The Use of Anti-VDAC2 Antibody for the Combined Assessment of Human Sperm Acrosome Integrity and Ionophore A23187-Induced Acrosome Reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca2+ increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca2+ transmembrane transport

Detection of Alpha-Fetoprotein Using Aptamer-Based Sensors

Alpha-fetoprotein (AFP) is widely-known as the most commonly used protein biomarker for liver cancer diagnosis at the early stage. Therefore, developing the highly sensitive and reliable method of AFP detection is of essential demand for practical applications. Herein, two types of aptamer-based AFP detection methods, i.e., optical and electrochemical biosensors, are reviewed in detail. The optical biosensors include Raman spectroscopy, dual-polarization interferometry, resonance light-scattering, fluorescence, and chemiluminescence. The electrochemical biosensors include cyclic voltammetry, electrochemical impedance spectroscopy, and giant magnetic impedance. Looking into the future, methods for AFP detection that are high sensitivity, long-term stability, low cost, and operation convenience will continue to be developed

Effect of anti-VDAC2 antibody on acrosome reaction.

<p>A: spermatozoa were incubated for 2 h, loaded with normal mouse IgG or anti-VDAC2antibody at different dilutions, and counted with or without A23187. B: spermatozoa were incubated for 4 h, loaded with normal mouse IgG or anti-VDAC2antibody at different dilutions, and counted with or without A23187. At least 200 spermatozoa were counted in each sample. Data were shown as mean ± SEM of six experiments.</p

Detection of Pb2+ in Tea Using Aptamer Labeled with AIEgen Nanospheres Based on MOFs Sensors

Tea is an important economic crop and health beverage in China. The presence of heavy metal ions in tea poses a significant threat to public health. Here, we prepared an aptamer biosensor labelled with AIEgen nanospheres to detect Pb2+ in tea. The dsDNA modified by amino and phosphoric acid was combined with the carboxylated AIEgen NPs to form AIEgen-DNA with a fluorescence group, which was then fixed to the surface of Zr-MOFs to quench the fluorescence of AIEgen NPs. At the same time, PEG was added to remove nonspecific adsorption. Then Pb2+ was added to cut the DNA sequences containing the cutting sites, and AIEgen NPs and part of the DNA sequences were separated from the Zr-MOFs surface to recover the fluorescence. By comparing the fluorescence changes before and after adding Pb2+, the detection limit of Pb2+ can reach 1.70 nM. The fluorescence sensor was applied to detect Pb2+ in tea, and the detection results showed that the tea purchased on the market did not contain the concentration of Pb2+ within the detection range. This study provides new insights into monitoring food and agriculture-related pollutants based on fluorescent biosensors

Effect of anti-VDAC2 antibody on acrosomal integrity.

<p>A: spermatozoa were incubated with normal mouse IgG or anti-VDAC2antibody at different dilutions and counted according to the different incubation time. B: spermatozoa were incubated for the different time and counted according to different treatments. At least 200 spermatozoa were counted in each sample. Data were shown as mean ± SEM of six experiments; *, P < 0.05 versus control.</p

Effect of anti-VDAC2 antibody on A23187-induced Ca²⁺ influx.

<p>Spermatozoa were incubated for 2 h, then loaded with anti-VDAC2 antibody (yellow), normal mouse IgG (blue), or without any antibody (red), and finally stimulated by A23187. The changes of fluorescence intensity reflecting intracellular Ca²⁺ concentration were recorded by the Microplate Reader.</p

FITC-PSA staining of human spermatozoa in different treatment groups.

<p>A–C: spermatozoa were separated and stained immediately. D–F: spermatozoa were incubated with anti-VDAC2 antibody. G–I: spermatozoa were incubated with normal mouse IgG. J–L: spermatozoa were incubated without any antibody. A, D, G and J: phase-contrast images; B, E, H and K: immunofluorescent images; C, F, I and L: merged images. Magnification was ×1000.</p

Antibody specificity and native VDAC2 in human spermatozoa analyzed by western blot.

<p>A: recombinant VDAC1 (lane 1), VDAC2 (lane 2) and VDAC3 (lane 3) proteins were probed with anti-VDAC2 antibody. B: the hydrophobic membrane protein extracts from human spermatozoa were separated by SDS-PAGE and probed with anti-VDAC2 antibody (lane 1) or the same antibody pre-absorbed with an excess of recombinant VDAC2 protein. The position of molecular weight standards (kDa) is indicated on the left.</p