Abstract Study question Does Ritalin (DAT antagonist) and dopamine (DAT agonist), influence human sperm parameters, and do these act via the DAT protein? Summary answer DAT is positively expressed on human sperm membranes with dopamine regulating sperm functions through mechanisms involving DAT whereas Ritalin does not affect sperm parameters. What is known already Spermatozoa exhibit a biphasic dose-response to dopamine: low concentrations enhance key sperm functions such as motility and viability, while high concentrations impair them. This biphasic response suggests the presence of a dopamine transporter (DAT), however its role in human sperm function remains under-investigated. Concerns regarding the unprescribed use of Ritalin, a dopamine reuptake inhibitor, are growing among men of reproductive age, given its potential to mimic dopamine’s negative effects on sperm function. Further research is crucial to elucidate the mechanisms and potential consequences of Ritalin exposure on male fertility. Study design, size, duration Twenty samples were collected and utilized to identify and localize DAT via indirect Immunofluorescence (IF) assays. Thirty samples were collected and sperm were treated at 37 °C to 100 nM, 10 μM, and 1 mM dopamine concentrations for 1, 3 and 6 hours; similarly sperm were exposed to 4 ng/mL, 30 ng/mL, and 100 ng/mL Ritalin concentrations for 1, 2 and 3 hours, whereafter the effects on basic and advanced sperm parameters were investigated. Participants/materials, setting, methods Participants were recruited from the Faculty of Medicine and Health Sciences at Stellenbosch University, Cape Town, South Africa. Male students aged 18-29 years donated a semen sample via masturbation. An indirect IF assay, adapted from Ramírez et al. (2009), was utilized to identify DAT. Computer-Aided Sperm Analysis (CASA) was employed to determine motility parameters. Viability was assessed via a dye-exclusion staining technique and the acrosome reaction was assessed via fluorescent microscopy. Main results and the role of chance Immunofluorescence confirmed the presence of the Dopamine Transporter (DAT) on human sperm, primarily localized in the mid-piece and tail regions. Interestingly, dopamine exhibited a biphasic effect on sperm function. Low doses offered protection, while high doses (1 mM) negatively impacted motility (total and progressive motility) and kinematic parameters (Curvilinear Velocity (VCL) and Average Path Velocity (VAP)) as early as 60 minutes, and significantly decreased viability after 3 hours. Additionally, the high dose prematurely triggered the acrosome reaction. Conversely, Ritalin, even at supraphysiological concentrations (100 ng/mL), showed no significant impact on basic or advanced sperm parameters. This suggests that, unlike high-dose dopamine, Ritalin exposure might not negatively affect sperm function within the tested parameters. This study highlights the complex role of dopamine in sperm function and emphasizes the need for further research to clarify potential consequences of high dopamine exposure or dopaminergic drugs such as Ritalin. Limitations, reasons for caution Ejaculated spermatozoa are transcriptionally and translationally silent, therefore the in vitro nature of this study is a limitation. The physiological relevance needs to be assessed in an in vivo system, additionally the treatments (dopamine plus Ritalin) should be investigated in combination to demonstrate their concurrent existence within a system. Wider implications of the findings These findings suggest a possible regulatory role for DAT inhibitory drugs on sperm parameters and cautions users thereof. Dopamine may be utilized as a physiological regulator of in vivo fertilization and should be further investigated with regards to Assisted Reproductive Techniques (ART’s). Trial registration number not applicable
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