm motility, capacitation status, Protein Kinase A activity (PKA), tyrosine phosphorylation, fertilization, and embryo development were assessed using computer-assisted sperm analysis, Combined Hoechst 33258/chlortetracycline fluorescence, Western blotting, and in vitro fertilization, respectively. AVPR2 was placed on the acrosome region and mid-piece in cauda epididymal spermatozoa, but the caput epididymal spermatozoa was mid-piece only. The high dDAVP treatment (10-8 and 10-5 M) significantly decreased sperm motility, intracellular pH and PKA substrates (approximately 55 and 22 kDa) and increased Ca2+ concentration. The highest concentration treatment significantly decreased PKA substrate (approximately 23 kDa) and tyrosine phosphorylation (approximately 30 kDa). VP detrimentally affected capacitation, acrosome reaction, and embryo development. Treatment with the lowest concentration (10-11 M) was not significantly different. Our data have shown that VP stimulates ion transport across sperm membrane through interactions with AVPR2. VP has a detrimental effect in sperm function, fertilization, and embryonic development, suggesting its critical role in the acquisition of fertilizing ability of mouse spermatozoa. These research finding will enable further to determine molecular mechanism associated with fertility in capacitation and fertilization. It is also an important pivotal precondition to the progress of diagnostic test to identify infertility and to apply male contraception.
Objective: To examine how VDAC regulates sperm function in capacitation conditions
Design: Experimental prospective study.
Setting: Academic basic research laboratory.
Animal(s): Male ICR and Female B6D2F1 / CrljOri mice (8-12 weeks)
Intervention(s): Female mice were superovulated with 5 IU of pregnant mare serum gonadotrophin (PMSG) given i.p., and 5 IU of human chorionic gonadotrophin (hCG) given i.p. 48 h later. Oocytes were applied to assess fertilization and embryo development.
Main Outcome Measure(s): Immunofluorescence Assay, Computer-Assisted Sperm Analysis, Hypo-osmotic Swelling Test, Combined Hoechst 33258/Chlortetracycline Fluorescence Assessment of Capacitation Status, Measurement of [Ca2+]i and [pH]i, Western Blotting, In Vitro Fertilization
Result(s): VDAC2 was localized on the acrosomal region and principal piece, while VDAC3 was localized on the acrosomal region and midpiece. Blocking VDAC with DIDS (500μM) significantly decreased motility, viability, acrosome reaction, capacitation, tyrosine phosphorylation, fertilization, and embryo development regardless of Ca2+. However, the most severe decreases were observed in the presence (+) of DIDS and absence (-) of Ca2+, respectively. A significant decrease in [Ca2+]i concentration was observed in (-) DIDS, while [pH]i was significantly increased in (-) DIDS regardless of Ca2+. However, a significantly elevated [pH]i was observed in (+) Ca2+.
Conclusion(s): Abnormal regulation of VDACs negatively affected sperm function. Thus, VDACs may be key regulators of the fertilization ability of spermatozoa.