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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Inhibitor dose-dependent effects of IODO and 3BP.(A) iSperm and (B) AMV from our paper-based MTT assay following treatment with 0, 0.25, 0.5, 1, and 2 mM of IODO at 37.5 °C. (C) motility of iSperm and (D) AMVof 3BP-treated and untreated samples. 3BP concentration was 0, 0.2, 0.4, 0.6, or 0.8 mM in this experiment. Error bars are standard deviation (SD) of three samples in each point (N = 3). Asterisks show the difference with the value of control group without inhibitor (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384208&req=5

f3: Inhibitor dose-dependent effects of IODO and 3BP.(A) iSperm and (B) AMV from our paper-based MTT assay following treatment with 0, 0.25, 0.5, 1, and 2 mM of IODO at 37.5 °C. (C) motility of iSperm and (D) AMVof 3BP-treated and untreated samples. 3BP concentration was 0, 0.2, 0.4, 0.6, or 0.8 mM in this experiment. Error bars are standard deviation (SD) of three samples in each point (N = 3). Asterisks show the difference with the value of control group without inhibitor (P < 0.05).

Mentions: iSperm and paper-based MTT assay results indicate that IODO and 3BP more strongly inhibited sperm motility and reduced AMV than other inhibitors. We investigated the dose-dependent effects of both IODO and 3BP and summarized our results in Fig. 3. When we exposed porcine sperm to 2 mM IODO, we found significant differences in motility between the inhibitor-treated and control samples from the start of detection (0 min), suggesting that this inhibitor reduced sperm motility immediately. Sperm motility and related enzyme function were sensitive to inhibitor, however detection of MTT reduction exhibited a duller response. Based on a comparison between Fig. 3A and B, when sperm motility was more than 50%, we could not find a difference in MTT reduction using our paper-based device. Soon after inhibitor treatments, decrease of MTT reduction was very small at 0 and 20 min because accumulated reductants such as NADH were not fully oxidized in sperm. With increasing treatment time, some of the accumulated reductants were oxidized by cell oxidants as sperm motility decreased. Therefore, sperm motility inhibition was observed immediately. However, detection of inhibitor function for MTT reduction requires a longer time interval than the time required for sperm motility decrease evaluation. Applications of 1 and 0.5 mM of IODO produced significant decreases in sperm motility, but only after 20 and 60 min, respectively as shown in Fig. 3A (P < 0.05). AMV, however, was only decreased significantly following 20 min of exposure to 2 mM IODO (P < 0.05) (Fig. 3B). AMV (and GAPDH activity) was not affected by treatment with 1 mM or 0.5 mM IODO. We postulate that our MTT assay may be less sensitive to inhibition response, because other enzymatic reactions still occur in IODO-treated samples.


Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices
Inhibitor dose-dependent effects of IODO and 3BP.(A) iSperm and (B) AMV from our paper-based MTT assay following treatment with 0, 0.25, 0.5, 1, and 2 mM of IODO at 37.5 °C. (C) motility of iSperm and (D) AMVof 3BP-treated and untreated samples. 3BP concentration was 0, 0.2, 0.4, 0.6, or 0.8 mM in this experiment. Error bars are standard deviation (SD) of three samples in each point (N = 3). Asterisks show the difference with the value of control group without inhibitor (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384208&req=5

f3: Inhibitor dose-dependent effects of IODO and 3BP.(A) iSperm and (B) AMV from our paper-based MTT assay following treatment with 0, 0.25, 0.5, 1, and 2 mM of IODO at 37.5 °C. (C) motility of iSperm and (D) AMVof 3BP-treated and untreated samples. 3BP concentration was 0, 0.2, 0.4, 0.6, or 0.8 mM in this experiment. Error bars are standard deviation (SD) of three samples in each point (N = 3). Asterisks show the difference with the value of control group without inhibitor (P < 0.05).
Mentions: iSperm and paper-based MTT assay results indicate that IODO and 3BP more strongly inhibited sperm motility and reduced AMV than other inhibitors. We investigated the dose-dependent effects of both IODO and 3BP and summarized our results in Fig. 3. When we exposed porcine sperm to 2 mM IODO, we found significant differences in motility between the inhibitor-treated and control samples from the start of detection (0 min), suggesting that this inhibitor reduced sperm motility immediately. Sperm motility and related enzyme function were sensitive to inhibitor, however detection of MTT reduction exhibited a duller response. Based on a comparison between Fig. 3A and B, when sperm motility was more than 50%, we could not find a difference in MTT reduction using our paper-based device. Soon after inhibitor treatments, decrease of MTT reduction was very small at 0 and 20 min because accumulated reductants such as NADH were not fully oxidized in sperm. With increasing treatment time, some of the accumulated reductants were oxidized by cell oxidants as sperm motility decreased. Therefore, sperm motility inhibition was observed immediately. However, detection of inhibitor function for MTT reduction requires a longer time interval than the time required for sperm motility decrease evaluation. Applications of 1 and 0.5 mM of IODO produced significant decreases in sperm motility, but only after 20 and 60 min, respectively as shown in Fig. 3A (P < 0.05). AMV, however, was only decreased significantly following 20 min of exposure to 2 mM IODO (P < 0.05) (Fig. 3B). AMV (and GAPDH activity) was not affected by treatment with 1 mM or 0.5 mM IODO. We postulate that our MTT assay may be less sensitive to inhibition response, because other enzymatic reactions still occur in IODO-treated samples.

View Article: PubMed Central - PubMed

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&thinsp;&lt;&thinsp;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.

No MeSH data available.