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BOR - Papers in Press, published online ahead of print October 17, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.007633
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BIOLOGY OF REPRODUCTION 68, 370–374 (2003)
DOI: 10.1095/biolreprod.102.007633
© 2003 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Estrogen Synthesis in Fetal Sheep Brain: Effect of Maternal Treatment with an Aromatase Inhibitor1

Charles E. Roselli2,a, John A. Reskoa, and Fredrick Stormshakb

a Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97201-3098 b Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331-6702


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8–1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02–0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (Km) for aromatase in the fetal sheep brain was 3–4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

aromatase, behavior, Cyp19, early development, estradiol, neuroendocrinology, sexual differentiation, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In many mammalian species, including sheep, exposure to testosterone (T) from the fetal testes permanently masculinizes and defeminizes adult sexual behavior, suppresses the surge mechanism for gonadotropin secretion, and advances the timing of puberty [1–3]. Our recent studies in rams suggest that prenatal exposure to T may also determine adult preferences regarding sexual partners [4]. The process that determines gender-specific brain function is referred to as sexual differentiation of the brain and normally accompanies the development of masculine genitalia, which is also controlled by testicular hormones during fetal development [5]. As initially determined by Short [6] and later refined by Clarke et al. [7–10], sexual differentiation in sheep occurs from approximately Days 30 to 100 of the 145-day gestation.

The action of T in the developing male is mediated, in part, through its aromatization to estradiol (E2) by cytochrome P450 aromatase and subsequent activation of the estrogen receptor [11]. The aromatization hypothesis was originally developed through research with rats and other short-gestation species, in which sexual differentiation occurs largely during the early postnatal period. Recent indirect evidence suggests that this mechanism may also apply to the sheep, an animal with a long gestation, in which masculinization occurs prenatally [12]. However, to our knowledge, it has never been demonstrated that the brain of the fetal sheep can aromatize androgens to estrogens.

Therefore, the present study was undertaken to test the hypothesis that the fetal sheep brain, especially the hypothalamus and preoptic area (HPOA), which are regions important for the control of neuroendocrine functions and sexual behavior, has the capacity to aromatize androgens to estrogens during the critical developmental period for sexual differentiation. We also determined whether administration of 1,4,5-androstatriene-3,17-dione (ATD), a potent aromatase inhibitor, could cross the placenta and act in the fetal brain to inhibit aromatization. If successful, this approach could allow us to probe the hormonal requirements for sexual differentiation of gonadotropin secretion and to determine whether the principles of sexual differentiation apply to the phenomenon of sexual orientation. Also, we compared male and female sheep to determine whether a sex difference in aromatase exists during the critical period when the brain becomes sexually differentiated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Eight mature Polypay ewes were bred for the present study. The sheep were maintained under standardized conditions at the sheep facility at Oregon State University. Animal husbandry and experimental protocols were conducted in accordance with the principles and procedures outlined by the National Institutes of Health and approved by the Institutional Animal Care Committee at Oregon State University. Four of the pregnant ewes were given s.c. implants containing the aromatase-inhibitor ATD. The other four ewes received sham surgeries and served as controls. Six constant-release Silastic implants, each containing 4 g of crystalline ATD, were placed s.c. in the axillae on Day 50 of pregnancy. Each packet released ATD at a rate of approximately 5 mg/day [13]. All surgeries were conducted under sodium pentothal anesthesia and maintained with halothane. Two weeks after implant placement or sham surgery (i.e., Day 64 of pregnancy), the ewes were again anesthetized, and the fetuses were delivered by cesarean section. Crown-rump lengths were equivalent for male and female sheep regardless of prenatal treatment (data not shown), indicating that ATD treatment did not adversely affect fetal growth. Brains were rapidly removed from the skull, dissected, and frozen on dry ice. Frozen brain tissue was stored at -80°C for subsequent determination of aromatase activity. During surgery, maternal jugular and umbilical venous and arterial blood were collected for subsequent hormone determinations. Five of the seven ewes carried twins, and two ewes, both treated with ATD, carried quadruplets. In total, the study included five ATD-treated male, five ATD-treated female, five sham-treated male, and three sham-treated female sheep.

Brain Dissection

Brains were dissected using ventral surface landmarks. The HPOA consisted of a block of tissue extending caudally from the anterior border of the optic chiasm to the posterior border of the mamillary bodies and dorsally to the roof of the third ventricle. The amygdala consisted of a block of ventromedial temporal lobe that had approximately the same rostrocaudal dimensions as the HPOA and that contained entorhinal cortex as well as the major cortical, medial, and basal amygdaloid nuclei. The midbrain extended from the posterior border of the mamillary bodies to the anterior border of the pons. The pons encompassed the entire pontine eminence, and the medulla extended from the caudal border of the pons to the caudal border of the olive. Brain areas were bisected along the sagittal plane, and half of the tissues were used to measure aromatase activity.

Aromatase Activity Assay

Aromatase activity was assayed in brain microsomes using a radiometric technique that quantifies the incorporation of tritium from [1ß-3H]androstenedione into 3H-labeled water. The technique was validated previously in our laboratory for sheep brain tissues [14]. The apparent Michaelis-Menten dissociation constant (Km) of aromatase in fetal amygdala as measured using 18–360 nM [1ß-3H]androstenedione was 3.8 nM. All measurements were performed in duplicate using a saturating concentration (300 nM) of substrate. The intra- and interassay coefficients of variation for the aromatase assay were 2.4% and 9.8%, respectively.

Steroid RIAs

Specific RIAs that have been described previously [15, 16] were used to measure steroid hormones in aliquots of serum (500 µl) that were extracted with ether and fractionated by Sephadex LH-20 (Sigma Chemical Co., St. Louis, MO) column chromatography. The mean percentages of recovery, water blanks, and intraassay coefficients of variability were as follows: T, 68.5%, 3.0 pg, and 5.3%, respectively; dihydrotestosterone (DHT), 81.4%, 4.2 pg, and 2.6%, respectively; androstenedione (A4), 71.0%, 19.4 pg, and 12.9%, respectively; E2, 80.3%, 1.9 pg, and 7.1%, respectively; estrone (E1), 83.7%, 4.2 pg, and 6.7%; and ATD, 93.6%, 2.0 pg, and 12.0%, respectively.

Data Analysis

Data were analyzed using the GB-STAT statistical package, version 7.0 (Dynamic Microsystems, Inc., Silver Springs, MD). Aromatase activity within each brain region was analyzed by two-way ANOVA (treatment x sex). Because no sex differences were present, the data for male and female sheep were combined, and the effect of treatment on each brain region was analyzed using individual Student t-tests. Hormone concentrations in the fetuses were analyzed by a three-way ANOVA (treatment x vessel x sex). The T and ATD data were analyzed nonparametrically with the Kruskal-Wallis H test, because their variances were heterogeneous. Hormone concentrations in the ATD-treated and control dams were compared using Student t-tests. Differences between groups were considered to be significant at P < 0.05 (two-tailed).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Aromatase Activity

Aromatase activity was widely distributed in the central nervous system of the fetal lamb at 64 days of gestation. No statistically significant sex differences were identified in any brain region. Therefore, the data from male and female sheep were combined according to treatment group to illustrate the distribution and treatment effect (Fig. 1). The highest concentrations of aromatase activity were measured in the HPOA and amygdala > midbrain > pons and frontal cortex > medulla > parietal cortex and olfactory bulb. Treatment of the pregnant dam with ATD for 14 days significantly suppressed aromatase activity in all fetal brain regions except the parietal cortex and olfactory bulb. This suppression was >75% in the HPOA and amygdala.



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  		FIG. 1. Microsomal aromatase activity in discrete brain regions from sheep fetuses (64 days of gestation) 2 wk after vehicle and ATD treatment. Two-way ANOVA (treatment x sex) was performed on the data for each area. Because no sex differences were found, the data from male and female sheep were combined according to treatment group to illustrate the distribution and effect of treatment. Values represent the mean (bars) ± SEM (vertical lines) for each brain area and treatment. The number of animals contributing to each mean is indicated at the base of each bar. *P < 0.05. AMYG, Amygdala; FCTX, frontal cortex; HPOA, hypothalamus-preoptic area; MB, midbrain; MD, medulla; OB, olfactory bulb; PCTX, parietal cortex

Fetal Serum Steroid Levels

After 2 wk of treatment, ATD was measurable in the serum of fetuses from treated dams but not in the serum from control fetuses. No differences in concentrations of ATD were observed between male and female fetuses (Fig. 2). The concentrations of ATD were equivalent in the umbilical artery and vein, suggesting that a steady-state equilibrium had been reached.



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  		FIG. 2. ATD concentrations in serum from the umbilical artery and vein of ATD-treated sheep fetuses on Day 64 of gestation, 2 wk after treatment had been initiated. The ATD was not detected in serum from control fetuses. See Materals and Methods for details of treatments. Data are presented as the mean (bars) ± SEM (vertical lines). The number of animals contributing to each mean is nine males and eight females. *P < 0.05

Treatment with ATD did not significantly affect serum T concentrations in the fetuses. Thus, the T data for each treatment were combined and presented according to sex and umbilical vessel. Male fetuses exhibited significantly greater circulating T than female fetuses, and T concentrations were significantly higher in the umbilical artery than in the umbilical vein (Fig. 3). Treatment with ATD did not significantly affect serum concentrations of E1, progesterone, T, DHT, or A4 in fetuses. However, serum E2 was lower in ATD-treated fetuses, but this difference failed to reach significance (F1,25 = 4.25, P = 0.05). Thus, the hormone data for each treatment were combined and presented according to sex and umbilical vessel. No significant differences were observed between male and female fetuses or between the arterial and venous umbilical blood (Fig. 4).



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  		FIG. 3. Concentrations of T in serum from control and ATD-treated sheep fetuses on Day 64 of gestation. See Materials and Methods for details of treatments. Because ATD treatment did not affect serum T concentrations, the data from ATD-treated and control groups were combined to allow comparisons between male (n = 9) and female (n = 8) sheep and umbilical vessels. See Figure 2 for other details



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  		FIG. 4. Gonadal hormone concentrations in serum from fetal sheep on Day 64 of gestation. E1, Estrone; E2, estradiol; P, progesterone; DHT, dihydrotestosterone; A4, androstenedione. See Figure 2 for other details

Maternal Serum Steroid Levels

Figure 5 presents the hormone data for control and ATD-treated ewes on Day 64 of pregnancy (i.e., 2 wk after treatments were initiated). Administration of ATD resulted in high systemic levels of the inhibitor in maternal blood, but it did not affect the profile of serum steroids at this stage of pregnancy. Low, but measurable, levels of androgens (T, DHT, and A4) and of estrogens (E1 and E2) were detected, whereas progesterone was present at substantially higher levels.



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  		FIG. 5. Gonadal hormone concentration in serum from control and ATD-treated pregnant ewes on Day 64 of pregnancy. See Figure 2 for details


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that high levels of cytochrome P450 aromatase are present in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation of the brain. Lower levels of aromatase activity were detected in the cortical areas and throughout the brain stem. Thus, the capacity for local estrogen synthesis is highest in areas of the brain thought to be involved in gonadotropin secretion and sexual behavior. However, the fact that activity is widespread within the brain stem and cortex supports the idea that androgen-derived estrogen may play a more global role during gestation in supporting neuronal growth and differentiation [17].

No major sex differences in aromatase activity were observed in any of the brain areas sampled from fetal sheep at 64 days of gestation. Because the critical period for sexual differentiation extends from approximately Day 30 to Day 100 of gestation in sheep, sex differences in brain aromatase could be expressed either earlier or later than Day 64. Previous investigations in several other long-gestation species that have been studied more extensively throughout their gestation have also failed to find sex differences in hypothalamic aromatase [18–21]. In contrast, sex differences in hypothalamic aromatase activity were detected in fetal ferrets [22], and in rats, sex differences appear to emerge at discrete times during the first week of life [23]. Nonetheless, the present results suggest that even if a sex difference in aromatase activity exists, it is not a constant feature throughout sexual differentiation in sheep. The observation that serum T concentrations are 10-fold higher in male than in female sheep indicates that the availability of substrate must also be considered an important factor limiting exposure of the developing sheep brain to estrogen. The much lower serum concentrations of A4 that were detected in both male and female sheep further suggest that T is the physiologically important substrate for aromatization at this time in development.

Of the steroid hormones measured in fetal serum, only T was clearly synthesized by the fetus. This is evident because T concentrations in male sheep were significantly higher in the umbilical artery than in the umbilical vein and significantly greater overall in males than in females. These results agree with earlier studies showing that the fetal testis begins to produce T by Day 30 of gestation. Concentrations of T in the serum of fetal male sheep peak initially at approximately Day 70, decline sharply between Days 80 and 90, and then rise again late in gestation [24, 25].

Surprisingly, treatment with ATD did not significantly suppress maternal serum estrogen levels and exerted only a trend toward suppression in the fetus. This observation suggests that ATD acts in a tissue-specific manner. This may be the result of differential uptake and metabolism of ATD or the effect of endogenous substrate competition. It could also indicate that different isozymes of aromatase are present in the sheep placenta and fetal brain. The pig (Sus scrofa) expresses three distinct isozymes of the aromatase protein that are specific for the gonads, placenta, and embryo [26]. Although aromatase isozymes in pigs share >90% amino acid sequence identity, they exhibit distinct catalytic properties and responses to different aromatase inhibitors [27]. To our knowledge, the possibility that sheep express isozymes of aromatase has never been explored.

Recent studies suggest that progesterone may play a pivotal role in sexual differentiation of the brain [28]. Our results demonstrate that moderately high levels of progesterone are present in the serum of fetal sheep during the critical period. It seems likely that the placenta is the source of progesterone, because no difference in concentration was found between the umbilical artery and vein. This is supported by the fact that pregnancy is maintained in ewes if ovariectomy is performed after Day 55 of gestation [29].

Aromatase activity was generally inhibited in the fetal brain after treating the mother for 2 wk with the suicide-inhibitor ATD, which is known to inactivate the enzyme. This effect did not appear to be secondary to alterations in systemic hormone levels in either the fetus or the mother. These results demonstrate that it is feasible to inhibit brain aromatase activity in utero by transplacental administration of ATD to study the hormonal requirements for sexual differentiation. This approach has been used successfully in ferrets and rats to deprive male fetuses of estrogen during the critical period [30, 31].

According to the aromatization hypothesis for sexual differentiation, estrogen derived from the local conversion of androgen in specific brain regions, such as the HPOA, act during the critical period of sexual differentiation to masculinize the brain. The critical period in rodents occurs mostly during the first few days after birth [11]. Exposure of rat pups to exogenous estrogen was more potent than T in inducing functional and morphological brain masculinization [1]. Subsequent studies showed that inhibition of aromatase activity or antagonism of the estrogen receptor interferes with masculinization of the brain, leading to feminized patterns of gonadotropin secretion, decreased mounting behaviors, increased lordosis, and altered preferences regarding sexual partners [1, 32–34]. In animals with a long gestation, such as sheep, ferrets, and primates, the critical period for sexual differentiation occurs prenatally and is difficult to manipulate. Nonetheless, Baum et al. [30, 35] demonstrated in ferrets that transplacental administration of ATD blocks development of the sexually dimorphic nucleus of the preoptic area/anterior hypothalamus and diminishes masculine sexual behavior.

The contribution of estrogen to brain sexual differentiation in sheep has not been fully determined. A recent study by Masek et al. [12] suggests that aromatization of prenatal T mediates differentiation of the surge control of LH from operating in male sheep, because exposure of fetal ewes to T, but not to the nonaromatizable androgen DHT, blocks the LH surge response to estrogen priming. In contrast, both T and DHT treatment advances the pubertal rise in LH, suggesting that the tonic mode of gonadotropin secretion is masculinized by androgen. In sheep, androgens also appear to masculinize juvenile play behavior [36] and to masculinize sexual behavior [7, 10, 37]. However, to our knowledge, nothing is yet known about the role of aromatization in these processes. The differential sensitivity to the organizational actions of different steroids and the high levels of aromatase found the fetal sheep brain strongly suggest that locally formed estrogens play an important organizational role during development. The demonstration that aromatase can be effectively inhibited in utero will make it possible to study the effect of estrogen deprivation on masculine sexual differentiation in sheep.


   ACKNOWLEDGMENTS
 
The authors gratefully thank Henry Stadelman, Sara Supancic, and Ericha Cross for technical assistance. Hormone assays were performed by the RIA core facility at the Oregon National Primate Research Center under the supervision of Dr. David Hess.


   FOOTNOTES
 
1 Supported by NIH grant RR14270 (C.E.R.). Back

2 Correspondence: Charles E. Roselli, Department of Physiology and Pharmacology L334, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. FAX: 503 494 4352; rosellic{at}ohsu.edu Back

Received: 22 May 2002.

First decision: 20 June 2002.

Accepted: 7 August 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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