Testosterone Propionate Increased The Expression Of VMAT2in Rats

The vesicular monoamine transporter type2(VMAT2) is atransmembrane glycoprotein and located in the plasmic membrane of synapticvesicles of monoaminergic neurons. It is constitutively expressed asglycosylated (75KDa), partially glycosylated (55KDa) and native (45KDa)forms. Glyco-VMAT2is the mature form, which uptakes cytosolicmonoamines into synaptic vesicles after monomine neurotransmitters aresynthesized from their precursors, and control the cytosolic level of freemonoamines. Decreased expression of VMAT2transporter expression wouldreduce the available monoamine neurotransmitters in synaptic vesicles, andlead to high level of cytosolic free monoamine neurotransmitters, thus,increases oxygen radicals. Increased reactive oxygen species interfere thefunction of mitochondrion, which results in monoaminergicneurodegeneration.Monoamine neurotransmitters include dopamine (DA), serotonin (5-HT),norepinephrine (NE), epinephrine and histamine. They all have a amino group,which can be oxidized by monoamine oxidase to produce oxygen radicals. It isshowed that monoaminergic neurons are involved in the regulations of neuralactivity in vivo, and their dysfunction could lead to neurological or psychiatricdiseases. Data from PET or SPECT studies revealed the abnormalligand-receptor imaging related to two or more monoaminergic systems insome patients afflicted with neuropsychiatric disorders, such as Parkinson'sdisease (PD). In addition to motor symptomes resulting from theneurodegeneration of dopaminergic neurons in substantia nigra (SN), PDpatients also have nonmotor symptoms, such as depression, which is related tothe impaired serotonergic neurons in dorsal raphe nucleus. It was found thatnigrostriatal VMAT2protein was significantly reduced in PD patients. It was hypothesized that reduced VMAT2resulted in the increased cytosolic freeDA/5-HT and oxygen radical, which would lead to the degeneration ofdopaminergic and serotonergic neurons. Up-regulation of VMAT2expressionwould prevent monominergic neurons from toxic effects of reactive oxygenspecies, especially when they synthesize much more monomineneurotransmitters in compensatory mechanisms.Besides reproductive function, androgen is also involved in the regulationof the neural structures and activities. Testosterone secretion decreasesgradually during aging, while some neuropsychiatric disorders mainly occurein aged men, e.g. PD. Whether their onset is related to androgen is stillunknown. Clinical data showed that testosterone supplement amelioratedmotor and nonmotor symptoms of PD patients, which might be related to theincreased expression of VMAT2by testosterone replacement, in addition tothe testosterone-enhenced activity of dopaminergic/serotoninergic systems.Therefore, in the present study, adult male rats were used either to be castratedor castrated and supplemented with testosterone propionate (TP) to analyzethe altered VMAT2expression.Objective: To study the influence of testosterone propionate uponVMAT2expression and analyze the efficacy of TP treatment on VMAT2expression by intranasal delivery or by subcutaneous injection of TP.Methods:60adult male Wistar rats were randomly divided intointranasal administration and subcutaneous injection groups.(1) Intranasaladministration group included sham-operated (Sham), gonadectomized (GDX)and gonadectomized supplemented with TP (GDX-TP) rats.(2)Subcutaneousinjection group consisted of sham-operated (Sham-sc), gonadectomized(GDX-sc) and gonadectomized supplemented with TP (GDX-sc.TP) rats. Halfof them in each group were used either for immunohistochemistry or forWestern blot.Testosterone propionate was administered either by intranasal delivery(GDX-TP) or subcutaneous injection (GDX-sc.TP) to gonadectomized rats.The same treatment using sesame oil as a vehicle to gonadectomized rats (GDX or GDX-sc). Testosterone propionate was given at a dose of2.0mg/kg,at5:00PM to6:00PM, once a day, for21days.The rats for immunohistochemistry were perfused transcardially with afixative containing4%paraformaldehyde. Brains were carefully removed andpostfixed for4h at4°C. Based on the rat brain in stereotaxic coordinates byPaxinos and Watson, the brain blocks that contain substantai nigra (SN)/vental tegmental area (VTA) or caudate-putamen (CPu), as well asaccumbens nucleus (Acb) were prepared, washed in PB, dehydrated in titratedethanol and cleared in xylene before being embedded in paraffin wax. Theblocks were sliced on a sliding microtome into consecutive5μm thick coronalsections for VMAT2immunohistochemistry. A computer-assisted imageanalysis system was used for the average optical density (AOD) measurementsof VMAT2immunoreactive intensity. After subtraction of the nonspecificstaining, the AOD of VMAT2-ir cells in SN/VTA, as well as terminals inCPu/Acb were measured. The averaged amount of the AOD of VMAT2-ircells in SN/VTA, as well as terminals in CPu/Acb was presented for each ratin the results.The rats in each group used for Western blot were sacrificed bydecapitation. The brains were removed quickly. The SN, VTA, CPu and Acbwere dissected on ice-cold plate. Dissected tissue pieces were frozen in liquidnitrogen and were homogenized in Radio Immuno Precipitation Assay buffer.After centrifugation, the supernatant was collected and the proteinconcentration of the supernatant was determined using the Bradford method.SN/VTA (80μg) and CPu/Acb (50μg) protein samples were diluted in2×sample buffer and heated for5min at95°C before SDS-PAGE, andsubsequently transferred to a PVDF membrane to detect VMAT2or GAPDHusing enhanced chemiluminiscence. The ratio of labeling densities for VMAT2with those of GAPDH was analyzed with the use of Gel-Pro AnalyserAnalysis software.Results:1The general observation of immunohistochemistry In substantia nigra (SN), ventral tegmental area (VTA) and dorsal raphenucleus nuclear the positive structure of VMAT2mainly included neuronsbody; and in caudate-putamen (CPu) and accumbens nucleus (Acb) thepositive structure scattered in the matrix of nuclear group, there was nopositive neurons body.Compared to control groups, immunoreaction intensity of VMAT2in SN,VTA, CPu and Acb decreased in GDX/GDX-sc rats, which would increasedafter InTP/ScTP treatment.2The influence of TP by intranasal administration on VMAT2(1) VMAT2in SN and CPu: The statistical analysis showed significantlyreduced AOD of VMAT2-ir intensity in GDX rats. The AOD in GDX rats was13%in SN,16%,11%,11%and17%in CPu-DM, CPu-VM, CPu-VL andCPu-DL less than that in sham rats respectively, which was restored to thelevel of sham rats after InTP treatment. The expression of glycol-VMAT2,partially glyco-VMAT2and native-VMAT2in SN, as well as in CPu of GDXrats was significantly reduced compared to sham rats,33%,27%and51%inSN, as well as54%,31%and26%in CPu lower than that in sham ratsrespectively, which was restored to the level of sham rats after InTP treatment.(2) VMAT2in VTA and Acb: The statistical analysis revealed significantlyreduced AOD of VMAT2-ir intensity in GDX rats. The AOD in GDX rats was15%in VTA,12%and11%in Acb-Core and Acb-Shell less than that in shamrats respectively, which was restored to the level of sham rats after InTPtreatment. The expression of partially glyco-VMAT2and native-VMAT2inVTA, as well as glycol-VMAT2, partially glyco-VMAT2and native-VMAT2in Acb of GDX rats was significantly reduced compared to sham rats,41%and46%in VTA, as well as19%,50%and47%in Acb lower than that insham rats respectively, which was restored to the level of sham rats after InTPtreatment. The expression of glyco-VMAT2in Acb was even more190%thanthe level of sham rats.3The influence of TP by subcutaneous injection on VMAT2(1) VMAT2in SN and CPu: The statistical analysis found significantly reduced AOD of VMAT2-ir intensity in GDX-sc rats, which was increased butstill lower than the level of sham-sc rats after ScTP treatment. The expressionof glycol-VMAT2, partially glyco-VMAT2and native-VMAT2in SN, as wellas in CPu of GDX-sc rats was significantly reduced compared to sham-sc rats,which was increased but still lower than the level of sham-sc rats after ScTPtreatment.(2) VMAT2in VTA and Acb: The statistical analysis revealed significantlyreduced AOD of VMAT2-ir intensity in GDX-sc rats, which was increased butstill lower than the level of sham-sc rats after ScTP treatment. The expressionof partially glyco-VMAT2and native-VMAT2in VTA was significantlyreduced compared to sham-sc rats, which was increased but still lower thanthe level of sham-sc rats after ScTP treatment. The expression ofglycol-VMAT2, partially glyco-VMAT2and native-VMAT2in Acb ofGDX-sc rats was significantly reduced compared to sham-sc rats, which wasincreased but still lower than the level of sham-sc rats after ScTP treatment(but there was statistical significance only in the expression of partiallyglyco-VMAT2).4The efficacy of TP effect on VMAT2expression by intranasal delivery andby subcutaneous injectionThe statistical analysis indicated that, the expression of VMAT2wasincreased after TP treatment to GDX/GDX-sc rats. Intranasal administration ofTP could restore the expression of VMAT2in GDX rats, however, it wasincreased but still lower than the level of sham rats in GDX-sc rats aftersubcutaneous TP treatment.Conclusions:1. The gonadectomy reduced the expression of VMAT2in SN, CPu, VTA andAcb of male rats.2. Testosterone propionate increased the expression of VMAT2in SN, CPu,VTA and Acb in GDX/GDX-sc rats.3. Intranasal route improved the reduced expression of VMAT2in SN-CPuand VTA-Acb better than subcutaneous injection.