Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

Koji Matsuura,Ming-Cheng Chen,Yu Chen,Han-Wei Huang,Chao-Min Cheng

doi:10.1038/srep46213

Abstract

Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

Highlights

Sperm motility and sperm motion velocities can be tracked using computer-assisted semen analysis (CASA) an approach that refers to a variety of automatic or semi-automatic, often image-tracking, approaches to indicate fertility in analyzed semen
Because mammalian sperm motility is expressed by motor protein function using adenosine triphosphate (ATP) catalysis, ATP shuttle system, and ATP production system[16,17], analyses of sperm cell chemical reactions may be used as potential indicators of sperm motility[17]
Because MTT reduction must be coupled with redox reaction during sperm ATP production, and this system includes redox mediator reactions such as nicotinamide adenine dinucleotide (NADH)[25,26], the effectiveness of an MTT reduction assay for sperm motility analysis can be strengthened based on simultaneous sperm motility analysis and an MTT assay examining inhibition of mitochondrial or glycolytic enzymatic functions

Summary

Introduction

Sperm motility and sperm motion velocities can be tracked using computer-assisted semen analysis (CASA) an approach that refers to a variety of automatic or semi-automatic, often image-tracking, approaches to indicate fertility in analyzed semen. Our paper-based MTT assay experimental results suggest that MTT color intensity changes by MTT reduction could be correlated to sperm motility, so this system may be applicable for fertility examinations[18,19]. These results were consistent with other research groups reporting on the use of paper-based MTT assays for human sperm motility[20]. Because MTT reduction must be coupled with redox reaction during sperm ATP production, and this system includes redox mediator reactions such as nicotinamide adenine dinucleotide (reduced form) (NADH)[25,26], the effectiveness of an MTT reduction assay for sperm motility analysis can be strengthened based on simultaneous sperm motility analysis and an MTT assay examining inhibition of mitochondrial or glycolytic enzymatic functions.

Methods

Results

Discussion

Conclusion