The identification of the Dopamine Transporter (DAT) and the effects of dopamine on human sperm parameters
Date
2021-03
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
Thesis (MMed)--Stellenbosch University, 2021.
Introduction: Catecholamines such as dopamine are found throughout the mammalian reproductive system, where they are hypothesized to play some role in fertilization. A dopaminergic phenotype has been discovered in the male spermatozoan of many mammalian species. Dopamine regulates a wide range of functions including capacitation, and the induction of sperm motility and the acrosome reaction through mechanisms involving dopamine receptor (D2) activation. A dose-dependent response to dopamine has been observed in sperm, whereby low concentrations were shown to be protective and high concentrations were observed to negatively impact sperm functions. This biphasic effect can only be explained by the existence of a transporter protein; however, the Dopamine Transporter (DAT) is yet to be well explored on human sperm membranes. Objectives: To identify DAT on human sperm membranes and determine the effects of dopamine, at high, medium, and low concentrations, on human sperm parameters. Methods: Utilizing an indirect immunofluorescence assay, spermatozoa were stained with a primary anti-DAT antibody against the fluorescent secondary Alexa Fluor® 488. Fluorescence was visualized on a confocal microscope. Western blot analyses were employed as a second assay to show the presence of DAT on human sperm membranes with rat brain tissue used as a positive control. The membranes were exposed to anti-DAT primary antibody against a Horseradish Peroxidase (HRP)-linked secondary antibody. Furthermore, the effect of dopamine on sperm parameters, at different dopamine concentrations were tested for three time periods (0, 1, 3 and 6 hours). Sperm were exposed to high (1 mM), medium (10 μM) and low (100 nM) dopamine concentrations at 37°C for 1, 3 and 6 hours, thereafter functional sperm parameters were analysed. Using CASA and the SCA® software system, total motility, progressive motility, and sperm kinematics were analysed. Sperm viability and the acrosome reaction were additionally assessed. Results: The immunofluorescence assay indicates a strong immunoreaction in the positive controls vs. the negative controls and it could conclude that DAT is positively expressed on human sperm membranes. Western blot analyses further confirmed the existence of DAT in human sperm membranes when compared to the positive brain controls. Protein bands of 62 kDa and 48 kDa appear in the Western Blot lanes loaded with sperm samples, thereby positively correlating with bands found in the brain tissue. High concentrations of dopamine were found to negatively affect motility parameters as early as 60 minutes (43.05% ± 12.72 vs. 18.75% ± 8.75). Sperm kinematics were also negatively affected by the addition of high concentrations of dopamine when compared to the controls. Sperm viability was significantly ecreased by high dopamine concentration after 3 hours of incubation (51.86% ± 7.220 vs. 34.36% ± 13.03). The high concentration of dopamine was found to elicit a premature acrosome reaction upon addition of treatment (26.88% ± 3.199 vs. 31.91% ± 2.85). Conclusion: It can be concluded that DAT is found on human sperm membranes and that dopamine regulates sperm functions through mechanisms involving the dopamine D2 receptor and DAT. These findings suggest a possible regulatory role for DAT inhibitory drugs such as Ritalin on sperm parameters.
Inleiding: Katekolamiene soos dopamien kom dwarsdeur die voortplantingstelsel van soogdiere voor, waar hulle veronderstel is om 'n rol in bevrugting te speel. 'n Dopaminerge fenotipe is in die spermatozoë van baie soogdierspesies ontdek. Dopamien reguleer 'n wye verskeidenheid funksies, insluitend kapasitering, en die induksie van spermmotiliteit en die akrosoomreaksie deur meganismes wat dopamienreseptoraktivering (D2) insluit. 'n Dosisafhanklike reaksie op dopamien is waargeneem in sperma, waardeur lae konsentrasies beskermend is en hoë konsentrasies spermfunksies negatief beïnvloed. Hierdie bifasiese effek kan slegs verklaar word deur die bestaan van 'n vervoerderproteïen; die Dopamien-vervoeder (DAT) moet egter nog goed ondersoek word op menslike spermmembrane. Doelstellings: Om DAT op menslike spermmembrane te identifiseer en die effek van dopamien teen hoë, medium en lae konsentrasies op menslike spermparameters te bepaal. Metodes: Met behulp van 'n indirekte immunofluoressensie toets is spermatozoa gekleur met 'n primêre anti-DAT-teenliggaam teen die fluoresserende sekondêre Alexa Fluor® 488. Fluoressensie is in 'n konfokale mikroskoop gevisualiseer. Western Blot ontledings is gebruik as 'n tweede toets om die teenwoordigheid van DAT op menslike spermmembrane met rotbreinweefsel as 'n positiewe kontrole te toon. Die membrane is blootgestel aan anti-DAT primêre teenliggaam teen 'n Horseradish Peroxidase (HRP)-gekoppelde sekondêre teenliggaam. Verder is die effek van dopamien op spermparameters by verskillende dopamienkonsentrasies vir drie tydperke (0, 1, 3 en 6 uur) getoets. Sperma is gedurende 1, 3 en 6 uur aan hoë (1 mM), medium (10 μM) en lae (100 nM) dopamienkonsentrasies by 37 ° C blootgestel, waarna funksionele spermparameters geanaliseer is. Met behulp van CASA en die SCA® sagtewarestelsel is totale beweeglikheid, progressiewe beweeglikheid en spermakinematika geanaliseer. Daarbenewens is die lewensvatbaarheid van die saad en die akrosoomreaksie beoordeel. Resultate: Die immunofluoressensie toets dui op 'n sterk immuunreaksie in die positiewe kontroles teenoor die negatiewe kontroles, en dit kan aflei dat DAT positief tot uitdrukking kom op menslike spermmembrane. Western Blot ontledings het die bestaan van DAT in menslike spermmembrane bevestig in vergelyking met die positiewe breinkontroles. Proteïenbande van 62 kDa en 48 kDa verskyn in die Western Blot-bane gelaai met spermmonsters, wat sodoende positief korreleer met bande wat in die breinweefsel voorkom. Daar is gevind dat hoë konsentrasies dopamien beweeglikheidsparameters negatief beïnvloed al in 60 minute (43,05% ± 12,72 teenoor 18,75% ± 8,75). Sperma-kinematika is ook negatief beïnvloed deur die toevoeging van hoë konsentrasies dopamien in vergelyking met die kontroles. Sperma- lewensvatbaarheid is aansienlik verminder deur hoë dopamienkonsentrasie na 3 uur inkubasie (51,86% ± 7,220 versus 34,36% ± 13,03). Daar is gevind dat die hoë konsentrasie dopamien 'n voortydige akrosoomreaksie uitlok by toevoeging van die behandeling (26,88% ± 3,199 versus 31,91% ± 2,85). Gevolgtrekking: Daar kan tot die gevolgtrekking gekom word dat DAT op menslike spermmembrane voorkom en dat dopamien spermfunksies reguleer deur meganismes wat die dopamien D2-reseptor en DAT insluit. Hierdie bevindings dui op 'n moontlike regulerende rol vir DAT-remmende middels soos Ritalin op spermparameters.
Introduction: Catecholamines such as dopamine are found throughout the mammalian reproductive system, where they are hypothesized to play some role in fertilization. A dopaminergic phenotype has been discovered in the male spermatozoan of many mammalian species. Dopamine regulates a wide range of functions including capacitation, and the induction of sperm motility and the acrosome reaction through mechanisms involving dopamine receptor (D2) activation. A dose-dependent response to dopamine has been observed in sperm, whereby low concentrations were shown to be protective and high concentrations were observed to negatively impact sperm functions. This biphasic effect can only be explained by the existence of a transporter protein; however, the Dopamine Transporter (DAT) is yet to be well explored on human sperm membranes. Objectives: To identify DAT on human sperm membranes and determine the effects of dopamine, at high, medium, and low concentrations, on human sperm parameters. Methods: Utilizing an indirect immunofluorescence assay, spermatozoa were stained with a primary anti-DAT antibody against the fluorescent secondary Alexa Fluor® 488. Fluorescence was visualized on a confocal microscope. Western blot analyses were employed as a second assay to show the presence of DAT on human sperm membranes with rat brain tissue used as a positive control. The membranes were exposed to anti-DAT primary antibody against a Horseradish Peroxidase (HRP)-linked secondary antibody. Furthermore, the effect of dopamine on sperm parameters, at different dopamine concentrations were tested for three time periods (0, 1, 3 and 6 hours). Sperm were exposed to high (1 mM), medium (10 μM) and low (100 nM) dopamine concentrations at 37°C for 1, 3 and 6 hours, thereafter functional sperm parameters were analysed. Using CASA and the SCA® software system, total motility, progressive motility, and sperm kinematics were analysed. Sperm viability and the acrosome reaction were additionally assessed. Results: The immunofluorescence assay indicates a strong immunoreaction in the positive controls vs. the negative controls and it could conclude that DAT is positively expressed on human sperm membranes. Western blot analyses further confirmed the existence of DAT in human sperm membranes when compared to the positive brain controls. Protein bands of 62 kDa and 48 kDa appear in the Western Blot lanes loaded with sperm samples, thereby positively correlating with bands found in the brain tissue. High concentrations of dopamine were found to negatively affect motility parameters as early as 60 minutes (43.05% ± 12.72 vs. 18.75% ± 8.75). Sperm kinematics were also negatively affected by the addition of high concentrations of dopamine when compared to the controls. Sperm viability was significantly ecreased by high dopamine concentration after 3 hours of incubation (51.86% ± 7.220 vs. 34.36% ± 13.03). The high concentration of dopamine was found to elicit a premature acrosome reaction upon addition of treatment (26.88% ± 3.199 vs. 31.91% ± 2.85). Conclusion: It can be concluded that DAT is found on human sperm membranes and that dopamine regulates sperm functions through mechanisms involving the dopamine D2 receptor and DAT. These findings suggest a possible regulatory role for DAT inhibitory drugs such as Ritalin on sperm parameters.
Inleiding: Katekolamiene soos dopamien kom dwarsdeur die voortplantingstelsel van soogdiere voor, waar hulle veronderstel is om 'n rol in bevrugting te speel. 'n Dopaminerge fenotipe is in die spermatozoë van baie soogdierspesies ontdek. Dopamien reguleer 'n wye verskeidenheid funksies, insluitend kapasitering, en die induksie van spermmotiliteit en die akrosoomreaksie deur meganismes wat dopamienreseptoraktivering (D2) insluit. 'n Dosisafhanklike reaksie op dopamien is waargeneem in sperma, waardeur lae konsentrasies beskermend is en hoë konsentrasies spermfunksies negatief beïnvloed. Hierdie bifasiese effek kan slegs verklaar word deur die bestaan van 'n vervoerderproteïen; die Dopamien-vervoeder (DAT) moet egter nog goed ondersoek word op menslike spermmembrane. Doelstellings: Om DAT op menslike spermmembrane te identifiseer en die effek van dopamien teen hoë, medium en lae konsentrasies op menslike spermparameters te bepaal. Metodes: Met behulp van 'n indirekte immunofluoressensie toets is spermatozoa gekleur met 'n primêre anti-DAT-teenliggaam teen die fluoresserende sekondêre Alexa Fluor® 488. Fluoressensie is in 'n konfokale mikroskoop gevisualiseer. Western Blot ontledings is gebruik as 'n tweede toets om die teenwoordigheid van DAT op menslike spermmembrane met rotbreinweefsel as 'n positiewe kontrole te toon. Die membrane is blootgestel aan anti-DAT primêre teenliggaam teen 'n Horseradish Peroxidase (HRP)-gekoppelde sekondêre teenliggaam. Verder is die effek van dopamien op spermparameters by verskillende dopamienkonsentrasies vir drie tydperke (0, 1, 3 en 6 uur) getoets. Sperma is gedurende 1, 3 en 6 uur aan hoë (1 mM), medium (10 μM) en lae (100 nM) dopamienkonsentrasies by 37 ° C blootgestel, waarna funksionele spermparameters geanaliseer is. Met behulp van CASA en die SCA® sagtewarestelsel is totale beweeglikheid, progressiewe beweeglikheid en spermakinematika geanaliseer. Daarbenewens is die lewensvatbaarheid van die saad en die akrosoomreaksie beoordeel. Resultate: Die immunofluoressensie toets dui op 'n sterk immuunreaksie in die positiewe kontroles teenoor die negatiewe kontroles, en dit kan aflei dat DAT positief tot uitdrukking kom op menslike spermmembrane. Western Blot ontledings het die bestaan van DAT in menslike spermmembrane bevestig in vergelyking met die positiewe breinkontroles. Proteïenbande van 62 kDa en 48 kDa verskyn in die Western Blot-bane gelaai met spermmonsters, wat sodoende positief korreleer met bande wat in die breinweefsel voorkom. Daar is gevind dat hoë konsentrasies dopamien beweeglikheidsparameters negatief beïnvloed al in 60 minute (43,05% ± 12,72 teenoor 18,75% ± 8,75). Sperma-kinematika is ook negatief beïnvloed deur die toevoeging van hoë konsentrasies dopamien in vergelyking met die kontroles. Sperma- lewensvatbaarheid is aansienlik verminder deur hoë dopamienkonsentrasie na 3 uur inkubasie (51,86% ± 7,220 versus 34,36% ± 13,03). Daar is gevind dat die hoë konsentrasie dopamien 'n voortydige akrosoomreaksie uitlok by toevoeging van die behandeling (26,88% ± 3,199 versus 31,91% ± 2,85). Gevolgtrekking: Daar kan tot die gevolgtrekking gekom word dat DAT op menslike spermmembrane voorkom en dat dopamien spermfunksies reguleer deur meganismes wat die dopamien D2-reseptor en DAT insluit. Hierdie bevindings dui op 'n moontlike regulerende rol vir DAT-remmende middels soos Ritalin op spermparameters.
Description
Keywords
Human sperm membranes, UCTD, Dopamine, Mammalian -- Reproductive system, Dopamine Transporter (DAT), Spermatozoa