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BOR - Papers in Press, published online ahead of print May 31, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.052795
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BIOLOGY OF REPRODUCTION 75, 469–476 (2006)
DOI: 10.1095/biolreprod.106.052795
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Sex Differences and the Development of the Rabbit Brain: Effects of Vinclozolin1

Erin S. Bisenius , D.N. Rao Veeramachaneni , Ginger E. Sammonds , and Stuart Tobet 2 

Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT

The preoptic/anterior hypothalamic area (POA/AH) is one of the most sexually dimorphic areas of the vertebrate brain and plays a pivotal role in regulating male sexual behavior. Vinclozolin is a fungicide thought to be an environmental antiandrogen, which disrupts masculine sexual behavior when administered to rabbits during development. In this study, we examined several characteristics of the rabbit POA/AH for sexual dimorphism and endocrine disruption by vinclozolin. Pregnant rabbits were dosed orally with vinclozolin (10 mg/kg body weight) or carrot paste vehicle once daily for 6 wk beginning at midgestation and continuing through nursing until Postpartum Week 4. At 6 wk, offspring were perfused with 4% paraformaldehyde and brains processed for immunocytochemical localization of tyrosine hydroxylase, calbindin, gonadotropin-releasing hormone (GnRH), or Nissl stain. There were significant sex differences in the distribution of calbindin in the POA/AH and the size of cells in the dorsal POA/AH (values greater in females than in males), but not in the number or distribution of tyrosine hydroxylase or GnRH neurons. In both sexes, exposure to vinclozolin significantly increased calbindin expression in the ventral POA/AH and significantly decreased number of GnRH neurons selectively in the region of the organum vasculosum of the lamina terminalis (OVLT) but not more caudally in the POA/AH. This is the first documentation of a sexually dimorphic region in the rabbit brain, and further supports the use of this species as a model for studying the influence of vinclozolin on reproductive development with potential application to human systems.

early development, gonadotropin-releasing hormone, hypothalamus, male sexual function, neuroendocrinology

INTRODUCTION

Vinclozolin is a dicarboximide fungicide that is widely used in agriculture and commercial horticulture. It is applied to many food crops, including lettuce, snap beans, and grapes, as well as to golf courses, ornamental plants, and lawn turf [1]. Thus it can be ubiquitously found in soil, water, and air, where humans can be exposed to it through a variety of modes/routes. Vinclozolin belongs to a family of endocrine disrupting compounds (EDCs) that are normally thought to act as antiandrogens. This family of compounds typically works by binding to androgen receptors, thus preventing the intended ligand from binding and eliciting expected responses, such as influencing gene expression [2].

Steroid hormones play a critical role in the processes of brain sexual differentiation and sexual development. Therefore, these processes may be particularly vulnerable to endocrine disruption by compounds that block or mimic endogenous hormones. Steroid hormones can organize neural systems perinatally which can later be activated by the same hormones, yielding sex differences in physiology and behavior as well as brain sexual dimorphism [3]. Exposure to EDCs during critical periods in fetal development can alter the organization or differentiation of reproductive organs, the neuroendocrine system, and subsequent sexual function and behavior. Perinatal vinclozolin exposure produces adverse effects in male rodents, including abnormal reproductive development, alterations in sexual differentiation, dysfunctional sexual behavior, and malformed reproductive/sex organs [2–6]. More specifically, these alterations manifest as female-like anogenital distance at birth, the formation of areolas and subsequent retention of nipples, suprainguinal ectopic testes (cryptorchidism), genital malformations (cleft phallus and hypospadias), hypoplastic or absent sex accessory organs, the retention of a blind vaginal pouch, abnormal ejaculation, and the formation of epididymal granulomas [3–6].

Brain targets for sexual differentiation in rabbits may parallel systems that are known as sexually dimorphic in other species, such as the preoptic area/anterior hypothalamus (POA/AH). The POA/AH is one of the most sexually dimorphic regions of the vertebrate brain and plays a key role in regulating male sexual behavior [7, 8]. Anatomical studies in adult animals from a number of species have defined sex differences in the POA/AH ranging from the detection of specific cell groups and their volumes to measures of cell number, cell size, dendritic characteristics, and details of synaptic ultrastructure [7, 8]. At the biochemical level, sex differences in peptides, receptors, and protein content and hormone influences on them have been repeatedly shown in the peripubertal and adult POA/AH [7, 9, 10]. While the POA volume in rats, gerbils, guinea pigs, ferrets, quail, and humans has been shown to be sexually dimorphic, no potentially sexually dimorphic parameters have been described in rabbits.

The underlying mechanisms of how endocrine disruptors exert their adverse effects in various processes, including reproduction, and particularly at the level of the hypothalamus, are little known. In humans and other primates, the hypothalamic-pituitary unit that governs gonadal function becomes operational during fetal and neonatal development, and final differentiation and maturation occurs as puberty approaches [11]. We have observed that developmental exposure of male rabbits to vinclozolin altered adult sexual behavior, compromised erectile or ejaculatory function, diminished FSH secretion, and impaired spermiogenesis [12], suggesting that the developing brain may be a significant target. The current study was conducted to determine sexually dimorphic characteristics in the rabbit brain and to assess whether they are influenced by vinclozolin. The results indicate selective sexual dimorphism in regions of the rabbit brain that are likely tied to neuroendocrine function. The results also show that developmental vinclozolin exposure influences the regions of the rabbit brain that are likely tied to neuroendocrine function, but that are not sexually dimorphic.

MATERIALS AND METHODS

Ten 6-mo-old, specific-pathogen-free, timed-pregnant female Dutch-Belted rabbits were procured from Myrtle's Rabbitry (Thompson Station, TN) and individually housed in standard stainless steel cages in the Colorado State University animal care facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. All procedures involving animals were conducted after approval by the Institutional Animal Care and Use Committee.

Housing was in a room maintained at 12L:12D cycle, at approximately 19–21°C and 40% humidity. Rabbits were fed Prolab Hi-Fiber rabbit diet (5P25, Purina Mills) and were provided water ad libitum. Beginning at midgestation (Gestation Day 15; gestation length = 30–31 days), rabbits were dosed orally with vinclozolin (10 mg/kg body weight, n = 5) or certified organic carrot puree vehicle (1ml/kg body weight, n = 5) once daily for 6 wk, until weaning at the end of Postnatal Week 4. Vinclozolin (PS-1049, ChemService Inc, West Chester, PA) was administered as a homogeneous suspension in the carrot puree (Earth's Best Organic First Carrots, Hain Celestial Group, Inc, Melville, NY). There was no adverse effect of vinclozolin treatment on maintenance of pregnancy to full term or growth of pups compared to controls. Due to the long developmental period in rabbits, which mimics human development, puberty does not begin until roughly 16 wk. Thus, it is likely that gonadal steroid levels were equally low in both control and treated male and female rabbits.

At birth, there were 24 live pups and 3 stillborn pups in 5 vehicle-treated control litters (litter size ranged from 4 to 10) and 33 live pups in 5 vinclozolin-treated litters (litter size ranged from 6 to 8). By Postnatal Week 1, two pups in one control litter and four pups in two vinclozolin litters died. There were no further deaths in either group. Two weeks after weaning, at 6 wk of age, 12 male pups and 7 female pups from control litters and 16 male pups and 10 female pups from vinclozolin litters were killed, each with an intravenous injection of 0.25 ml Beuthanasia-D Special (390 mg pentobarbital sodium/ml; Schering-Plough, Union, NJ). The pups were perfused with 100 ml heparinized saline followed by 200 ml 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, postfixed overnight in 4% paraformaldehyde, and refrigerated in 0.1 M phosphate buffer until sectioning. Brains were sectioned 60 µm thick using a vibrating microtome (Leica VT1000S) and collected into chilled PBS, beginning at the point where the anterior commissure initially appears from the rostral diagonal band region and ending eight sections after the disappearance of the anterior commissure. The brain sections were stored at 4°C until use. Seven male and six female pups from five vinclozolin-treated litters and seven male and six female pups from four control litters were processed for immunocytochemical analyses according to previously published procedures [13, 14].

Briefly, free-floating sections were cut into 0.05 M PBS at 4°C, followed by incubation in 0.05 M PBS with 0.1 M glycine for 30 min. The slices were then rinsed in PBS, bathed for 15 min in 0.5% sodium borohydride, and then rinsed again in PBS. Sections were incubated for 30 min in 5% normal goat serum (NGS) (Sigma-Aldrich, St. Louis, MO) as a blocking agent with 0.3% Triton-X/PBS and 1% H2O2, followed by incubation at 4°C for 48 h in primary antibody (anti-GnRH mouse monoclonal at a dilution of 1:500, QED Bioscience (San Diego, CA); antityrosine hydroxylase mouse monoclonal at a dilution of 1:4000, Immunostar (Hudson, WI); and anticalbindin mouse monoclonal at a dilution of 1:20 000, Sigma-Aldrich.

After incubation in primary antisera, section processing for secondary antisera began with rinses in 0.05 M PBS with 1% NGS and 0.02% Triton-X at room temperature. This was followed by incubation with secondary antibody (anti-mouse raised in goat at 1:2500 dilution, Jackson ImmunoResearch (West Grove, PA) for 2 h at room temperature. The tissue was then rinsed in 0.05 M PBS/0.02% Triton-X, incubated in Vectastain Elite ABC reagent (1µl/ml each of A and B reagent 30 min prior to use), and rinsed in 0.05 M Tris buffer (TBS) at pH 7.5. To visualize the reaction, brain sections were incubated for 5 min in 0.025% DAB/0.02% nickel (Sigma-Aldrich) with 0.02% H2O2 in TBS, followed by a final set of washes in TBS and cold storage in TBS until mounting on gel-coated slides. After collecting the tissue slices on slides, coverslips were mounted with Permount for morphometric analyses.

Data Analysis

Immunocytochemical localization of tyrosine hydroxylase and calbindin was mapped in selected regions of the POA/AH that corresponded to regions of interest based on sex differences in other species; i.e., the region of the anteroventral periventricular (AVPV) preoptic area for tyrosine hydroxylase [15] and the caudal preoptic area for calbindin [16]. Digital images were captured using the 10x objective on an Olympus microscope fitted with bright field optics and a Diagnostic Instruments Spot Insight QE camera. Using a grid overlay (each box represents 100 x 100 µm) anchored by the anterior commissure, third ventricle, and optic chiasm, differences in the number of immunoreactive cells based on position were quantified (Fig. 1). As described previously for the mouse preoptic area and hypothalamus [14, 17], we used a grid overlay to describe cell positions in a consistent manner anchored by objective landmarks (anterior commissure dorsally, optic chiasm ventrally, and third ventricle medially). This was particularly important for the rabbit brain in the current study, since there were no discernible nuclear groupings in the region (other than the suprachiasmatic nucleus) to orient the reader (Fig. 1A). For ease of identification and in combination with Dorsal-Ventral and Medial-Lateral neuroanatomical designations, the grid overlay is based on equally spaced 100-µm distances from dorsal regions just beneath the anterior commissure stretched ventrally to the optic chiasm, and from medial to lateral equally spaced 100-µm distances beginning at the margin of the third ventricle (Fig. 1, B and C). GnRH neurons were counted manually in each section. Sections for each animal in a group were aligned in sequence by the location of the organum vasculosum of the lamina terminalis (OVLT). The rostral OVLT region (4 sections) consisted of cell counts from the section rostral to the OVLT section, including 2 sections caudal to the OVLT. The more caudal POA/AH region was counted as the next 4 caudal sections. Cell counts were analyzed using SPSS statistical software (SPSS Inc., Chicago, IL).


Figure 1
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  		FIG. 1. Digital images depict the quantitative approach to analyzing differences in the number of immunoreactive elements based on their positions. A) Nissl-stained section through the POA/AH showing the absence of cell grouping other than the suprachiasmatic nucleus (SCN) at the base of the POA/AH. B) Note the cell grouping in the middle region of the POA/AH (see arrow). This grouping of immunoreactive calbindin cells is in a similar position to that noted as being sexually dimorphic in rats. Also note that the SCN is calbindin negative. C) The same section as in B. Grid lines are 100 µm apart. AC, anterior commissure; OC, optic chiasm; SCN, suprachiasmatic nucleus; V, third ventricle

For Nissl analyses, every fourth brain section from the OVLT region through the caudal POA/AH was inspected for evidence of reliably discernible nuclear groupings. In the absence of reliable nuclear groupings, sections were selected from a specified region in the caudal preoptic area of each animal for cell size analysis, and high-magnification images were captured using an Olympus microscope fitted with a 40x objective and a Diagnostic Instruments Spot camera. Images were then analyzed using IP Lab software (Scanalytics Inc., Rockville, MD) on a Macintosh G4. This approach was adapted from the one used in ferrets to maximize size measurement reliability and within section controls for tissue processing [18]. For each animal, 12 cells were traced to provide estimates of individual cell areas from each of 3 image fields from 2 regions (dorsal versus ventral). The value taken for analysis was the average of the mean cell size from the three regions in each animal.

RESULTS

Gonadotropin-Releasing Hormone

Vinclozolin treatment resulted in a region-specific decrease in the immunoreactive GnRH-1. Confirming previous reports, neurons immunoreactive for GnRH-1 were spread across a broad region stretching rostrally from the diagonal band of Broca (DBB) caudally to regions of the basal hypothalamus. There was no sex difference in the number of immunoreactive GnRH neurons from the DBB to the caudal POA. However, perinatal exposure to vinclozolin resulted in 22% fewer immunoreactive-GnRH neurons overall in both sexes (Fig. 2, A–C; P < 0.01). Perhaps more striking was the strong trend for the treatment effect to be limited to the rostral OVLT region (36% difference between control and vinclozolin-treated) compared to the more caudal POA/AH (10% difference) (Fig. 2D). Thus virtually all of the statistical significance of the overall influence of vinclozolin was due to the difference in the more rostral OVLT region. Ratings of GnRH-1 immunoreactive fiber density in the region of the OVLT failed to detect effects of sex or vinclozolin (data not shown).


Figure 2
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  		FIG. 2. The number of GnRH-immunoreactive neurons was reduced in rabbits exposed to vinclozolin perinatally. Digital images of coronal sections through the OVLT region of the hypothalamus of a 6-wk-old control male (A) and vinclozolin-exposed male (B) show notably fewer immunoreactive neurons in the vinclozolin-exposed rabbit. C) Graph showing the mean cell number ± SEM by both sex and region, demonstrating a treatment difference (P < 0.01) but no sex difference. D) Graph showing the mean cell number ± SEM in the OVLT and POA regions, demonstrating the significantly lower overall numbers in vinclozolin-treated rabbits (P < 0.01) and a strong indication that the effect is due to differences in the more rostral OVLT region. Bars = 100 µm. V, opening of the third ventricle

Calbindin

Vinclozolin exposure increased calbindin immunoreactivity in both males and females in the ventrolateral region of POA/AH; calbindin immunoreactivity was sexually dimorphic in the ventral region adjacent to the third ventricle. Calbindin immunoreactivity in the ventral POA/AH was significantly increased (P < 0.05) in both males and females exposed to vinclozolin (Fig. 3, small boxes). This effect was restricted to the lateral portion (200–400 µm lateral from the third ventricle) and ventral portion of the POA/AH (the region 600–800 µm ventral to the anterior commissure), suggesting that vinclozolin may not have been acting as a pure antiandrogen, since its effect was in a region where there were no sex differences (Fig. 4).


Figure 3
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  		FIG. 3. Digital images show calbindin immunoreactivity in the POA/AH in control female (A), vinclozolin female (B), control male (C), and vinclozolin male (D) rabbits at 6 wk of age. In the medial large boxed region (500 µm tall x 200 µm wide), the number of immunoreactive calbindin neurons was sex-dependent (P < 0.05, quantitative graph in figure 4) with females (A and B, large boxes) having more cells than males (C and D, large boxes). In the more lateral small boxed region (300 µm tall x 200 µm wide), vinclozolin-exposed rabbits had increased calbindin immunoreactivity (B and D) compared to controls (A and C) in both males and females (P < 0.05, quantitative graph in figure 4). AC, anterior commissure; OC, optic chiasm; V, third ventricle


Figure 4
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  		FIG. 4. Graphs depict quantitative analyses of immunoreactive calbindin neurons dorsal to ventral (A) and medial to lateral (B). A) Notable differences in cell number (cell number ± SEM) were seen 300–800 µm ventral to the anterior commissure for sex and 700 to 900 µm ventral to the anterior commissure for vinclozolin. B) Across the medial lateral dimension, significant differences in cell number (cell number ± SEM) were easier to discern and were located medially 100 to 200 µm from the third ventricle for sex and 200 to 400 µm from the third ventricle for the influence of vinclozolin. AC, anterior commissure

Calbindin-immunoreactive neurons were distributed throughout the POA/AH of the rabbit brain. Interestingly, the suprachiasmatic nucleus was devoid of immunoreactive calbindin. In the medial POA/AH a cluster of immunoreactive calbindin cells (Fig. 3) occupied a similar position to those of the well-documented sexually dimorphic cell grouping in rodents [16, 19]. We found a significant sex difference in number and distribution of calbindin-immunoreactive cells in rabbits (P < 0.05). However, in contrast to rodents, there were more calbindin-immunoreactive cells in the female rabbit POA/AH (Fig. 3, A and B, large boxes) than in males (Fig. 3, C and D, large boxes). In the rabbit, these sex differences were restricted to the ventromedial portion of the POA/AH: within 200 µm of the third ventricle and the region 400–800 µm ventral to the anterior commissure.

Nissl Stain

Cell size was sexually dimorphic in the dorsal POA/AH. Nissl-stained brain sections rostrally from the region of the OVLT to caudal POA/AH regions were analyzed for sex and treatment differences in cell grouping and cell size using computer-assisted image analysis. Throughout the POA/AH, there were no obvious medial cell groupings similar to those found in rats or humans [20], and thus there were no conspicuous sex differences in cell grouping. As previous studies in ferrets [18] and mice [21] have shown significant sex differences in the size of cells located in the dorsal POA/AH, we also measured cell size in this region of rabbits. Cell size measurements were taken from the region directly below the anterior commissure (dorsal cells) and a region 200 µm more ventral (ventral cells) (Fig. 5). The size of ventral cells was measured to control for artifacts of tissue processing, and the ratio of dorsal:ventral was used as the dependent measure [18]. After controlling for tissue processing, dorsal cell size in males was significantly smaller than in females (P < 0.05). The ratio of dorsal:ventral cell size was 1.45 + 0.06 for females (n = 12) and 1.31 + 0.04 for males (n = 12). There was no effect of vinclozolin exposure on cell size in males or females (P > 0.10).


Figure 5
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  		FIG. 5. Nissl staining in the POA/AH shows sexual dimorphism in cell size. Digital images of coronal sections through male POA (A) show the locations of dorsal (B) and ventral (C) cell size measurements (indicated by D and V, respectively in A). Digital images of coronal sections through female POA (F) show the comparable locations of dorsal (C) and ventral (E) cell size measurements (indicated by D and V, respectively in F). G) The graph shows a significant sex difference (P < 0.05) in the cell size ratio (dorsal/ventral). The error bars are ± SEM

Tyrosine Hydroxylase

Immunoreactivity of tyrosine hydroxylase showed no sex or treatment differences. Cells containing immunoreactive tyrosine hydroxylase were distributed throughout the rabbit POA/AH, similar to other species. We investigated the influence of sex and vinclozolin in two groups of cells containing immunoreactive tyrosine hydroxylase in the rabbit hypothalamus: a group located near the opening of the third ventricle in a region potentially homologous to the AVPV in rodents (Fig. 6A), and a group located more caudally in the POA/AH where there was also high calbindin expression (Fig. 6B). Surprisingly, we found no significant sex or vinclozolin effects on the number or distribution of tyrosine hydroxylase-immunoreactive neurons in either region (P > 0.50).


Figure 6
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  		FIG. 6. Tyrosine hydroxylase expression in the hypothalamus of a control 6-wk-old female rabbit. A prominent group of immunoreactive cells was noted near the base of the third ventricle (V) in all animals both rostrally in the region of the putative AVPV (A) and more caudally in the POA (B). There were no effects of sex or vinclozolin on tyrosine hydroxylase immunoreactivity. Bar = 100 µm. OC, optic chiasm; AC, anterior commissure

DISCUSSION

In the current study using a relatively low dose of vinclozolin, we found modifications of brain organization as demonstrated by altered calbindin and GnRH-1 immunoreactivity. Interestingly, even though vinclozolin is known to be an antiandrogen and it affected regions of the brain associated with reproductive function, it did not modify sexually dimorphic characteristics of the rabbit brain. Vinclozolin is a potentially hazardous compound that is ubiquitous in our environment. A link has been proposed between human in utero exposure to vinclozolin and latent sexual dysfunction, including decreased sperm counts, infertility, erectile dysfunction, and urogenital malformations (hypospadias, cleft phallus, undescended testes) [5, 22]. It is estimated that children are exposed to vinclozolin at 0.167 mg/kg body weight/day [1, 23], which exceeds the benchmark doses associated with altered juvenile play behavior and erectile dysfunctions in our existing animal models [1]. The dosing paradigm used in our study may have yielded exposure levels in a range comparable to that commonly found in circulation and tissues of neonates following environmental exposure.

There are two hydrolytic products of vinclozolin, 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (Metabolite M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (Metabolite M2) formed in rabbits [24] as in rats [25]. Although we did not measure concentrations of vinclozolin and its metabolites in the current study, it is reasonable to assume that they were present in circulation during critical periods of sexual differentiation (in fetuses via transplacental exposure) and postnatal development (in pups via lactational exposure) and effectively competed with endogenous androgens for androgen receptor (Ki vs. R1881 (methyltrienolone) of >700, 92 and 9.7 µM for vinclozolin, M1 and M2) [25]. Based on studies in other species, the POA/AH is rich in androgen receptors [26]. To the best of our knowledge, however, there are no published studies in the localization of androgen receptors in the rabbit brain, so it is not possible at this point to guess whether vinclozolin could act directly in the cells that were visualized in the current study. In addition, little is known about whether vinclozolin cross-reacts with other steroid receptors, such as estrogen receptors. Even less is known about nonsteroidal alternative effects that have been attributed to vinclozolin action, such as on glutathione synthesis [27], that might impact neurotransmitter function [28].

Maternal treatment with vinclozolin led to male and female offspring having significantly decreased numbers of neurons containing immunoreactive GnRH-1. This decrease was particularly obvious in the region immediately surrounding the OVLT as compared to a more caudal region in the POA/AH. At the current time, there are few known intrinsic differences between neurons located in different portions of the GnRH neuronal population in any species examined [29] and androgen receptors were not found in GnRH neurons in rats [30]. Distinct functional subgroups within the GnRH neuron population have been postulated based on physiological responses following gonadectomy [31], mRNA fluctuations [32], progesterone receptor colocalization [33], and cFos induction [34]. A hierarchy of importance has been proposed whereby information flows from a small core group of GnRH neurons outward to more peripheral subgroups. If this is an accurate depiction, then interfering with a subpopulation of GnRH neurons, perhaps located around the OVLT in rabbits, could have important and widespread functional consequences [33]. Important to the current results, a previous study of developmental exposure to vinclozolin demonstrated diminished secretion of FSH (although not LH) in male rabbits [12]. Basal serum concentrations of FSH as well as pituitary secretion/discharge of FSH after exogenous GnRH were significantly lower than those of controls at 8 and 20 wk after cessation of vinclozolin exposure. This may be one indication of a potential functional consequence to the 36% decline in the number of detectable GnRH neurons in the region of the OVLT in rabbits of the current study.

There are several potential mechanism(s) responsible for a selective decrease in GnRH neurons. Possibilities include altering GnRH neuron generation or death, a cell's ability to synthesize GnRH-1 (and thereby our ability to detect it), or the migration of GnRH neurons (placing neurons in the regions for us to detect them). Future studies are needed to examine GnRH expression over a wider expanse of the migratory path to distinguish among these possibilities. Due to the longer migratory path of GnRH neurons in rabbits compared to rodents [35–37], it is possible that vinclozolin exposure may have caused GnRH neurons to migrate aberrantly during development, such that there are not actually fewer neurons, but rather they are located in a different region along the migratory path. The fact that the effect was regionally selective is consistent with a migratory disruption. Because GnRH-1 is the master hormone controlling the reproductive axis and essential to reproduction in all vertebrates, such a disruption in GnRH neuronal number or location might have profound implications on the development of subsequent reproductive abilities. Embryonic exposure to vinclozolin in Japanese quail altered GnRH expression in the POA and median eminence in male offspring and elicited abnormal male sexual behavior [38]. Thus, chemicals affecting migration and/or the development of GnRH neurons could cause significant endocrine disruption [39].

Developmental exposure to vinclozolin also led to an increase in the number of immunoreactive calbindin neurons ventrolaterally in the region of the POA/AH in both juvenile males and females. Interestingly, it did not target the subset of this region that is sexually dimorphic. In the current study, calbindin served as a biomarker for sexual dimorphism in the POA/AH with females having more calbindin immunoreactivity than males. Calbindin is a strong marker of sexual dimorphism in the rat sexually dimorphic nucleus of the preoptic area (SDN-POA) [16, 19], where calbindin expression is localized to a subset of the SDN-POA that mirrors the SDN-POA in its sexually dimorphic size (larger in males than females), and also by its being influenced by exposure to gonadal steroid hormones [16]. While calbindin immunoreactivity was sex-dependent in the medial POA/AH in the current study, the direction of the difference was opposite to that found in rodents: females had more than males. Calbindin is a neuron-specific calcium-binding protein, postulated to serve a neuroprotective role by buffering against calcium-induced neurotoxicity, thus enhancing cell survival [16, 40, 41]. Studies in humans corroborate this claim, revealing that calbindin levels in the hippocampus and substantia nigra decrease in Parkinson's disease, yet substantia nigra cells expressing calbindin are proportionally spared [40]. However, literature regarding the role of calbindin and other calcium-binding proteins is confounding, for some studies report it as neuroprotective while others report it as neurotoxic [42, 43]. For example, the calbindin-D28k-null mouse exhibits enhanced cell survival after ischemic insults [42].

In a number of circumstances calbindin expression has been influenced, either directly or indirectly, by gonadal steroid hormones [16, 19, 41, 44], in line with findings of sex differences in expression. Recent work has suggested that gestational exposure to genistein, a compound with estrogenic activity found in many soy products, increased the volume of a calbindin-D28k-immunoreactive subset of neurons in the SDN-POA in male rat offspring [19]. Estradiol has been linked with increased calbindin expression in various regions of rat brain [19], as has testosterone [44]. It is possible that vinclozolin, mimicking a steroid hormone, is acting directly to influence calbindin gene transcription. Data from rat studies suggest that vinclozolin may work as an antiandrogen [1, 4–6, 45]. However, because of the critical role steroid hormones play in nervous system development, particularly sexual differentiation, if vinclozolin was mimicking or blocking one of these hormones, it might be expected to exert effects on brain characteristics that are sexually dimorphic. This was not the case in the current study. However, all antiandrogenic compounds may not induce the same spectrum of lesions. This is evident from studies in male rabbits that were exposed to flutamide or p,p'-DDE ([1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene], a metabolite of the pesticide p,p'-DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane]), both of which are known androgen receptor antagonists. Both chemicals impaired testicular descent; however, germ cell atypia resembling carcinoma in situ was observed in undescended testes of p,p'-DDE-exposed rabbits, but not those of flutamide-exposed rabbits [46]. Likewise, sexual dysfunction and diminution of FSH secretion were observed in rabbits exposed developmentally to vinclozolin [12] but not in those similarly exposed to p,p'-DDT (Veeramachaneni, unpublished data).

Vinclozolin may have influenced GnRH-1 or calbindin immunoreactivity in the current study by acting through intermediary mechanisms. Selected neurotransmitter systems may modulate GnRH-1 or calbindin neuronal function in adulthood and in development [47, 48]. In the current study, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme for both dopamine and norepinephrine synthesis. For rabbits exposed to vinclozolin, we found no disruption of either fibers or cells in the regions where GnRH neurons were found nor anywhere else in the POA/AH. Immunoreactive tyrosine hydroxylase expression was neither sexually dimorphic nor affected by vinclozolin in rabbits. Similar to other species, tyrosine hydroxylase-immunoreactive cells were present throughout the rabbit hypothalamus. There was a group of immunoreactive tyrosine hydroxylase cells near the opening of the third ventricle in rabbits in a region similar to the AVPV in rodents, as well as a group of cells located more caudally in the region of high calbindin expression (POA/AH). In rodents, tyrosine hydroxylase is a biomarker for sexual dimorphism in these regions [49]. The current data in rabbits, however, are more in agreement with data in another induced ovulator, the ferret [50], where there were no sex differences in immunoreactive tyrosine hydroxylase in this region. Given that there may be sex differences in the activation of noradrenergic systems by mating stimuli in rabbits [51] and that other neurotransmitter systems may be important for male behaviors, additional systems (e.g., GABA [52]) or neuronal nitric oxide synthase [53–56] or functional information (e.g., turnover measurements) may be needed to distinguish further sex differences and vinclozolin actions in this region.

Nissl staining also revealed a sexual dimorphism in cell size in the dorsal region of the caudal POA/AH. Female rabbits had significantly larger cells than males in this region, a trend which is opposite to that seen in most other circumstances [7, 57], although not all [21]. Changes in somal size are often accompanied by changes in the extent of dendritic arborization, the amount of membrane with synaptic contacts, and the number of gap junctions between motoneurons [7]. Regardless of the functional correlate, the sexual dimorphism in the POA/AH continues to highlight this region as a prime target for sexual differentiation in rabbits as in other species. Importantly, however, in the current study cell size was not influenced by prenatal exposure to vinclozolin.

In summary, the current study has examined potential neural consequences of developmental exposure to a known endocrine disrupting chemical, vinclozolin. Furthermore, this work provides the first descriptions of sex differences in the rabbit brain. Results indicate that vinclozolin does influence brain development in cells or regions known to be important for neuroendocrine function. Surprisingly, vinclozolin did not influence specific, sexually dimorphic characteristics of the rabbit brain. Since vinclozolin is capable of acting as an antiandrogen, the results suggest that either the sexual dimorphisms identified in rabbit brain are estrogen-dependent or vinclozolin is capable of acting via alternative nonsteroidal mechanisms (i.e., glutathione and GABA).

ACKNOWLEDGMENTS

We thank Jennifer Palmer for technical assistance.

FOOTNOTES

1 Supported in part by NIH MH61376, USEPA STAR R826131, USEPA STAR R829429, and NIEHS ES013810. Back

2 Correspondence: Stuart Tobet, Department of Biomedical Sciences, W224 Anatomy Building, 1617 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1617. FAX: 970 491 7909; stuart.tobet{at}colostate.edu Back

Received: 28 March 2006.

First decision: 11 April 2006.

Accepted: 23 May 2006.

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