The dopamine D2-like receptor and the Y-chromosome gene, SRY, are reciprocally regulated in the human male neuroblastoma M17 cell line

expression


Introduction
Dopamine (DA) is an important neurotransmitter that regulates a wide range of central functions including voluntary movement, motivation and reward-based learning and cognitive and emotive processes, as well as peripheral functions such as regulation of hormonal and immune systems (Bjorklund and Dunnett, 2007;Iversen and Iversen, 2007).In turn, abnormal regulation of DA levels underlies a wide range of motor, cognitive and neuroendocrine disturbances that are found in brain disorders such as schizophrenia, attention-deficit hyperactive disorder (ADHD) and Parkinson's disease (PD) (Bjorklund and Dunnett, 2007).DA exerts its actions through two classes of DA receptors, the D1-like receptors (D1R; D 1 and D 5 receptors) which are coupled to G stimulatory G-proteins or the D2-like receptor (D2R; D 2, D 3, and D 4 receptors) which are coupled to G inhibitory G-proteins (Beaulieu and Gainetdinov, 2011;Missale et al., 1998).
Whilst sex differences in the dopaminergic pathway have been largely attributed to the effects of sex hormones (Dluzen, 2005;Gillies and McArthur, 2010), growing evidence indicate that sex chromosome genes also contribute (Loke et al., 2015;McCarthy and Arnold, 2011).Indeed, neuromodulatory effects of oestrogen on female DA pathway is well established, as oestrogen act directly on striatal DA nerve terminals to increase tyrosine hydroxylase (TH) activity (Pasqualini et al., 1995), enhance basal DA release (Becker, 1990;McDermott et al., 1994), and inhibit DA reuptake (Disshon et al., 1998).However, emerging evidence demonstrate that genes on the sex chromosomes exert cell-specific and sex-specific effects in the brain, independent of sex hormones (Carruth et al., 2002;Dewing et al., 2006).In support, embryonic midbrain cellscultured prior to expression of gonadal hormones -yielded more TH-positive neurons when cultures were composed of XY male cells than XX female cells (Beyer et al., 1992;Carruth et al., 2002).Whilst the precise sex chromosome genes that drive these sex differences are still unclear, recent evidence indicate that the Y-chromosome gene, SRY, directly regulates DA biosynthesis, uniquely in males (Dewing et al., 2006;Lee et al., 2019;Wu et al., 2009).
SRY (Sex-determining Region on the Y) encodes a transcriptional factor that initiates male sex determination by directing the development of the embryonic bipotential gonads to testes rather than ovaries (Koopman et al., 1990;Sekido and Lovell-Badge, 2008;Sinclair et al., 1990).SRY is also expressed in non-reproductive tissues of adult males including the heart, adrenal glands, lungs, kidneys, and brain (Clepet et al., 1993;Dewing et al., 2006).In the male brain, SRY protein is expressed in DA-abundant regions, such as the SNc and ventral tegmental area (VTA), where it colocalizes with DA neurons (Czech et al., 2012;Dewing et al., 2006).Consistent with the expression in midbrain DA neurons, SRY positively regulates transcription of DA pathway genes such as tyrosine hydroxylase (TH), monoamine oxidase-A (MAOA), DOPA decarboxylase (DDC) and D2R in human and rodent cell lines (Czech et al., 2012;Milsted et al., 2004;Wu et al., 2009).Moreover, suppression of nigral SRY expression in male rats, via antisense oligonucleotide infusion, led to reductions in nigral DA pathway gene expression and striatal DA level (Lee et al., 2019), as well as motor deficits (Dewing et al., 2006;Lee et al., 2019).These studies indicate that SRY transcriptionally regulates multiple components of the DA pathway to modulate DA levels and consequently DA-dependent functions, uniquely in males.
Here, we show that dopamine receptor signalling reciprocally regulates SRY expression in a human male neuroblastoma cell line, adding a new dimension to the interaction between SRY and dopaminergic pathway in males.Specifically, we demonstrate that i) physiological levels of dopamine increase SRY expression via dopamine receptor signalling, ii) D1R and D2R signalling bidirectionally regulate SRY expression via phospholipase C signalling and iii) reduction of SRY expression decreases D2R, but not D1R, expression, indicating a positive feedback loop between SRY and D2R signalling, uniquely in males.

Cell culture
Human neuroblastoma BE (2)-M17 (M17) cell line used in our studies was derived from the American Type Culture Collection (CRL-2267; ATCC, Manassas, VA, USA, RRID:CVCL_0167).The M17 cell line was chosen as it is a male neuroblastoma cell line that express endogenous SRY as well as the cellular machinery typical of DA neurons, including TH, MAO-A, DAT and D1R and D2R (Czech et al., 2012).M17 cells are composed of a subpopulation of TH-positive (70% of total) and TH-negative (30% of total) cells (Fig. S1), as reported previously (Haycock, 1993).Cells were cultured using Dulbecco's modified Eagle's medium (DMEM) with Ham's F12 nutrient in a 50:50 ratio supplemented with Glutamax, 10% Foetal Bovine Serum (FBS), 1% penicillin/streptomycin and maintained at 5% CO 2 and 37 • C in a Galaxy 170 S incubator.M17 cells were subcultured when a confluence of greater than 70% was reached twice per week.Cells were washed with 1% PBS and aspirated, released with 0.01% versene (Gibco), and resuspended in a 1:4 dilution in fresh medium.The cell line was maintained in sterile 75 cm 2 cell culture flasks.Cells were seeded with either 5 × 10 5 per well in 6-well plates (qRT-PCR) or 3 × 10 4 per well in 48-well plates (immunocytochemistry) with each well corresponding to a treatment group.

siRNA gene interference
Human SRY siRNA (sc-38443, Santa Cruz, CA, USA) or control nonsense siRNA (sc-37007) was transfected into M17 cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), as previously published (Czech et al., 2012;Wu et al., 2009).Briefly, 3 × 10 4 cells were seeded per well in 48-well plates in 300 μL of fresh medium without antibiotics, the next day cells were transfected using a mixture of siRNA-oligomer (10 μM) and Lipofectamine 3000 reagent diluted in reduced-serum medium (Opti-MEM, Gibco) which was added to the 300 μL of culture media.The mixture was incubated for 24 h at 37 • C, 5% CO 2 then replaced with fresh media after which cells were treated with either vehicle (1% DMSO/saline), SKF38393 (10 μM) or ropinirole hydrochloride (20 μM) for a further 6 h.At the end of the drug treatment, cells were processed for immunocytochemistry for assessment of protein expression.

Quantitative RT-PCR
Total RNA (0.5-1 μg) was isolated from M17 cells using the PureLink RNA Mini Kit (12183018 A, 12183025, Invitrogen) according to the manufacturer's instructions.cDNA was synthesised using SuperScript III First-Strand Synthesis System (18080-051, Invitrogen) as per manufacturer's instructions.Real time quantification of mRNA levels was conducted using Power SYBR Green PCR Master Mix (4367659, Applied Biosystems) and the QuantStudio 6 Flex Real-Time PCR systems as per manufacturer's instructions.Primer design as follows (F-forward, Rreverse), SRY F: TTC CCC GCA GAT CCC GCT TCG GTA CTC TG, R: TTT TTT TTT TTT TTT GAA ATG AAT AA-G, GAPDH, F: GTA AAG TGG ATA TTG TTG CC, R: GGG TGG AAT CAT ATT GGA A-C, GADD45G, F: ACT AGC TGC TGG TTG ATC GC, R: CAA CTC ATG CAG CGC TTT C. The relative level of mRNA was extrapolated from a standard curve prepared by serially diluting the cDNA reaction.Specificity of PCR product formation was confirmed by monitoring melting peaks.All quantitative PCR reactions were conducted in triplicates.The mRNA levels were normalised relative to the housekeeping gene GAPDH mRNA, and the fold changes for each mRNA were calculated using the comparative CT (2 − ΔΔCt ).The mean value of the untreated vehicle group was set to 1; the values of other groups were normalised to the vehicle group and D.-H.Kim et al. expressed as a fold change of vehicle to account for inter-assay variability.

Confocal microscopy
Immunolabelled cells were imaged with a 60× objective using a confocal laser scanning microscope (Nikon A1R) and UV, GFP, Texas Red, Cy5 filter sets were used to discriminate labelling with the Alexa Fluor 405, 488, 594 and 647 fluorophores, respectively.For fluorescent intensity analysis, images were acquired using identical pinhole, exposure, brightness, and gamma levels for all cells.The laser intensity was adjusted to a level that removes background noise and photobleaching/ saturation of fluorophores, while still ensuring adequate signal for both the nuclear and cytoplasmic fluorescence from the final confocal image.The immunolabelling intensity of SRY, TH, D1R or D2R for each cell was calculated using Fiji-ImageJ v1.53c software (NIH, RRID:SCR_003070) by calculating the mean pixel intensity of the nuclear or cytoplasmic cell fluorescence.The Hoechst-33342 stain was used to delineate the boundary of the nucleus from the cytoplasm.The erode/dilate function was also used to decrease noise and false positives as well as achieve precise boundaries.Nuclear or cytoplasmic SRY or TH intensity (i.e.immunoreactivity) for each sample were calculated from an average of ten TH-positive or ten TH-negative cells.The mean nuclear or cytoplasmic SRY or TH intensity of each sample was expressed as a fold change relative to a vehicle group (0 h) to account for unwanted sources of variation.All fluorescence intensity data were analysed by one author (JT) in a randomised manner blind to treatment allocation.

Data and statistical analysis
The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018).Experiments were designed to obtain randomized groups of equal sizes and independent values with less than 20% variance were chosen to compose the groups.All values are expressed as the mean ± S.E.M with at least 5 biological replicates.No data were removed, and all outliers were included in data analysis.All data was analysed using tools within Prism 9.2 software (GraphPad Software, RRID:SCR_002798).Comparisons of two experimental groups were performed using two-tailed unpaired Student's t-test.Multiple comparisons of more than two experimental groups were performed using one-way ANOVA and Tukey's post hoc tests.Multiple comparisons of the treatment groups across different time points assessed were analysed by two-way ANOVA, followed by Tukey's post hoc test.The exact P-values of the ANOVAs are given in the figure legends.Probability level of 5% (P < 0.05) was considered significant for all statistical tests.

Physiological levels of DA upregulate SRY expression via a GADD45G independent mechanism in vitro
Previous studies demonstrate that SRY transcriptionally regulates multiple components of the DA pathway in vitro D2R (Czech et al., 2012;Milsted et al., 2004;Wu et al., 2009) and in vivo (Dewing et al., 2006;Lee et al., 2019), uniquely in males.To assess whether DA receptor signalling reciprocally regulates SRY expression, we assessed the effect of DA treatment on SRY mRNA and protein expression at varying concentrations and times post-treatment in human M17 cells (Fig. 1 and Fig. S2).

Stimulation of DA receptor signalling increases SRY expression in vitro
To confirm that SRY-upregulation induced by DA treatment was via DA receptor signalling, we assessed the effect of direct (apomorphine) or indirect (amphetamine) stimulation of DA receptors on SRY expression.We also assessed the converse effect of reducing DA receptor signalling by treatment with α-methyl-para-tyrosine (αMPT), a TH inhibitor which reduces DA synthesis, on SRY expression.

D1R and D2R signalling oppositely regulate SRY expression in vitro
Given that DA exerts its actions via the D1-like or the D2-like receptor, we assessed the effect of selective D1-or D2-like receptor agonists or antagonists on SRY expression.
Treatment with sulpiride alone reduced nuclear and cytosolic SRY-IR in TH-negative cells (0.81-and 0.79-fold vs vehicle, P = 0.0125, P = 0.0064, Fig. 4K and L).Collectively, these results demonstrate that activation of D2-like receptor signalling increases SRY expression.

D1R and D2R regulates SRY expression via the phospholipase C signalling pathway in vitro
To identify the downstream signalling pathways involved in SRY regulation, we assessed the effect of D1R or D2R agonists on SRY expression in the presence of protein kinase A (PKA), phospholipase C (PLC) or mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) inhibitors.

SRY regulates basal D2R expression and ropinirole-induced D1R and D2R upregulation in vitro
To determine potential feedback mechanism(s) between SRY and D1R and/or D2R signalling in M17 cells, we assessed the consequence on SRY inhibition, via SRY siRNA pre-treatment, on SKF38393 or ropinirole-mediated regulation of SRY, D1R or D2R expression.
In TH negative cells only, SKF38393 treatment alone reduced nuclear and cytosolic SRY expression (P < 0.0001 vs control siRNA/vehicle, Figure D), which were paralleled by reductions in cytosolic D1R-IR and D2R-IR (P = 0.048, P < 0.0001 vs control siRNA/vehicle, Fig. 7E and F, respectively).However, SRY siRNA pre-treatment did not further reduce SKF38393-induced downregulation of cytosolic D1R-IR and D2R-IR (Fig. 7E and F respectively), indicating that SRY does not regulates D1R or D2R expression during D1R-stimulated conditions in TH-negative cells.
Taken together, these results demonstrate i) a positive feedback loop between D2R signalling and SRY under basal conditions and ii) a SRYmediated regulation of D1R and D2R expression during D2R activated conditions, potentially as a negative feedback mechanism, in both THpositive and TH-negative cells.

Discussion
We and others showed that the Y-chromosome gene, SRY, is expressed in midbrain DA neurons where it transcriptionally regulates multiple DA pathway genes in vitro (Czech et al., 2012;Milsted et al., 2004;Wu et al., 2009) and in vivo (Dewing et al., 2006;Lee et al., 2019),   uniquely in males.Here, we assessed whether DA receptor signalling reciprocally regulates SRY expression in a human male neuroblastoma cell line.We found that D1R-downregulates SRY expression via PLC signalling and D2R upregulates SRY via PLC-ERK signalling.We also demonstrate that SRY specifically regulates basal D2R, and not D1R, expression, highlighting a novel feedback loop between SRY and D2R signalling, uniquely in males.
Here, we show for the first time that stimulating DA receptor signalling can directly upregulate SRY expression in human M17 cells.We found that treatment with 1 and 10 μM DA transiently increases SRY protein expression at 6 h post-treatment, without affecting GADD45γ mRNA expression or cell viability.This contrasts with the elevation of SRY following treatment with the DA toxin, 6-hydroxydopamine (Czech et al., 2014;Lee et al., 2019), or supraphysiological levels of 1 mM DA, which were associated with marked increases in GADD45γ expression and pronounced cues of cell death.Moreover, stimulation of endogenous DA release by amphetamine increased SRY expression, whilst treatment with αMPT, which blocks endogenous DA synthesis, had the opposite effect in TH-positive cells, demonstrating a positive relationship between endogenous DA levels and SRY expression.Taken together, these results demonstrate a novel DA receptor-dependent regulation of SRY expression, independent of the MAPK-GADD45γ signalling pathway and cellular stress.
Upon further examination with selective D1R and D2R agonists and antagonists, we found that D1R and D2R signalling, in fact, exerts opposite effects on SRY expression.Indeed, treatment with the D1R agonist, SKF38393, reduced SRY expression, whilst treatment with either D2R agonists, bromocriptine or ropinirole, elevated SRY expression.Additionally, pre-treatment with the D1R antagonist, SCH23390 potentiated the SRY upregulation induced by DA, whilst the D2R antagonist, sulpiride, blocked the SRY upregulation, demonstrating an inhibitory role for D1R and a stimulatory role for D2R on regulation of SRY.This contrasting effect of D1R and D2R stimulation on SRY expression is consistent with the opposing actions of D1R and D2R on intracellular signaling, cellular activity and physiological processes such as voluntary movement (Beaulieu and Gainetdinov, 2011;Missale et al., 1998).We also found that D2R activation increased SRY expression in both TH-positive and TH-negative cells, whilst D1R activation only reduced SRY expression in TH-negative cells.Given that D1R and D2R are expressed in both TH-positive and TH-negative M17 cells, these results may reflect differences in D1R density and/or affinity between TH-positive and TH-negative cells.
It is well established that both D1R and D2R can exert their actions via the canonical adenylyl cyclase/PKA-dependent signalling pathway or non-canonical pathways such as PLC and MAPK/ERK signalling pathways (Beaulieu and Gainetdinov, 2011).We found that pre-treatment with the PKA inhibitor, H-89, had no effect on either the SKF38393-induced SRY downregulation or ropinirole-induced SRY upregulation.In contrast, pre-treatment with the PLC inhibitor, U73122, abolished SKF38393-induced SRY downregulation in TH-negative cells and ropinirole-mediated SRY upregulation in TH-positive and TH-negative cells, demonstrating that D1R-and D2R-mediated regulation of SRY is via the PLC signalling pathway.Indeed, DA receptor-mediated regulation of SRY expression were observed 3-6 h post treatment, which is consistent with the slower-onset and longer-lasting effects (Kim et al., 2006;Undieh, 2010) mediated by the PLC pathway.In addition to the D2R-PLC-mediated regulation of SRY, ropinirole-induced SRY upregulation was also blocked in the presence of the MAPK/ERK inhibitor, PD98059.Given that D2R can regulate gene expression by PLC-mediated activation of the MAPK/ERK signalling pathway (Kolch et al., 1993;Yan et al., 1999) or by direct activation of the Src/Ras/ERK pathway (Oak et al., 2001;Wang et al., 2005;Zhen et al., 2001), the D2R-mediated SRY-upregulation may be via either a PLC-MAPK-dependent and/or PLC-MAPK-independent pathway.
We previously showed that SRY positively regulates multiple DA machinery genes and consequently DA levels in vitro (Czech et al., 2012) and in vivo (Lee et al., 2019).We extend these findings to show that SRY regulates basal D2R (and not D1R) expression, which is reciprocated by D2R-mediated regulation of SRY expression in both TH-positive and TH-negative cells.This positive feedback loop between SRY and D2R signalling is consistent with the positive feedback interaction between dopaminergic transcription factors, such as Nurr1 and Pitx3, and D2R signalling (Jacobs et al., 2009(Jacobs et al., , 2011;;Kim et al., 2006;Tseng et al., 2000).Whilst SRY did not regulate basal D1R expression, reducing SRY expression suppressed ropinirole-induced upregulation of D1R expression, indicating that SRY regulates D1R expression during conditions of D2R activation.Given that ropinirole also increases SRY and consequently D2R expression, SRY-mediated regulation of D1R expression may represent an inhibitory mechanism to "switch off" the positive feedback loop between SRY and D2R signalling.Overall, these findings represent a novel feedback loop between SRY and D2R signalling, perhaps as a slow-onset and longer lasting genetic mechanism to regulate DA levels and/or DA receptor expression, uniquely in males.
The human male neuroblastoma cell line, M17, has served as a useful model to assess the molecular mechanism(s) underlying the regulation (Czech et al., 2014) and downstream signalling targets of SRY (Czech et al., 2012;Wu et al., 2009).However, the current findings require further investigation in whole animal in vivo studies with intact nigrostriatal or mesoaccumbal pathways to demonstrate broader physiological significance.Given the co-expression of D1R and D2R in M17 cells, the possibility of D1R/D2R heterodimerization and/or crosstalk (Lee et al., 2000(Lee et al., , 2004) also needs to be considered in the interpretation of the current findings.Further studies assessing the effect of ligands selectively targeting D1/D2 heterodimers and/or in vivo studies utilising D1R or D2R knockout mice will be critical in delineating the relationship between SRY and D1R, D2R or D1R/D2R heterodimers.
The current findings may have significant implications for the development of novel male-specific therapeutic strategies for DAmediated disorders such as PD, schizophrenia and ADHD.Indeed, anti-parkinsonian drugs (levodopa, apomorphine) or psychostimulants (amphetamine, methylphenidate), which all stimulate DA receptor signalling, exhibit significant differences in efficacy and response between males and females (Kompoliti et al., 2002;Manza et al., 2021;Milesi-Hallé et al., 2007;Savageau and Beatty, 1981;Walker et al., 2006).Similarly, anti-psychotics such as haloperidol, clozapine, aripiprazole and amisulpride, which largely act to inhibit D2R signalling, exhibit significant sex differences in efficacy and response (Bouvier et al., 2020;Hoekstra et al., 2021;Walker et al., 2006).Thus, better understanding the interplay between SRY and D2R signalling may reveal therapeutic strategies aimed at delivering optimal efficacy and/or response to DAergic medications in male patients.
In summary, our study builds upon previous work establishing a role for SRY in male DA neurons, where it transcriptionally regulates DA biosynthesis (Czech et al., 2012;Milsted et al., 2004;Wu et al., 2009) and voluntary movement (Dewing et al., 2006;Lee et al., 2019).The current study extends these findings to demonstrate that D2R signalling reciprocates regulation of SRY expression via the PLC-ERK pathway, highlighting a novel feedback mechanism for slow-onset regulation of DA biosynthesis and signalling, uniquely in males.This ancillary genetic mechanism may contribute to male-specific DA-dependent functions such as male sexual arousal or male-biased physiological processes such the fight-flight response (Lee and Harley, 2012).Similarly, interplay between SRY and D2R signalling may contribute to male-specific response to dopaminergic agonists and antagonists and/or male vulnerability to DA-related disorders such as PD, ADHD, and schizophrenia (Davies, 2014;Gillies et al., 2014;Lee et al., 2019).Although the consequence of SRY regulation may differ by cell type and brain region, a thorough understanding of the anatomical and functional relationship between DA receptor signalling and SRY expression in vivo will be an important next step for revealing potential therapeutic avenues optimal for males.

Fig. 1 .
Fig. 1.Physiological levels of DA increases SRY expression via a GADD45γindependent mechanism in M17 cells.(A, B) Effect of DA (1 or 10 μM) on SRY (A) or GADD45γ (B) mRNA expression at 3, 6 and 12 h s post-treatment.All SRY and GADD45γ mRNA expressions were normalised as a fold change of vehicle at 0 h s relative to the housekeeping gene, GAPDH (n = 6/group).(C-F) Effect of DA (1 or 10 μM) on SRY (green) and TH (red) immunoreactivity (IR) at 3, 6 and 12 h s post-treatment in TH-positive (C, D) and TH-negative (E, F) cells.Nuclear (n) or cytoplasmic (c) SRY or TH intensities for each sample were calculated from an average of ten TH-positive or ten TH-negative cells.All SRY-and TH-IR were normalised as a fold change relative to vehicle (n ≥ 5/group).Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01, ****P < 0.0001, significantly different from the vehicle group; two-way ANOVA with Tukey's post hoc test.Dashed line represents baseline levels.Scale bar = 10 μm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .
Fig. 2. Stimulation of DA receptor signalling increases SRY expression in M17 cells.(A-C) Effect of apomorphine (APO) (A), amphetamine (AMPH) (B), or α-methylp-tyrosine (αMPT) (C) on SRY mRNA expression at 3 and 6 h post treatment.All SRY mRNA expressions were normalised as a fold change of vehicle at 0 h s relative to the housekeeping gene, GAPDH (n = 5/group).(D-G) Effect of APO on SRY (green) and TH (red) immunoreactivity (IR) at 3 and 6 h post treatment in TH-positive (D, E) and TH-negative (F, G) cells.(H, I) Effect of AMPH on SRY-and TH-IR at 3 and 6 h post treatment in TH-positive cells.(J, K) Effect of αMPT on SRY-and TH-IR at 3 and 6 h post treatment in TH-positive cells.Nuclear (n) or cytoplasmic (c) SRY or TH intensities for each sample were calculated from an average of ten TH-positive or ten TH-negative cells.All SRY-and TH-IR were normalised as a fold change relative to vehicle (n ≥ 5/group).Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, significantly different from the vehicle group; two-way ANOVA with Tukey's post hoc test.Dashed line represents baseline levels.Scale bar: 10 μm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .
Fig. 3. D1-like receptor activation reduces SRY expression in TH-negative M17 cells.(A-D) Effect of SKF38393 (SKF) on SRY (green) and TH (red) immunoreactivity (IR) at 6 h post-treatment in TH-positive (A, B) and TH-negative (C, D) cells.(E-H) Effect of pre-treatment with the D1-like receptor antagonist, SCH23390, on DAmediated regulation of SRY and TH-IR at 6 h post-treatment in TH-positive (E, F) and TH-negative (G, H) cells.Nuclear (n) or cytoplasmic (c) SRY or TH intensities for each sample were calculated from an average of ten TH-positive or ten TH-negative cells.All SRY-and TH-IR were normalised as a fold change relative to vehicle (n ≥ 5/group).Data are shown as mean ± S.E.M. *P < 0.05, ****P < 0.0001, significantly different from the vehicle group; ####P < 0.0001, significantly different from the DA group; One-way ANOVA with Tukey's post hoc test.Dashed line represents baseline levels.Scale bar: 10 μm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 .
Fig. 4. D2-like receptor activation increases SRY expression in M17 cells.(A-D) Effect of bromocriptine (bromo) on SRY (green) and TH (red) immunoreactivity (IR) at 6 h post-treatment in TH-positive (A, B) and TH-negative (C, D) cells.(E-H) Effect of ropinirole on SRY-and TH-IR at 6 h post-treatment in TH-positive (E, F) and TH-negative (G, H) cells.(I-L) Effect of the D2-like receptor antagonist, sulpiride, on DA-mediated regulation of SRY and TH-IR at 6 h post-treatment in TH-positive (I, J) and TH-negative (K, L) cells.Nuclear (n) or cytoplasmic (c) SRY or TH intensities for each sample were calculated from an average of ten TH-positive or ten THnegative cells.All SRY-and TH-IR were normalised as a fold change relative to vehicle (n ≥ 5/group).Data are shown as mean ± S.E.M. *P < 0.05, **P < 0.01, ****P < 0.0001, significantly different from the vehicle group; ####P < 0.0001, significantly different from the DA group; ^^^^ P < 0.0001, significantly different from the sulpiride/DA group; two-way ANOVA with Tukey's post hoc test and unpaired t-test.Dashed line represents baseline levels.Scale bar: 10 μm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 .
Fig. 6.D2R-mediated SRY upregulation is via the PLC-extracellular regulated kinase (ERK) signalling pathway.(A-D) Effect of the protein kinase A inhibitor, H-89, on ropinirole-mediated regulation of SRY (green) and TH (red) immunoreactivity (IR) at 6 h post-treatment in TH-positive (A, B) and TH-negative (C, D) cells.(E-H) Effect of the PLC inhibitor, U73122, on ropinirole-mediated regulation of SRY-and TH-IR at 6 h post-treatment in TH-positive (E, F) and TH-negative (G, H) cells.(I-L) Effect of the ERK inhibitor, PD98059 on ropinirole-mediated regulation of SRY-and TH-IR at 6 h post-treatment in TH-positive (I, J) and TH-negative (K, L) cells.Nuclear (n) or cytoplasmic (c) SRY or TH intensities for each sample were calculated from an average of ten TH-positive or ten TH-negative cells.All SRY-and TH-IR were normalised as a fold change relative to vehicle (n ≥ 5/group).Data are shown as mean ± S.E.M. **P < 0.01, ***P < 0.001, ****P < 0.0001, significantly different from the vehicle group; ###P < 0.001, ####P < 0.0001, significantly different from the ropinirole group; one-way ANOVA with Tukey's post hoc test.Dashed line represents baseline levels.Scale bar: 10 μm.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)