Effects of insulin and leptin on human spermatozoa function and their cross-talk with nitric oxide and cytokines

[摘要] ENGLISH ABSTRACT: In recent years there has been an increase in obesity and diabetes mellitus (DM).These conditions have for a long time been associated with infertility. Obesity ischaracterized by high levels of circulating leptin and cytokines as well as insulinresistance. Type I DM is associated with low or no insulin whereas, Type II DM ischaracterised by insulin resistance. As the prevalence of obesity and DM continuesto rise, it is likely that the incidence of infertility associated with these pathologicalconditions will likewise increase. The effects of insulin and leptin on malereproductive function have been reported on the endocrine and spermatogenesislevel, but their effects on cellular level of human ejaculated spermatozoa are yet tobe elucidated.This study presents data on the role of insulin and leptin on human ejaculatedspermatozoa and their interaction with cytokines and nitric oxide. In the first part ofthe study, we established the suitable concentrations of glucose, insulin and leptinthat could be administered to human spermatozoa in vitro. Glucose concentration of5.6 mM was chosen as the suitable concentration to be administered to humanspermatozoa because it has previously been reported in the literature; furthermore, itis within the range of the physiological glucose levels found in the blood of fastinghumans. Insulin and leptin concentrations of 10 μIU and 10 nmol were chosenrespectively because they gave much improved sperm function and this was withinthe range of insulin and leptin levels previously measured in human ejaculatedspermatozoa. This was followed by investigating the signalling pathway of insulin andits beneficial effects on human spermatozoa function. Endogenous insulin secretionfrom human ejaculated spermatozoa was blocked by nifedipine and its receptortyrosine phosphorylation effects were inhibited by erbstatin while phosphatidylinositol3-kinase (PI3K) phosphorylation activity was inhibited by wortmannin. Exogenousinsulin administration significantly increased human sperm motility parameters aswell as the sperm ability to acrosome react. The inhibition of endogenous insulinrelease from spermatozoa as well as the inhibition of the insulin receptor substrate(IRS) tyrosine phosphorylation significantly decreased motility parameters and theability of spermatozoa to acrosome react.The study also investigated the effects of insulin and leptin on human sperm motility,viability, acrosome reaction and nitric oxide (NO) production. Both insulin and leptinsignificantly increased sperm motility parameters, acrosome reaction and NOproduction. The NO production induced by insulin and leptin was via PI3K signallingas evidenced by a reduction in NO levels when PI3K activity was inhibited bywortmannin. To investigate whether insulin and leptin could improve motilityparameters of asthernozoospermic and teratozoospermic spermatozoa, thespermatozoa were separated into two fractions by means of a double densitygradient technique. The gradient system was able to separate spermatozoa into highmorphologically abnormal and less motile spermatozoa similar to that ofasthernozoospermic and teratozoospermic patients as well as a more motile fraction.Insulin and leptin significantly increased the motility parameters of spermatozoa fromthe immature and less motile fraction.The fourth part of the study was aimed at investigating the effects of the cytokines,tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), on human spermmotility, viability, acrosome reaction and NO production. The study shows that TNF-αand IL-6 significantly reduced motility parameters and acrosome reaction in a dose4and time-dependent manner. These cytokines were also shown to significantlyincrease NO production from human spermatozoa. The decreased motilityparameters induced by these cytokines could be attributed to their ability to induceexcessive NO production. It is not yet clear how they inhibit spermatozoa to undergothe acrosome reaction.The fifth part of the study was to investigate the expression and localization ofglucose transporter 8 (GLUT8) in human spermatozoa. This study shows that GLUT8is constitutively expressed and located in the midpiece region of the humanspermatozoa. The study also showed that stimulating spermatozoa with insulin led toan increase in GLUT8 expression as well as translocation to the acrosomal region.In the last part of the study we wanted to investigate why the increase in NOgeneration by spermatozoa due to insulin and leptin stimulation is accompanied withincreased sperm function whereas NO increased due to TNF-α and IL-6 stimulationis accompanied with decreased sperm function. We observed that TNF-α and IL-6not only increased NO production but also ROS production. This study speculatesthat the decrease in sperm motility and acrosome reaction when TNF-α and IL-6were administered was due to the concomitant high increase in NO and ROS theyinduced.In conclusion, this study has established in vitro beneficial effects of insulin and leptinin normozoospermic and asthernozoospermic human sperm function. Thesehormones influence sperm function via the PI3K signalling pathway in two ways.Firstly, by increasing GLUT8 expression and translocation thereby possiblyincreasing glucose uptake and metabolism and secondly, by increasing NOproduction. The study has also established that TNF-α and IL-6 have detrimentaleffects on human spermatozoa in a dose and time dependent manner. These effectsare mediated via their ability to stimulate both NO and ROS production in humanspermatozoa. This study reports that GLUT8 is expressed in the midpiece region ofhuman spermatozoa and that insulin stimulation upgrades its expression and leads toits translocation to the acrosomal region.

[发布机构] Stellenbosch University