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Endocrinology, doi:10.1210/en.2007-0454
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Endocrinology Vol. 148, No. 9 4450-4457
Copyright © 2007 by The Endocrine Society

The Ovine Sexually Dimorphic Nucleus of the Medial Preoptic Area Is Organized Prenatally by Testosterone

Charles E. Roselli, Henry Stadelman, Reed Reeve, Cecily V. Bishop and Fred Stormshak

Department of Physiology and Pharmacology (C.E.R., H.S.), Oregon Health and Science University, Portland, Oregon 97239; and Department of Animal Sciences (R.R., C.V.B., F.S.), Oregon State University, Corvallis, Oregon 97331

Address all correspondence and requests for reprints to: Dr. Charles E. Roselli, Department of Physiology and Pharmacology L334, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97239-3098. E-mail: rosellic{at}ohsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A sexually dimorphic nucleus that can be identified in adult sheep by its characteristic pattern of cytochrome P450 aromatase mRNA exists in the preoptic/anterior hypothalamic area and is called the ovine sexually dimorphic nucleus (oSDN). In other species, male-typical sexually dimorphic preoptic nuclei develop under the influence of gonadal testosterone exposure. Thus, we hypothesized that the oSDN develops before birth in the sheep and is organized by exposure to testosterone. To test this, we determined whether an identifiable oSDN is present in the fetal lamb brain at d 130–140 gestation (term ~150 d). In situ hybridization for aromatase mRNA revealed a cell group in the caudal preoptic area that corresponded to the oSDN in adults. Quantitative analysis showed that the mean volume of the oSDN in late-gestation fetuses was significantly larger in male than in female lamb fetuses. We next treated a group of pregnant ewes with testosterone propionate (TP) from d 30–90 gestation and measured oSDN volumes in TP-exposed and control fetuses on d 135 gestation. The mean volume of the oSDN in female fetuses exposed to TP was significantly larger than in control females and also larger than in control and TP-exposed males. Taken together, these data demonstrate that testosterone acts during a prenatal critical period to organize the development of aromatase-expressing neurons into the male-typical oSDN in sheep.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OVINE SEXUALLY dimorphic nucleus (oSDN) is a group of aromatase-expressing neurons that occupy the central component of the medial preoptic nucleus (MPNc) of the sheep brain (1). The volume of the oSDN is 2- to 3-fold greater in rams that are sexually attracted to ewes (female-oriented rams) than in either ewes or in rams that are sexually attracted to other rams (male-oriented rams). Thus, the preference of individual rams for male vs. female mounting partners correlates directly with the volume of the oSDN suggesting that the size oSDN and the number of aromatase-expressing neurons it contains contribute in some unknown way to sexual attraction in rams.

Studies using ferrets and rats have more directly linked sexual preference to the function of the medial preoptic-anterior hypothalamic nucleus (MPOA/AH) (2). Bilateral lesion of the sexually dimorphic MPOA/AH of male ferrets causes them to exhibit a female-like partner preference, i.e. an attraction for other males rather than for sexually receptive females when they were treated with estrogen as adults (3, 4). Similar results were observed in rats after bilateral lesion of the MPOA/AH (5). Damage to the MPOA/AH in either female ferrets (3) or rats (5) did not alter their male-oriented partner preference. Recently it was demonstrated that the female-typical partner preference of MPOA/AH-lesioned males reflects the subjects’ attraction to male body odors and correlates with an increase in the ability of soiled male bedding to induce a Fos response in the MPOA (6). Earlier studies in rats clearly demonstrated that sexual partner preferences and Fos responses to pheromonal stimulation are sexually differentiated in response to brain estrogen synthesis during early neonatal life (7, 8). Taken together, these results in sheep, ferrets, and rats indicate that male-typical preference for an estrous female depends on some sexually differentiated functional characteristic of intact MPOA/AH neurons, perhaps related to hormone sensitivity or connectivity, that appears to be essential for sexually dimorphic processing of afferent sensory cues.

In short-gestation mammals, such as rats and gerbils, the sexually dimorphic MPOA/AH is organized during a critical developmental period by perinatal exposure to testosterone (T) produced by the developing testis, which masculinizes adult sexual behavior and the morphology of the MPOA/AH (9, 10, 11). In guinea pigs and ferrets, however, masculinization of the MPOA/AH depends on exposure to T predominantly or entirely during fetal development (12, 13).

Sexual differentiation of the sheep brain occurs before birth sometime between embryonic d 30–100 of a 147-d gestation period. Systemic concentrations of T are significantly higher in male fetuses than in female fetuses during this period (14, 15). Females born to mothers treated prenatally with T are behaviorally masculinized and defeminized. They fail to show sexual receptivity but instead display increased mounting and aggressive behavior as adults (16, 17, 18, 19). Prenatal exposure to T also blunts estrogen positive feedback on LH secretion, decreases LH sensitivity to progesterone (P) suppression, and advances the onset of puberty (20, 21).

To better understand the process of brain sexual differentiation in sheep, we first determined whether the oSDN was present in late-gestation fetuses (d 130–140 gestation). Once established, we then examined the effect of prenatal T exposure from d 30–90 gestation on the organization of this nucleus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Eighteen timed pregnant ewes (Ovis aries) of mixed Western breeds were purchased from local farmers or bred at the sheep facility at Oregon State University. Animal care was conducted in accordance with the principles and procedures outlined by the National Institutes of Health. The Institutional Animal Care and Use Committees at Oregon Health and Science University and Oregon State University approved all experimental protocols.

Experiment 1: identification of oSDN in late-gestation fetuses
Late gestation male (n = 11) and female (n = 9) lamb fetuses obtained from pregnant ewes between gestational days (GD) 130–140 (mean gestation age ± SEM = 135 ± 0.8 d) were evaluated for the presence of the oSDN by the pattern of aromatase mRNA expression within sequential coronal sections of the preoptic area/anterior hypothalamus. Accompanying Nissl-stained sections were used to confirm the presence and location of the oSDN.

Experiment 2: prenatal T propionate (TP) treatment
Lamb fetuses (n = 8) were exposed to exogenous androgens by giving twice-weekly injections of TP (Steraloids Inc., Newport, RI) to their mothers (100 mg in 2 ml corn oil, im) from d 30–90 of pregnancy. TP is a long-acting 17-alkylated derivative of T, which is hydrolyzed before acting (22). This treatment regimen has been used previously to androgenize/masculinize female lambs (23, 24). Control lamb fetuses (n = 8) were from mothers untreated during gestation.

Jugular blood samples were collected twice a week from the TP-treated dams throughout the treatment period and for 2 wk after treatment ended. After about 7 wk of TP treatment, a series of frequent blood samples (i.e. every 2 h for the first 12 h, then at 24, 28, 32, and 48 h after TP injection) were drawn from the dams to obtain a profile of serum T for pharmacokinetic analysis.

To determine what fraction of T from the maternal circulation reached the fetus, three pregnant ewes were treated with TP twice weekly from d 30–85 of pregnancy as described above. Lamb fetuses (n = 6, a single male, twin males, and a triplet set of two males and a female) were delivered on GD 85 about 26 h after the mother received the second TP injection of the week. This time was chosen because it approximated the elimination half-life of T in the maternal circulation.

Tissue and blood collection
Ewes were euthanized with an iv overdose of sodium pentobarbital (Euthasol; Virbac Corp., Fort Worth, TX). The ewe’s abdomen and uterus were opened surgically, exposing the deeply anesthetized fetus. Blood samples were taken from the umbilical artery and vein. Heparin (10,000 U) was given through the umbilical vein, followed by 10 ml saturated KCl to euthanize the fetus and arrest the heart in diastole. The umbilical cord was cut, and the lamb fetus was weighed. The sex of the fetus was established by direct visual inspection of the gonads. The brain was removed from the fetal skull, and a diencephalic block that extended from the anterior margin of the optic chiasm to the mammillary bodies was dissected. This tissue contained the major nuclei of the preoptic area, anterior hypothalamus, and medial basal hypothalamus. Tissue blocks were immersed in 4% paraformaldehyde overnight at 4 C, cryoprotected in 20% sucrose, rapidly frozen in isopentane at –55 C, and then stored at –80 C.

In situ hybridization
Fixed tissues were sectioned coronally (40 µm thick) into four parallel series and stored in RNase-free cryoprotectant (30% ethylene glycol plus 20% glycerol in 0.025 M phosphate buffer) at –20 C until mounted onto microscope slides after which these were desiccated under a vacuum and stored frozen at –80 C. Expression of cytochrome P450 aromatase mRNA was detected using a sheep-specific 33P-labeled riboprobe and procedures described previously (25). An adjacent series of brain sections were stained with thionin for anatomical verification.

Image analysis
The volume of the oSDN and the anteroventral periventricular nucleus (AVPV) and the height of the preoptic area (i.e. distance between the optic chiasm and anterior commissure) were determined with NIH Image software (version 1.62). Two investigators unaware of the sex or age of the animals made measurements independently. The mean of these measurements were used for statistical comparisons.

Steroid hormone measurements
Serum was harvested from blood clotted overnight at 4 C and centrifuged at 1000 x g for 15 min. T, estradiol (E2), and P were measured in serum aliquots from umbilical artery blood by RIA after extraction with ethyl ether and fractionation on Sephadex LH-20 column chromatography as described previously (26). In pregnant dams treated with TP, T was measured in maternal blood after ether extraction but without prior chromatography. The antibody used to measure T has 67% cross-reactivity for dihydrotestosterone (26); however, preliminary studies found that dihydrotestosterone concentrations are negligible throughout pregnancy in sheep (<20 pg/ml). The mean percentage of recovery, water blanks, and intraassay coefficients of variation were as follows: T, 76.6%, 3.8 pg/ml, and 8.8%; E2, 63.9%, 1.5 pg/ml, and 13.8%; and P, 64.2%, 11.8 pg/ml, and 13.0%.

Statistical analysis
In experiment 1, comparisons of nuclear volumes and hormone levels between sexes were analyzed by Student’s t tests. In experiment 2, comparisons by sex and treatment group were analyzed by two-way ANOVA followed by post hoc Newman-Keuls test when appropriate. Maternal T levels were analyzed by one-way ANOVA for repeated measures. Noncompartmental pharmacokinetic analysis of T elimination half-life was performed with Winnonlin Analysis Program version 4.1 (Pharsight Corp., Mountain View, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: the oSDN is present in late-gestation fetuses
In situ hybridization for aromatase mRNA revealed a cell group in the preoptic area of late-gestation sheep fetuses (GD 130–140) that corresponded in location to the oSDN in adults (Fig. 1Go). The oSDN was evident in Nissl-stained sections, and individual neurons appeared to be heavily labeled with P450 aromatase cRNA probe (Fig. 2Go).


Figure 1
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  		FIG. 1. Autoradiographic images of coronal sections through the POA and hypothalamus of a late-gestation male sheep fetus (GD 133) showing the expression of cytochrome P450 aromatase mRNA. A–F are organized in a rostral to caudal order at approximately 420-µm intervals through the hypothalamus. ac, Anterior commissure; oc, optic chiasm; PVpo, periventricular preoptic nucleus; v, third ventricle.

 

Figure 2
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  		FIG. 2. Photomicrographs showing the dark-field distribution of aromatase mRNA in the oSDN (left) in a fetal ram lamb (GD 134 d) and the brightfield histology (middle) from the adjacent section (x10 magnification). Right panelshows high-magnification (x100) bright-field photograph of aromatase mRNA signal (silver grains) over neurons (arrows) counterstained with hematoxylin.

 
In addition to oSDN, the autoradiographic images in Fig. 1Go also illustrate that aromatase mRNA is abundantly expressed within the AVPV and principal component of the bed nucleus of the stria terminalis (BSTpr). High levels of hybridization signal were also detected in the caudal part of the MPN (MPNcp), the hypothalamic paraventricular nucleus (PVN), and ventrolateral part of the ventromedial nucleus of the hypothalamus (VMNvl). Lower levels of expression were found in the periventricular preoptic nucleus and the MPN. Only background levels of signal were present in tissue hybridized with sense probe (data not shown).

Quantitative analysis of aromatase mRNA expression (Fig. 3AGo) showed that the mean volume of the oSDN in late-gestation fetuses was approximately 2-fold larger in male than in female lamb fetuses (P < 0.01). This sex difference was specific to the oSDN and not associated with general morphological differences in the MPOA of male and female fetal lambs, because no sex differences were seen in the volume of the nearby AVPV (Fig. 3BGo) or in the height of the preoptic area (mean ± SEM: male, 4.29 ± 0.08 mm; female, 4.40 ± 0.12 mm). There were no significant sex differences in the concentration of T (male, 197.5 ± 37.9 pg/ml, n = 6; female, 159.4 ± 18.8 pg/ml, n = 10), E2 (male, 48.2 ± 20.5 pg/ml, n = 6; female, 24.8 ± 7.4 pg/ml, n = 10), or P (male, 1.5 ± 0.2 ng/ml, n = 6; female, 2.0 ± 0.4 ng/ml, n = 10) in the serum from the umbilical arteries of the fetal lambs studied.


Figure 3
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  		FIG. 3. Sex comparisons of oSDN (A) and AVPV (B) volumes in late-gestational sheep fetuses (GD 135 ± 0.8 SEM). Data are presented as means ± SEM.

 
Experiment 2: prenatal exposure of females to exogenous TP increased the size of the oSDN
Pregnant ewes were injected twice weekly with TP and bled at regular intervals after each injection to monitor the consistency of T exposure over the 60 d of treatment. Figure 4AGo shows that the mean concentration of T in maternal serum 24 h after the first injection (F8,32 = 0.72; P = 0.67) or 36 h after the second injection (F7,28 = 1.93; P = 0.10) was unchanged statistically over wk 5–12 of treatment, indicating the relative consistency of exposure produced by this treatment. The elimination half-life of T in maternal serum calculated using a noncompartmental model was 30.0 ± 5.0 h (Fig. 4BGo).


Figure 4
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  		FIG. 4. A, Serum concentrations (mean ± SEM) of T in pregnant sheep treated with TP twice weekly from GD 30–90 (n = 4). Bleed 1 represents T levels 24 h after the first weekly injection, and bleed 2 represents T levels 36 h after the second weekly injection. B, Pharmacokinetic profile of the serum concentration of T (mean ± SEM) after a single injection of TP on wk 3 of treatment. The elimination half-life is 30.0 ± 5.0 h.

 
The concentration of T in GD-85 fetuses measured about 26 h after injection of TP into the mother was significantly higher in the umbilical vein (695 ± 81 pg/ml) than in the artery (571 ± 62 pg/ml) reflecting that the source of T was derived from injection of the mother. These concentrations were two to three times higher than levels measured in the umbilical artery of age-matched control male fetuses (278 ± 133 pg/ml, n = 4; P < 0.05 vs. TP-exposed fetuses) and represented 5.2 ± 0.6% (vein) and 4.2 ± 0.5% (artery) of average maternal serum T concentration (13.5 ± 0.9 ng/ml).

Prenatal exposure to T completely masculinized the external genitalia of the female lambs and increased the ano-genital distance consistent with males (data not shown). T-exposed female lamb fetuses were easily identified by the presence of ovaries and uterus and the absence of testes in the scrotum.

Two-way ANOVA revealed a significant effect of prenatal TP exposure on oSDN volume (F1,12 = 36.2; P < 0.0001; Fig. 5AGo). There was no overall effect of sex (F1,12 = 0.04; P = 0.8), but there was a significant interaction between sex and TP exposure (F1,12 = 62.8; P < 0.0001). Newman-Keuls post hoc comparisons revealed the significant interaction was due to the fact that oSDN was significantly larger in control males than in control females but larger in TP-exposed females than in control and TP-exposed males. There was no difference in oSDN volume between control males and TP-exposed males.


Figure 5
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  		FIG. 5. Effect of prenatal T exposure on oSDN (A) and AVPV (B) volumes. Pregnant ewes received injections of TP twice a week from GD 30–90 so that their fetuses were exposed to elevated systemic levels of T during the critical period for sexual differentiation. Control animals were not injected (see Materials and Methods for additional details). Data are presented as means ± SEM (n = 4 animals per group). Bars with different superscript letters differ at P < 0.05.

 
There was a significant overall effect of TP exposure on the volume of AVPV (F1,12 = 5.2; P = 0.04; Fig. 5BGo) reflecting the approximately 20% smaller volume of AVPV in both TP-exposed male and female fetuses vs. the controls. However, there was no overall effect of sex (F1,12 = 0.2; P = 0.7) and no interaction between treatment and sex (F1,12 = 0.03; P = 0.9). There were no significant effects of sex or T exposure on POA height or brain weight (Table 1Go). However, our results agree with a previous study (27), in which fetal body weight was found to be significantly lower in fetuses exposed to exogenous T (Table 1Go).


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  		TABLE 1. Effect of prenatal T exposure from d 30–90 of gestation on POA height, brain weight, and body weight of fetal lambs at d 135 of gestation

 
There were no significant effects of prenatal TP exposure or sex on the concentrations of T and P in the serum from the umbilical arteries of control and TP-exposed fetuses (Table 2Go). However, there was an overall effect of TP exposure on E2 concentrations in umbilical artery serum (F1,12 = 11.2; P < 0.01), which appeared to be due to a suppression of E2 concentrations in both male and female fetuses exposed to TP earlier in gestation (i.e. GD 30–90).


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  		TABLE 2. Effect of prenatal T exposure from d 30–90 gestation on steroid concentrations in serum from umbilical artery of fetal lambs at d 135 gestation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate that there is a cluster of aromatase mRNA-expressing cells in the caudal POA that anatomically defines the MPNc in late-gestation fetal lambs. We further found that the volume of this nucleus is approximately 2-fold larger in males than in females, suggesting that it is the fetal equivalent to the oSDN described previously in adult sheep (1). Additional evidence that this is the nascent oSDN was provided by our demonstration that prenatal TP exposure was effective in promoting its cellular organization, such that nuclear volume in TP-exposed females resembled the volume in control males and was significantly larger than in control females. These results suggest that T exposure during midgestation is sufficient to morphologically masculinize the oSDN.

Our results are similar to previous findings in guinea pigs (13) and ferrets (28), which demonstrated that normal development of the sexually dimorphic preoptic area takes place prenatally during the time when there are marked and consistent sex differences in androgen levels. In ferrets, the sexually dimorphic male nucleus in the POA/AH is identifiable as early as embryonic d 37 of a 41-d gestation and is organized by estrogen derived from aromatization of systemic T (12, 28). The SDN-POA in rats is also organized by androgen-derived estrogen, but in contrast to ferrets, the first statistical difference in volume occurs on d 1 of postnatal life and continues to increase in the male over the next 7–10 d of postnatal life (29). Aromatase mRNA is expressed in rat SDN as early as d 16 gestation but does not appear to be sexually dimorphic until postnatal life (30). The homologous sexually dimorphic area of the POA pars compacta in gerbils is present at birth in both sexes but disappears over the next 2 wk of life in females (10). The neural organization of the sexually dimorphic area of the POA pars compacta is not completely under the control of either neonatal exposure to T or estrogen and requires prenatal exposure to T for complete masculinization (31). Sexual differentiation of the human hypothalamus is poorly understood. Three independent groups of investigators (32, 33, 34) agree that the third interstitial nucleus of the anterior hypothalamus (INAH3) is significantly larger in heterosexual men than in women. In contrast, Swaab et al. (35) found a different sexually dimorphic nucleus in the human hypothalamus that appears to share some similarities in localization, cytoarchitecture, and neurotransmitter content with the SDN-POA of the rat. Allen et al. (32) gave this nucleus the name INAH1, but neither Allen et al. (32) nor LeVay (34) nor Byne et al. (36) found a sex difference in the SDN-POA/INAH1. Unfortunately, no data exist that link fetal/neonatal differences in T exposure to the differentiation of male-typical SDN-POA/INAH1 or INAH3 volume in humans, although Swaab and Hofmann (37) have reported that the SDN-POA/INAH1 is not sexually dimorphic until after 4 yr of age.

The high level of aromatase mRNA expression in the oSDN makes it likely that estrogen is involved in some aspect of neural differentiation as demonstrated for rats and ferrets. For example, locally produced estrogens could enhance neuronal survival, specify neuronal phenotype, stimulate neurite outgrowth, and promote synaptogenesis during development, all of which are believed to contribute to the development of sexually dimorphic circuits in the mammalian forebrain (38). Recently we demonstrated that mRNA for estrogen receptor {alpha} (ER{alpha}), as well as androgen and P receptors, are expressed in the fetal sheep POA at GD 64 during the critical period for sexual differentiation (39). However, experiments have not yet been performed to determine whether estrogenic metabolites of T are essential for organizing the oSDN.

The possibility that differentiation of the oSDN continues after birth, as it does in other species, was not addressed in the present study but should be a focus for future research along with a more detailed description of its prenatal ontogeny. In addition, although there were 45 d between when TP exposure ended and when oSDN volume changes were measured, the current study does not demonstrate that the effects observed are permanent and would persist into adulthood. Nonetheless, the fact that a sex difference in oSDN exists at such a young age before the period of early social interactions raises the possibility that it exerts a sexually dimorphic function during postnatal life when sexual behaviors and attractions are being formulated.

We did not observe a sex difference in AVPV volume in fetal lambs despite the high levels of aromatase found in this nucleus. In rats, the AVPV is larger in females than in males and is a critical component of the circuit regulating estrogen-induced positive feedback on gonadotropin secretion (40, 41). Structural sexual dimorphisms in the AVPV of the rat are organized perinatally by gonadal steroids through an ER-dependent mechanism but do not appear until around the time of puberty (42, 43). This may explain why AVPV is not sexually dimorphic in fetal lambs. It is also evident, however, that the site of estrogen-induced positive feedback in sheep is within the medial basal hypothalamus and that AVPV may not have a sexually dimorphic function in this species (44).

The present results show that T organizes the oSDN morphologically during the same period that it also influences behavioral sexual differentiation. Prenatal exposure to T from approximately GD 30–90 defeminizes the sexual behavior of ewes (18, 19). Adult ewes that were previously exposed to androgen in utero as fetuses do not express female sexual receptivity when left intact or after exogenous E2 treatment and show signs of altered gonadotropin secretion in response to E2 and P (18, 20, 23). Moreover, androgen exposure prenatally was reported to masculinize ewes by enhancing spontaneous mounting behavior and attraction to other females (16, 17, 18, 45), although it has also been argued that this may more accurately represent an extension of the juvenile state in which male-like patterns of behavior appear independent of hormones (19).

The role aromatization plays in the developmental organization of sheep sexual behavior remains unresolved. Masek et al. (45) suggested that aromatization of T mediates behavioral sexual differentiation in sheep because exposure of fetal ewe lambs to T, but not to the nonaromatizable androgen dihydrotestosterone, facilitated the expression of male sexual behaviors and inhibited female mating behaviors in these animals when they attained adulthood. In contrast, rams that were exposed to the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) prenatally from GD 50–80 exhibited only a modest decrease in mounting behavior and did not display altered sexual partner preferences or enhanced female receptivity in response to P and E2 treatment as adults (46). These results indicate that either prenatal aromatization of T is not an absolute requirement for the masculinization and defeminization of sheep behavior or that sufficient estrogen synthesis remained for defeminization to occur even in the presence of the aromatase blocker (15). However, it is also possible, as suggested for long-gestation ferrets (47), that the fetal actions of estrogens produced by brain aromatization are involved in the initiation of sexual differentiation and that continued exposure to elevated levels of T during gestation and early postnatal life are needed to complete this process. Indeed, recent research has provided strong evidence that postnatal steroid exposure is needed to completely defeminize the estrogen positive-feedback mechanism in the sheep (48). Thus, further research is needed to determine the timing and hormonal requirements for behavioral and brain sexual differentiation in the sheep.

The present study provides the first detailed information regarding the anatomical distribution of aromatase mRNA expression in the hypothalamus of fetal lambs. As in adult sheep, the greatest expression of aromatase mRNA was found in the BSTpr and MPN, including the MPNc or oSDN (25). Based on work in several species, this interconnected neural circuit is essential for the expression of sexual attraction and copulatory behaviors in males (49, 50, 51, 52). The expression of aromatase mRNA levels in VMNvl appeared to be considerably higher in the fetus than in the adult, although quantitative comparisons were not performed. Evidence in adult rams implicates VMN as an important site at which estrogen acts to suppress LH secretion in males (52). Expression of aromatase mRNA was also evident in the dorsolateral part of the PVN, where neurons expressing ER{alpha} and ERß are also localized (53). Aromatase expression in PVN was not observed in previous studies of fetal and adult mammals (54, 55, 56) Although the function of aromatase in PVN is not known, perhaps it is also involved in the steroidal control of copulation because parvocellular oxytocinergic neurons in the PVN express ERß and project to lumbar spinal cord where they influence copulatory behavior and seminal emission (57, 58). Taken together, this pattern of distribution suggests that in the sheep fetus, aromatase plays a role in establishing neural circuits that will be important for the central coordination of reproductive behaviors and neuroendocrine function.

In rats, aromatase-expressing neurons have been categorized into three distinct groups according to their peak expression during developing and adult stages (59). Although we have examined only a limited period in sheep development, it is apparent that aromatase mRNA expression throughout the steroid-sensitive preoptic-hypothalamic-limbic circuitry is expressed at higher levels during late gestation in fetuses than in adults.

Systemic concentrations of gonadal steroids did not differ between the sexes during late gestation in fetal sheep. However, T concentrations were consistently higher and more variable in males than in females. In contrast, serum T concentrations are significantly higher in males than in females when sampled during midgestation at GD 64 (15). Our results agree with an earlier study that demonstrated the greatest sex difference in fetal sheep occurs during midgestation when sexual differentiation of the brain presumably occurs in sheep (14). Although there are species variations in the developmental profile and magnitude of sex differences in systemic T concentrations (60), generally the greatest sex difference in systemic T is observed during the critical period for sexual differentiation. In the current study, TP was administered to pregnant ewes to masculinize genetic female fetuses. We found that this treatment regimen acutely elevated T in fetuses to levels that were twice as high as age-matched control males. Moreover, although this treatment did not appear to have any lasting effects on systemic T and P concentrations, it did produce a persistent and significant reduction in E2 concentrations measured in both male and female fetuses. This reduction in E2 cannot explain the morphological differences observed; however, it may affect other aspects of endocrine development and should be further studied.

In summary, the present study shows that T acts during the prenatal critical period to organize the development of aromatase-expressing neurons into the male-typical oSDN in the sheep fetus. It remains to be determined what contribution aromatase makes to this process.


   Acknowledgments
 
We gratefully acknowledge Dr. Kent Thornburg (K.T.) for providing the control fetal tissues used in experiment 1 and to Drs. Samantha Louey and Sonnett Junker for help with surgeries.


   Footnotes
 
This work was supported by National Institutes of Health Grants R01 RR014270 (C.E.R.) and P01 HD034430 (K.T.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 31, 2007

Abbreviations: AVPV, Anteroventral periventricular nucleus; BSTpr, principal component of the bed nucleus of the stria terminalis; E2, estradiol; ER{alpha}, estrogen receptor {alpha}; GD, gestation day; INAH3, third interstitial nucleus of the anterior hypothalamus; MPNc, central component of the medial preoptic nucleus; MPOA/AH, medial preoptic-anterior hypothalamic nucleus; oSDN, ovine sexually dimorphic nucleus; P, progesterone; PVN, paraventricular nucleus; T, testosterone; TP, testosterone propionate; VMNvl, ventrolateral part of the ventromedial nucleus of the hypothalamus.

Received April 9, 2007.

Accepted for publication May 22, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Roselli CE, Larkin K, Resko JA, Stellflug JN, Stormshak F 2004 The volume of a sexually dimorphic nucleus in the ovine medial preoptic area/anterior hypothalamus varies with sexual partner preference. Endocrinology 145:478–483[Abstract/Free Full Text]
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L. M. Jackson, K. M. Timmer, and D. L. Foster
Sexual Differentiation of the External Genitalia and the Timing of Puberty in the Presence of an Antiandrogen in Sheep
Endocrinology, August 1, 2008; 149(8): 4200 - 4208.
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