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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Eur J Neurosci. 2015 Mar 18;41(9):1157–1166. doi: 10.1111/ejn.12871

Sex Differences and Effects of Prenatal Exposure to Excess Testosterone on Ventral Tegmental Area Dopamine Neurons in Adult Sheep

Erinna C Z Brown 1, Casey J Steadman 2, Theresa M Lee 3, Vasantha Padmanabhan 4, Michael N Lehman 5, Lique M Coolen 1,2,5
PMCID: PMC4673679  NIHMSID: NIHMS665562  PMID: 25784297

Abstract

Prenatal testosterone (T) excess in sheep results in a wide array of reproductive neuroendocrine deficits and alterations in motivated behavior. The ventral tegmental area (VTA) plays a critical role in reward and motivated behaviors and is hypothesized to be targeted by prenatal T. Here we report a sex difference in the number VTA dopamine cells in the adult sheep with higher numbers of tyrosine hydroxylase (TH)-immunoreactive cells in males than females. Moreover, prenatal exposure to excess T during either gestational days 30–90 or 60–90 resulted in increased numbers of VTA TH- immunoreactive cells in adult ewes compared to control females. Stereological analysis confirmed significantly greater numbers of neurons in the VTA of males and prenatal T-treated ewes, which was primarily accounted for by greater numbers of TH-immunoreactive cells. In addition, immunoreactivity for TH in the cells was denser in males and prenatal T-treated females, suggesting that sex differences and prenatal exposure to excess T affects both numbers of cells expressing TH as well as the protein levels with dopamine cells. Sex differences were also noted in numbers of TH-immunoreactive cells in the substantia nigra, with more cells in males than females. However, prenatal exposure to excess T did not affect numbers of TH-immunoreactive cells in the substantia nigra, suggesting that this sex difference is organized independently from prenatal actions of T. Together, these results demonstrate sex differences in the sheep VTA dopamine system which are mimicked by prenatal treatment with excess T.

Introduction

The effects of prenatal testosterone (T) on reproductive physiology and behavior have been documented in several species including rat (Wolf et al., 2002; Wu et al., 2010), rhesus macaque (Goy et al., 1988; Abbott et al., 2005), human (Merke & Bornstein, 2005), and sheep (Wood & Foster, 1998; Robinson et al., 2002; Padmanabhan et al., 2006; Bormann et al., 2011; Hogg et al., 2011). For sheep, the critical period of sexual differentiation of the hypothalamic-pituitary-gonadal (HPG) axis spans gestational days 30–90 of their 147-day gestational period (Clarke et al., 1976; Wood & Foster, 1998; Robinson et al., 2002; Padmanabhan et al., 2006). Females treated with T during gestational days 30–90, are born with virilized genitalia (Wood & Foster, 1998), and multiple reproductive neuroendocrine and ovarian disruptions (Wood & Foster, 1998; Padmanabhan et al., 2006). Female sheep treated with T during gestational days 60–90, are born with normal feminine external genitalia (Steckler et al., 2007) and ovarian cycles in the first breeding season (Savabieasfahani et al., 2005). However, they display masculinized urinary posture, altered expression of genes coding for components of steroidogenesis in the developing ovary (Hogg et al., 2011), delayed estradiol positive feedback response (Savabieasfahani et al., 2005; Steckler et al., 2007), and a masculinized sexually dimorphic nucleus of the preoptic area (Alexander et al., 2011; Roselli et al., 2011).

In addition, prenatal T excess treatment alters motivated behaviors. Sheep live in stable social groups where dominance hierarchies arise from the competition for resources (e.g., food and mates) and males are at the top of the dominance hierarchy. Prenatal T treatment significantly increases the ranking of females (Roberts et al., 2009). and masculinizes sexual behavior (Roberts et al., 2008; Roberts et al., 2009). The mesolimbic system plays a critical role in motivated behaviors including food intake (Palmiter, 2007), and sexual behavior (Fields et al., 2007; Frohmader et al., 2010). The mesolimbic system consists of dopamine producing neurons located in the ventral tegmental area (VTA) and their projections (Fields et al., 2007). Studies in rodents showed that VTA dopamine neurons are sexually dimorphic (Engele et al., 1989; McArthur et al., 2007). However to what extent prenatal steroid hormones contribute to the sexual dimorphism is unclear, as sex differences in midbrain dopamine neurons can occur in the absence of steroid hormones and may be caused by sex chromosome complement(s) (Engele et al., 1989; Carruth et al., 2002; Arnold, 2014). VTA dopamine neurons express androgen receptor (AR) (Kritzer, 1997) and estrogen receptor (ER) β (Kritzer, 1997; Creutz & Kritzer, 2002) and tyrosine hydroxylase (TH), the rate-limiting enzyme of the dopamine biosynthesis pathway, can be activated by androgen (Jeong et al., 2006) and estrogen (Ivanova & Beyer, 2003; Serova et al., 2004; Maharjan et al., 2005) receptor activation. Therefore, we hypothesize that excess prenatal T treatment alters the VTA dopamine neurons in the sheep mesolimbic system. To address this hypothesis, we characterized the distribution and presence of sex differences of midbrain dopamine neurons in male and female sheep, and examined the effect of prenatal T treatment of female sheep during gestational days 60–90 or 30–90 on the number and size of midbrain dopamine neurons.

Materials and Methods

Animals and General Care

Female and male Suffolk ewes were used for all experiments. All animals were raised at the Sheep Research Facility at the University of Michigan (Ann Arbor, MI, 42° 18′N). Animal care, including husbandry and nutrition were conducted as previously described (Manikkam et al., 2004; Jackson et al., 2013). Briefly, adult Suffolk ewes with proven fertility were purchased in the local Michigan area, housed under a natural photoperiod in the pasture, and group fed daily. After birth, each mother and her lambs were individually housed for the first 3 days, then group housed in a barn, and lambs were weaned at 8 weeks of age. After weaning, female lambs were kept in their same-age cohort, always able to freely interact with each other. All experimental procedures were approved by the University of Animal Care and Use Committee at the University of Michigan and are consistent with National Research Council’s Guide for the Care and Use of Laboratory Animals.

Experimental Groups

Experimental females were exposed prenatally to excess T between either gestational days 60 to 90 (T60–90) of the 147 days gestational period, or for a longer prenatal period between gestational days 30 to 90 (T30–90); in two separate studies. These two experimental groups each had age-matched controls that were reared and housed simultaneous with the corresponding T-treated groups (C60–90 and C30–90 resp). For generation of prenatal T-treated females, pregnant Suffolk ewes of known conception dates received twice weekly 2 ml intramuscular injections of 100 mg testosterone propionate (~1.2mg/kg; Sigma-Aldrich Corp., St. Louis, MO, USA) in cottonseed oil for 30 or 60 days, between gestational days 60 to 90 (T60–90) or 30 to 90 (T30–90) of the 147 day gestational term. The dose and mode of T propionate treatment has been shown to achieve a concentration of T comparable to adult males in pregnant ewes and levels of T in female fetuses that are comparable with that of male fetuses (~0.6 ng/mL in the umbilical arterial blood) (Veiga-Lopez et al., 2011). Control females did not receive vehicle since previous studies have demonstrated no differences in reproductive attributes between vehicle-treated and non-vehicle controls in a previous study (Veiga-Lopez et al., 2008).

In order to control steroid levels between the groups of females, all ewes were ovariectomized at two years of age, were sequentially treated with progesterone implants (CIDR; Inter AG, Hamilton, Waikato, New Zealand) and estradiol (E2; Silastic implants; 16 hours after removal of CIDR), in a regimen previously demonstrated to artificially mimic late follicular phase levels (Jackson et al., 2013). All animals were euthanized 20 hours after E2 (approximately two months following ovariectomy) and brains collected. One group of control males was added for comparison of sex differences. These males were bred on the farm and did not receive any prenatal treatments. They were raised with either group of females, but were combined as one group and served as control Male group for both studies. These males were gonadally intact, vasectomised at 3.5 months, sexually experienced, and euthanized at 10–12 months of age.

Tissue Collection

Prior to sacrifice, animals received two intravenous injections of heparin (25,000 U given 10 min apart; Abraxiz Pharmaceutical Products, Schumberry, IL), were anesthetized with an intravenous administration of sodium pentobarbital (2–3g; Sigma-Aldrich), and sacrificed with rapid decapitation. Each head was perfused via both internal carotids with 6L of 4% paraformaldehyde in 0.1M sodium phosphate buffer (PB, pH 7.3) mixed with 0.1% sodium nitrate and 10 U/mL heparin. After perfusion, the brains were removed; a block of tissue containing the midbrain was dissected out and placed in 4% paraformaldehyde in PB overnight for postfixation at 4°C. The block was then transferred into 30% sucrose in PB at 4°C to complete infiltration. Frozen coronal sections were cut into 10 parallel series (45μm thick slices) using a freezing microtome (Microm HM400R, Walldorf, Germany) and stored in cryoprotective solution (30% ethylene glycol, 0.1% sodium azide, 30% sucrose in PB) at -20°C until being processed for immunohistochemistry.

Immunohistochemistry General Methods

For each experiment, tissue sections from all groups were processed simultaneously as described below. All steps were performed at room temperature with gentle agitation. Free floating sections were rinsed thoroughly in 0.1M phosphate buffered saline (PBS) between incubations. Sections were incubated with 1% hydrogen peroxide (H2O2, Fisher Scientific, Pittsburgh, PA) in PBS for 10 min to eliminate the endogenous peroxidase activity. Unless otherwise specified, tissue sections were blocked for 1 h with incubation solution containing PBS, 0.4% Triton X-100 (Sigma-Aldrich) and 4% normal goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) and all primary antibody incubations were performed overnight at room temperature in the incubation solution. After staining, the sections were washed several times with 0.1M PB, mounted on glass slides with 0.3% gelatin in ddH2O, air dried and coverslipped with DPX (Electron Microscopy Sciences, Fort Washington, PA).

Immunohistochemistry for Tyrosine Hydroxylase

Tissue sections from the midbrain containing the VTA were incubated overnight with monoclonal antibody to tyrosine hydroxylase (TH; 1:50,000; MAB5280; Millipore/Chemicon International, Temecula, CA) followed by 1 hour incubations in biotinylated goat anti-mouse IgG (1:500; Vector Laboratories, Burlingame, CA) and avidin-horseradish peroxidopaminese complex (ABC-elite; 1:500 in 0.1M PBS; Vector Laboratories). TH-immunoreactivity was visualized using diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) in PB containing 0.012% hydrogen peroxide, resulting in a reddish-brown reaction product. Sections were counterstained using cresyl violet (0.625% cresyl violet acetate (Sigma-Aldrich) and 0.0625% sodium acetate trihydrate (Fisher) in ddH2O) only for Stereological analysis and Neurolucida Mapping. Antibody controls: The antibody used for TH has been previously characterized for use in rodent brain (Balfour et al., 2004). Omission of the primary antibody prevented all staining, and western blot analysis using sheep midbrain protein revealed a single band at the appropriate weight (64 kDa). Finally, the distribution of the TH-immunoreactive cells appeared similar to these described in other mammalian species (see results and discussion).

Neurolucida Mapping of TH-immunoreactive cell distribution

The neuroanatomical localization of the TH-ir neurons was first analyzed in control males (n=8) and females (C60–90; n=9). Using Neurolucida mapping software (MicroBrightfield Bioscience, Williston, VT, USA) and a CCD camera (Microfire, Optronics, Goleta, CA, USA) attached to a Leica microscope (DM5000B, Leica Microsystems; Wetzlar, Germany) images were taken and contour drawings were made for one representative control male and female (C60–90), using Cresyl Violet counterstained sections. Contour drawings were made of three sections, representative of the rostral, middle and caudal levels in the VTA and substantia nigra (SN), and the location of the TH-ir neurons were noted (Figure 1). For the SN, the standard area of analysis was dorsal and lateral to the VTA and did not discriminate between the SN pars compacta or reticulata (Figure 1). Major anatomical landmarks, were used to delineate the VTA (based on delineations in rat VTA (Balfour et al., 2004), including the fasciculus retroflexus (FR), cerebral peduncle (CP), central aquaduct (AQ), dorsal tegmental bundle (DTB), posterior commissure (PC), red nucleus (RN), central grey (CG), oculomotor nerve bundles (OM), oculomotor nucleus (ON) (Figure 1). The medial tip of the CP was defined as the boundary between the VTA and SN. The rostral level of the VTA was characterized as having the FR dorsal to the VTA. The FR then descends ventrally as the VTA moves more caudally (Figure 1a–d). The middle level of the VTA was determined as the level when the RN appears and when there are abundant numbers of oculomotor nerve bundles (Figure 1e–h). At what was determined as the caudal level of the VTA TH-ir cells, the red nucleus has enlarged and the numbers of oculomotor nerve bundles have decreased and the oculomotor nucleus has appeared in the central grey (Figure 1i–l).

Figure 1. Distribution of TH-ir neurons in the sheep midbrain.

Figure 1

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Representative images and contour map drawings of the male rostral (A, B), middle (E, F), and caudal (I, J) levels and female rostral (C, D), middle (G, H) and caudal (K, L) levels of the VTA and SN. Contour drawings illustrate the location of central aquaduct (AQ), dorsal tegmental bundle (DTB), fasiculus retroflexus (fr), cerebral peduncle (CP), posterior commissure (PC), red nucleus (RN). Scale bar 2mm.

TH cell Count Analysis

Using a Leica microscope (DM5000B, Leica Microsystems; Wetzlar, Germany) attached to a CCD camera (Microfire, Optronics, Goleta, CA, USA) and Neurolucida software (MicroBrightfield Bioscience, Williston, VT, USA), the number of TH-ir neurons was counted in the standard area of analysis (700 X 800μm). Two sections from each of the rostral, middle and caudal levels of the VTA and SN were analyzed and an average was calculated for each animal for each of the three rostral to caudal levels. In addition, averages across the three rostral to caudal levels were calculated for each animal. Differences between prenatal T-treated females (either T60–90 or T30–90), control males and females were compared using Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn’s method for all pairwise multiple comparisons for the rostral VTA and one-way ANOVA and Holmes-Sidak post hoc tests for middle and caudal VTA and SN, using 5% significance levels.

Perimeter and optical density analysis

The perimeter and optical density was analyzed in the VTA of a subset of control females (C60–90); n=7), males (n=5) and T60–90 females (n=5). For each animal, 15 images of the VTA were captured using a CCD camera (DFC 420, Leica, Wetzlar, Germany) attached to a Leica microscope (DM5000B, Leica Microsystems; Wetzlar, Germany), using identical camera settings (10x objective) for all images. Images were imported into ImageJ (National Institutes of Health, USA) and not altered in any way. For each animal, the perimeter of 45–90 TH-ir neurons (average 75) was measured and within each neuron the number of pixels above threshold were calculated as percentage of analyzed area above threshold. The threshold value was calculated based on the average grey value of non-stained areas of all images and fixed for all images during analysis. For each animal, the average perimeter and average percentage of area above threshold were calculated. The group averages of T60–90 females, control males and females (C60–90) were compared using Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn’s method with 5% significance levels.

Unbiased Stereology Analysis

Using StereoInvestigator software (MicroBrightField, Williston, VT, USA) and the optical fractionator method, unbiased stereological estimations of the total number of TH-IR, non-TH-ir/cresyl violet-stained cell bodies, and total numbers of all neurons in the VTA as well as total volume of the VTA in sections from males (n=3), control females (C30–90; n=5), and T30–90 females (n=6). Sections stained for TH with DAB used in the cell counting analysis described above, were counterstained with chresyl violet and imaged using a Leica microscope (DM5000B, Leica Microsystems; Wetzlar, Germany) attached to a CCD camera (Microfire, Optronics, Goleta, CA, USA) and a motorized stage controlled by StereoInvestigator software (MicroBrightField Inc). The cerebral peduncle and the diameter of the central aquaduct were used as landmarks to determine the most rostral end of the VTA and thus the location to initiate the stereological analysis. The seven subsequent sections were chosen for analysis in a rostral-caudal manner, for a total of eight tissue sections per animal. Prior to immunoprocessing, the sections were 45 μm thick and every tenth parallel section was included in the analysis. Section thickness was determined by recording the distance between the planes at which the first object within the area of analysis to come into focus, and the plane at which the last object went out of focus. The average final section thickness was 28.2 μm, accounting for shrinkage. The region of interest (i.e. the VTA) was delineated at 5x magnification. A grid measuring 700 × 350 μm was then positioned over this contour tracing; the individual counting frame used measured 190 × 190 μm with a dissector height of 10 μm. Individual cell bodies were identified by an observer blinded to treatment groups at 40x magnification, based on the identification of the nucleus of each neuron. Cells stained for TH with a visible nucleus were marked TH-ir, while cells stained with cresyl violet but not TH were marked as non-TH-ir. The optical fractionator method was used to estimate the total numbers of neurons. VTA regional volume was determined using the Cavalieri Estimator Protocol. The coefficient of error for all samples was ≤0.13. One Way ANOVA and Homs-Sidak tests were used for statistical comparisons between groups.

Photographs

Digital images for Figures were captured using a CCD camera (DFC340FX or DFC420, Leica, Wetzlar, Germany) attached to a Leica microscope (DM5000B). Digital images for Fig. 2.2 and Fig. 2.7 were captured using a CCD camera (Microfire, Optronics, Goleta, CA, USA) attached to a Leica microscope (DM5000B, Leica Microsystems; Wetzlar, Germany). All images were imported into Adobe Photoshop software (Adobe Systems, San Jose, CA, USA) and were not altered in any way except for adjustment of brightness.

Results

Characterization of the midbrain dopaminergic neurons in sheep

There is virtually no published information about the VTA dopamine system in sheep; therefore; the neuroanatomical localization of the TH-ir neurons was first analyzed in control males and females (C60–90). The distributions of TH-ir midbrain neurons in a representative male and control female are illustrated in Figure 1. In both sexes, TH-ir neurons were observed in the midbrain, in locations corresponding to the VTA and the SN with very similar anatomical landmarks to those described in rodents (Balfour et al., 2004) (Figure 1). Even though there were no apparent differences in the general localization of TH-ir neurons between males and females, apparent differences in TH-ir and cell number were observed (Figure 2).

Figure 2. Representative images of TH-ir neurons.

Figure 2

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of male (A, B, C), control female (C60–90; D, E, F) and prenatal T-treated female (T60–90; G, H, I) in the middle VTA at 5x (A, D, G), and 20x (B, E, H) and in the middle SN at 20x (C, F, I). Scale bars: 250μm (5x) or 50μm (20x).

Sex differences and effects of prenatal T60–90

Numbers of TH-ir cells in VTA

Sex differences and effects of prenatal T were observed in the numbers of TH-ir cells in the VTA. Statistical analysis revealed an overall difference between groups at all rostral-caudal levels of the VTA (F(2,22)=31.34–33.225; p<0.001). Specifically, males had significantly greater numbers of TH-ir cells than control females (C60–90) at middle (p<0.001) and caudal (p<0.001) levels of the VTA (Figure 3). Females with excess prenatal T treatment from GD 60–90 (T60–90 females) had significantly more TH-ir neurons compared to control females in all levels of the VTA (p<0.05, 0.005, 0.001; Figure 3) but fewer TH-ir neurons than males in the middle (p<0.001) and caudal (p=0.002) regions of the VTA (Figure 3).

Figure 3. Quantitative analysis of TH-immunoreactive neurons in T60–90.

Figure 3

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Mean number (± SEM) of TH-ir cells in the rostral, middle and caudal levels of the VTA (A) and SN (B) in control males (black; n=8), control females (C60–90; white; n=9) and prenatal T-treated females (T60–90; grey, n=6). * indicates statistically significant difference compared to male controls. # indicates statistically significant difference compared to female control C60–90 group.

Numbers of TH-ir cells in SN

Sex differences were also observed in TH-ir in the SN, as control males had significantly more cells compared to control females (C60–90) in the middle (p<0.001) and caudal (p<0.001) levels, but not rostral level (Figure 3). However, prenatal T treatment from GD 60–90 (T60–90) did not affect numbers of TH-ir neurons in the SN as T60–90 females were not significantly different from control females and had significantly fewer TH-ir neurons than males at the middle (p=0.007) and caudal (p=0.002) levels (Figure 3).

Optical Density Analysis of TH-ir

Differences between groups in intensity of TH-ir were observed in the VTA, with more extensive dendritic and soma labeling in males and T60–90 females compared to control females (Figure 2). Analysis of the optical density of TH-ir within the soma of VTA neurons demonstrated that males had significantly greater TH-ir optical density within the neurons compared to control females (p<0.001; Figure 4). T60–90 females had significantly greater TH-ir optical density in cells compared to control females (p=0.003) and did not differ from control males (Figure 4). Analysis of the soma size or perimeter of TH-ir neurons in VTA revealed no significant differences between groups (Figure 4).

Figure 4. TH-ir density and soma perimeter in T60–90.

Figure 4

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(A) Percent area above threshold (± SEM) of TH-ir in soma and mean (± SEM) perimeter of TH-ir cell bodies of control males (black; n=5), control females (C60–90; white; n=7) and prenatal T-treated females (T60–90; grey; n=5) in the VTA (A). * indicates statistically significant difference from males and T60–90 females.

Effects of prenatal T 30–90

To test the effect of prenatal T during a longer gestational period (GD 30–90), the numbers of TH-ir neurons were compared among control males (subgroup of males in study above; the mean of VTA TH-immunoreactive cells in this subgroup was similar to the mean of the entire group), control female (C30–90), and T30–90 females.

VTA

There was an overall difference between groups at the middle and caudal levels of the VTA, with a trend in the rostral VTA (Table 1; Rostral: F(2,16)=3.296; p=0.067, Middle: F(2,16)=15.07; p<0.001, Caudal: F(2,16)=12.49; p<0.001). Since effects of prenatal T in the VTA were not specific to a rostro-caudal subregion (Table 1), from here on data were analyzed and presented in Figure 5 as the average of the entire VTA (Figure 5; F(2,16)=16.51; p<0.001). Sex differences were observed, and consistent with the results described above, males had significantly greater numbers of TH-ir cells than control females (p<0.001, Figure 5). T30–90 females had significantly increased number of TH-ir cells when compared to control females (p<0.001) and did not differ from control males (Figure 5). Moreover, differences in TH-ir were again noted within the TH-positive neurons (Figure 5C–D) with darker immunoreactivity in T30–90 females than control females.

Table 1.

Summary of numbers of TH-ir neurons in three rostral-caudal levels in VTA

Group Rostral Middle Caudal
Male 81.3 ± 4.3 176.5 ± 18.3 # 230.3 ± 16.1 #
Female 65.9 ± 4.4 106.5 ± 6.1 * 146.9 ± 8.9 *
T30–90 84.0 ± 8.07 155.4 ± 7.21 # 217.7 ± 18.0 #
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Mean number (± SEM) of TH-ir cells in the rostral, middle and caudal levels of the VTA in control males (Male; n=4), control C30–90 females (Female; n=8), prenatal T-treated females (T30–90; n=5).

*

indicates significant difference from male and T30–90 groups (p<0.05).

#

indicates significant difference from control females (p<0.05).

Figure 5. Quantitative analysis of TH-immunoreactive neurons in T30–90.

Figure 5

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Mean number (± SEM) of TH-ir cells in the VTA (A) and SN (B) in control males (n=4), control females (C30–90; n=7) and prenatal T-treated females (T30–90; n=5). * indicates statistically significant difference compared to male controls and T30–90 females. C and D show representative images of TH-immunoreactivity in VTA of control female (C30–90; C) and prenatal T-treated female (T30–90; D. Scale bar indicates 25 μm.

SN

In the SN, sex differences were again noted at all three rostral-caudal levels (Table 2) and data are therefore also presented in Figure 5 as an average across the three levels. Analysis showed that control males had significantly greater number of TH-ir neurons than control females (p=0.001; Figure 5). In contrast, prenatal T 30–90 treatment did not affect the number of TH-ir in the SN as T30–90 females did not significantly differ from control females and had significantly fewer TH-ir neurons than males (p=0.002; Figure 5), confirming the results described above.

Table 2.

Summary of numbers of TH-ir neurons in three rostral-caudal levels in SN

Group Rostral Middle Caudal
Male 66.6 ± 1.5 160.0 ± 7.5 202.0 ± 11.0
Female 67.7 ± 5.3 120.3 ± 6.7 * 156.7 ± 6.2 *
T30–90 70.1 ± 6.2 120.7 ± 11.6 * 146.9 ± 6.9 *
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Mean number (± SEM) of TH-ir cells in the rostral, middle and caudal levels of the SN in control males (Male; n=4), control C30–90 females (Female; n=8), and prenatal T-treated females (T30–90; n=5)

*

indicates significant difference from males (p<0.05).

Stereological Analysis

The findings thus far demonstrate that prenatal T (60–90 or 30–90) increased the numbers of TH-ir neurons in the VTA and also increased immunoreactivity within the TH neurons, similar to males. To further test whether the sex differences and/or the effect of prenatal T were specific for TH-ir cells, or were also present in non-TH-ir neurons in the VTA, a stereological analysis was conducted in control males, control females (C30–90), and T30–90 females. And secondly, it was tested if the volume of the VTA was affected by prenatal T or by sex differences. Statistical analysis confirmed an overall difference in numbers of TH-ir neurons between groups (F (2,11)= 26.78; p<0.001). Specifically, control males had higher number of TH-ir neurons than control females (p<0.001). Prenatal T-treated females (T30–90) had significantly more TH-ir and total numbers of cells in VTA compared to control females (p<0.001) and did not significantly differ from control males. In contrast, there were no sex differences or effects of prenatal T30–90 in the numbers of non-TH-ir neurons in the VTA and in the total volume of the VTA. Hence, effects of prenatal T and sex differences were specific to TH-ir neurons in the VTA. Moreover, prenatal T increased the total numbers of neurons in the VTA, which was mostly attributed to increases in TH-ir neurons.

Discussion

The results of the present study demonstrated that the adult sheep midbrain dopamine neurons of the VTA are sexually dimorphic, with more TH-ir neurons in males compared to females. Prenatal T excess during gestational days 60–90 partially masculinized the VTA dopamine system, while exposure to a longer period of prenatal T during gestational days 30–90 completely masculinized the VTA dopamine neurons. This was reflected by changes in both the number of dopamine neurons and the density of TH-ir. Moreover, stereological analysis showed that non TH-ir cells in the VTA or VTA volume were not affected by prenatal T. Together these results suggest that exposure to excess prenatal T during gestational days 30–90 results in increases in TH-ir neuron number and expression in VTA of adult ewes, mimicking sex differences in adult sheep VTA.

The findings of this study revealed a sex difference in the expression of TH-ir neurons in the VTA and SN of the sheep, with males having a significantly greater number of dopamine neurons than females in both regions. The general distributions of TH-ir neurons in the VTA and SN of male and female sheep did not differ between sexes or from those previously described in a variety of mammals, including rat (Balfour et al., 2004; Johnson et al.; Johnson et al.), hamster (Vincent, 1988), pig (Ostergaard et al., 1992), sheep (Tillet & Thibault, 1989), human (Pearson et al., 1983), and primates (McGeer et al., 1971). However, the finding that ewes have significantly fewer TH-ir neurons in the VTA and SN compared to males is in apparent contrast with previous findings in the female rat, showing significantly greater number of TH-ir neurons in the caudal VTA in females than in males (McArthur et al., 2007). Moreover, no sex differences in TH-ir neurons were detected in California Mice (Campi et al., 2013).

These conflicting findings may in part be explained by species differences and/or by procedural differences in the control of steroidal milieu in the adult females. In the present study, all females were ovariectomized and received estradiol to eliminate differences in estradiol levels between groups. In contrast, the afore mentioned rodent studies utilized gonadally intact females. Since estradiol has been shown to increase TH-ir cells in the female rat VTA (Johnson et al., 2010b), controlling for differences in the activational effects of estradiol is critical (Arnold, 2014). However, a caveat of the present study is the lack of control for activational effects of steroids in the adult male group. Even though testosterone in male rats has been shown to cause a decrease in TH-ir neurons in the VTA (Johnson et al., 2010a), it is possible that the present finding of higher numbers of TH-ir neurons in VTA in males may be partially due to the activational actions of testosterone.

In the SN, it has been reported that male rats have significantly greater number of dopamine than females (Dewing et al., 2006; McArthur et al., 2007). Moreover, the specific expression of the Y chromosome-linked male-determining Sry gene appears to mediate the increased expression of TH in the SN, without any mediation of steroid hormones (Dewing et al., 2006). Dewing and co-workers demonstrated that Sry is expressed in TH-expressing neurons in the SN and that Sry down regulation using oligonucleotide antisense treatment caused a decrease in numbers of TH expressing neurons in the SN. This is consistent with our findings in the sheep that males had significantly greater number of dopamine in the SN, but that prenatal T treatment for either 30 or 60 days did not result in increased SN dopamine neuron number in females, and suggest that the sex differences observed in the SN of the sheep may be independent of the actions of prenatal steroids and rather a result of sex chromosome compliments (Arnold, 2014). However, prenatal T treatment in the ewe did appear to affect the expression of TH protein within SN neurons. Hence, a combination of gene-specific and hormonal mediation may regulate TH expression and cell number in the sheep SN.

In the VTA, prenatal T treatment during either gestational days 30–90 or 60–90 resulted in a significant increase in the number of TH-ir neurons and the intensity of TH-ir. However, the perimeter of the dopamine neurons was not affected by prenatal T exposure, which is consistent with studies in rodents, in which sex, steroid treatment or regional analysis revealed differences in TH-ir cell number, but not size (McArthur et al., 2007; Johnson et al.; Johnson et al.). The current results suggest that the increase in the number of neurons observed in the current study in males and prenatal T-treated females is possibly a result of an increase in TH protein level expression, as evidenced by increased levels of imunoreactivity such that more cells expressed TH at levels above the limit of detection by the immunohistochemical technique. Further evidence for this possibility arises from a study in sheep that examined effects of prenatal T treatment on neuropeptide expression in the arcuate nucleus of the hypothalamus. In a population of cells that co-express three neuropeptides: kisspeptin (KISS), neurokinin B (NKB) and dynorphin (DYN), prenatal T treatment from gestational days 30–90 resulted in a decrease in NKB and DYN; hence fewer cells expressing NKB or DYN were observed. However, the expression of KISS, or the number of KISS cells was unchanged (Cheng et al., 2010). Thus, prenatal T did not result in cell loss per se, but rather affected expression of two out of three neuropeptides co-expressed by the same cell population. However, the results of the present study also suggest that prenatal T exposure alters processes of cell death and/or proliferation during development, as stereological analysis revealed an increase in total numbers of cells in prenatal T-treated females and males, which was primarily accounted for by an increase in TH-ir cells. Effects of prenatal exposure to T in rodents on VTA cell survival has not yet been examined, but studies in rat observed that activational effects of T in adulthood cause a decrease in midbrain dopamine cell survival (Johnson et al., 2010a; Johnson et al., 2010b).

The findings of the current study demonstrate that prenatal T organizes the VTA dopamine system of adult sheep. It is currently unclear whether in sheep, T has these organizational effects via androgenic or estrogenic actions. Previous studies in rodents have shown that TH gene expression can be regulated by both T and estradiol, which can act directly by regulating promoter activity or indirectly by altering levels of other regulator factors (Simerly et al., 1989; Thanky et al., 2002). T can be converted to DHT and act on androgen receptors (AR), while AR activation is able to directly increase TH expression (Jeong et al., 2006). Moreover, DHT can also be metabolized to a compound, 5α-androstan-3β,17β-diol, which exerts its actions through estrogen receptor β (ERβ) (Handa et al., 2008). Evidence from a study using the TH promoter-LacZ transgenic mouse model suggests that estradiol regulates TH expression at the transcriptional level (Thanky et al., 2002). Moreover, TH transcriptional regulation by estradiol depends on the ERα subtype (Maharjan et al., 2005). In cultured rodent midbrain dopamine neurons, estrodial can increase TH mRNA and protein levels (Ivanova & Beyer, 2003) and stimulate neurite growth and plasticity (Beyer & Karolczak, 2000). Female mice that lack the gene coding aromatase, the enzyme responsible for the conversion of T to estradiol, have lower SN dopamine neuron numbers compared to wild-type animals (Morale et al., 2008) and removal of estradiol by ovariectomy in adult rats decreased VTA and SN dopamine neurons, an effect mediated by estrogen receptors (Johnson et al., 2010b). Thus, in rodents T can influence TH expression by actions on AR, ERβ, or ERα receptors. Expression of steroid receptors in sheep VTA dopamine neurons is currently untested. However, in the rat, AR and ERβ are expressed in midbrain dopamine neurons (Kritzer, 1997). In rodents, estrogen receptors are present in the midbrain with ERβ having a more prominent and widespread distribution than ERα, which is not co-expressed in dopamine neurons of the VTA (Kritzer, 1997; Creutz & Kritzer, 2002). In addition, TH neurons in VTA and SN also co-express AR (Kritzer & Creutz, 2008) (Creutz & Kritzer, 2004). Together these results suggest that in the rodent, sex differences in the midbrain dopamine neurons are a result of the estrogenic actions of T. However, this remains to be determined in the sheep and will be the focus of future studies.

Together these studies demonstrate that there are sex differences in the sheep VTA dopamine system with greater numbers of TH-ir neurons in males than females. Moreover, these findings demonstrate that T masculinizes the VTA dopamine during prenatal life, increasing TH-ir neurons in the adult ewe. These differences in dopamine expression are the result of the organizational effects of T during the prenatal period, since the endogenous steroid hormones during adult life were equal in prenatal T-treated females and control females. Even though in the discussion above we have favored the hypothesis that prenatal T directly affected VTA dopamine neurons, we did not test whether the observed changes in the adult animal were evident immediately after birth, in early life. Hence, the possibility that prenatal T-induced changes in behavior or other functions and experiences during the animal’s life could in turn influence the mesolimbic dopamine system to contribute to the observed changes cannot be eliminated and remains to be tested.

Figure 6. Stereological analysis in T30–90.

Figure 6

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Graphs show numbers of VTA TH-ir cells (A), numbers of VTA non-TH-ir cells (B), total numbers of VTA cells (C), and total VTA volume (in cubic millimeters; D) in control males (N=3), control females (C30–90; N=5), and prenatal T-treated females (T30–90; N=6). Data are presented as mean ± SEM. * indicates statistically significant difference from male control. # indicates statistically significant difference from female control.

Acknowledgments

This research was supported by NIH P01 HD44232 awarded to LC, TM, MNL and VP; NIH HD41098 awarded to VP; and NSERC PGSD to ECZB

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